Biomedicine & Pharmacotherapy 87 (2017) 145152

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Preliminary report

Anti-diabetic effect of three new norditerpenoid alkaloids in vitro and

potential mechanism via PI3K/Akt signaling pathway
Dan Tanga , Qi-Bin Chena,b , Xue-Lei Xina,* , Haji-Akber Aisaa,*
a
The Key Laboratory of Plant Resources and Chemistry of Arid Zone and State Key Laboratory Basis of Xinjiang Indigenous Medicinal Plants Resource
Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi 830011, Peoples Republic of China
b
University of Chinese Academy of Sciences, Beijing 100039, Peoples Republic of China

A R T I C L E I N F O A B S T R A C T

Article history:
Received 10 October 2016 Diabetes is a metabolic disease with the characteristic of high blood glucose (hyperglycemia). In our
Received in revised form 2 December 2016 previous study, we found that nigelladines AC (compounds AC), three norditerpenoid alkaloids from
Accepted 14 December 2016 the seeds of Nigella glandulifera Freyn (Ranunculaceae) exhibited protein of tyrosine phosphatase 1B
(PTP1B) inhibitory activity in vitro. In the present study, we further investigated their anti-diabetes
Keywords: activities in L6 moytubes and illuminated the mechanisms of action of compounds AC. Several
Diabetes parameters of glucose metabolism such as glucose consumption, glycogen content and hexokinase
Norditerpenoid alkaloids activity were increased by compounds AC. The results suggested that compounds AC improved glucose
Anti-diabetic activity
metabolism through promoting synthesis of glycogen. Expression of PTP1B protein was inhibited by
Glucose metabolism
compounds AC in L6 moytubes. PI3K-dependent Akt phosphorylation was found to be activated by
Protein tyrosine phosphatase 1B
PI3K/Akt signaling pathway compounds A-C and completely blocked by wortmannin (a PI3K inhibitor). Moreover, the insulin-
mediated induction of insulin receptor substrate-1 (IRS-1) and glycogen synthase kinase-3b (GSK-3b)
were also suppressed by wortmannin. Western blot results indicated that compounds AC-induced IRS-
1/Akt activation was likely a consequence of PTP1B inhibition. Compounds AC promoted glycogen
synthesis through Akt-mediated GSK3 phosphorylation. Therefore, activation of PI3 K/Akt insulin
signaling pathway and suppression of PTP1B is the molecular mechanism that contributes to the anti-
diabetic effect of compounds AC in cellular models. The three alkaloids potentially serve as lead
compounds for the development of antidiabetic drugs.
2016 Elsevier Masson SAS. All rights reserved.

1. Introduction Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator

Type 2 diabetes mellitus (T2DM) has a high prevalence all over
the world and the incidence will be doubled by 2025 [1]. T2DM is a
chronic disease accompanied by a series of metabolic disorders
due to insulin deciency or impaired insulin action [2]. Insulin
plays an important role in the regulation of plasma glucose
homeostasis, and promotes glucose consumption, glycogenesis
and protein synthesis of skeletal muscle tissue through the
tyrosine kinase receptor pathway [3]. Impairment of insulin
signaling pathway induces insulin resistance and leads to
hyperglycemia and hyperlipidemia [4]. Therefore, it is very
important to develop a drug for improving glucose metabolism
and insulin resistance in the treatment of T2DM.
* Corresponding authors.
E-mail addresses: xinxl@ms.xjb.ac.cn (X.-L. Xin), haji@ms.xjb.ac.cn (H.-A. Aisa).
of insulin signaling pathway, which inhibits insulin-stimulated
tyrosine phosphorylation of insulin receptor (IR) and insulin
receptor substrate-1 (IRS-1) [5]. PTP1B has been a potential drug
target in the treatment of T2DM [6]. Insulin binds to its receptor to
activate the tyrosine kinase activity of receptor through dimeriza-
tion. IRS-1 is phosphorylated at tyrosine residues by the insulin
receptor (IR) [7], which leads to recruitment and activation of
phosphatidylinositol 3-kinase (PI3K) [8]. PI3K-dependent Akt
phosphorylation is increased and then leads to activation of
signaling cascades involving PI3 K/Akt/GSK-3b, which promotes
glycogen synthesis and glucose utilization for the metabolic
activities in skeletal muscle [911].
The current therapies of T2DM are very limited by the few
choices of existing drugs and the side effects of synthetic drugs.
Thus traditional Chinese herbal and ethnic medicines draw more
and more attention, particularly medicinal plants and natural
products, for the treatment of T2DM [12,13]. Natural products
extracted from medicinal plants are characterized by their

http://dx.doi.org/10.1016/j.biopha.2016.12.058
0753-3322/ 2016 Elsevier Masson SAS. All rights reserved.
146 D. Tang et al. / Biomedicine & Pharmacotherapy 87 (2017) 145152

chemical diversity and special bioactivities, and used as a primary
source for synthesized drugs [14]. Seeds from Nigella glandulifera
Freyn (Ranunculaceae) have been used as a traditional Uighur
medicinal plant and food component in Xinjiang of China [15].
Chemical constituents of the Nigella plants have been investigated
for their antidiabetic and antioxidant potentials in the treatment of
diabetes [16,17]. Although the chemical composition of N.
glandulifera has been investigated in several studies, the mecha-
nism of action is not fully understood [18]. In our earlier study,
nigelladines AC (compounds AC) were found to possess a good

Fig. 1. HPLC results of nigelladines (AC). (A): nigelladine A; (B): nigelladine B; (C): nigelladine C. Experimental conditions: Phenomenex Gimini 5 mm C18 110
(250 mm
4.6 mm i.d.) column; column temperature: 30  C; ow rate: 1 ml/min; detection: 365 nm; injection volume: 10 ml; mobile phase: MeOH0.05% TEA in water, 0
10 min 40%60%, 1030 min 60%75% 3037 min 75% 3740 min 75%100% MeOH, 4047 min 100% MeOH.
 D. Tang et al. / Biomedicine & Pharmacotherapy 87 (2017) 145152 147

PTP1B inhibitory activity, but it was not clear how glucose
metabolism was promoted by compounds AC. The current study
evaluated the in vitro antidiabetic activity of compounds AC and
then investigated their underlying mechanism of action in a
cellular model. The results of this study revealed that compounds
AC increased insulin sensitivity and promoted glucose metabo-
lism in L6 moytubes. These ndings suggest that the three new
norditerpenoid alkaloids (compounds AC) are lead compounds
for novel hypoglycemic drugs.
2. Materials and methods
2.1. Plant materials and reagents
Seeds of N. glandulifera were purchased from Xinjiang Uyghur
Pharmaceutical Co., Ltd in China. in October 2011. The specimen
was authenticated by Prof. De Min of Xinjiang Uyghur Autonomous
Region Food and Drug Inspection Ofce. A voucher specimen
(XTIPCNG-051011) was deposited at the Key Laboratory of Plant
Resources and Chemistry of Arid Zone and State Key Laboratory
Basis of Xinjiang Indigenous Medicinal Plants Resource Utilization,

Fig. 2. Effects of compounds AC on glucose consumption and lactic acid production. L6 skeletal muscle cells were serum-starved overnight and treated with 12.5 mM AC
and/or insulin at a nal concentration 100 nM for 24 h. (A) Dose-dependent changes of glucose consumption. (B) Dose-dependent changes of lactic acid concentration. (C)
Effect of compounds AC on glycogen content (D) Effect of compounds AC on hexokinase activity in L6 rat skeletal muscle cells in the absence or presence of 100 nm insulin.
Data represent mean  SD of three independent experiments. Basal represents insulin-free. *P < 0.05, **P < 0.01 vs the control.
148 D. Tang et al. / Biomedicine & Pharmacotherapy 87 (2017) 145152
Xinjiang Technical Institute of Physics and Chemistry, Chinese
Academy of Sciences, P. R. China.
The compounds AC were analyzed by Dionex P680 HPLC
apparatus at 365 nm for purity as described by Chen et al. [19]. L6
rat skeletal myoblasts were obtained from the Type Culture
Collection of the Chinese Academy of Sciences, Shanghai, China.
Fetal bovine serum (FBS), Bovine serum albumin (BSA), antibiotics,
Dulbeccos modied Eagles medium (DMEM) and Trizol reagent
were purchased from Gibco1 Life Technologies (Carlsbad, CA,
USA). Specic antibodies to phospho-Akt (p-Akt), phospho-GSK-
3b (p-GSK-3b), phospho-AMPK (p-AMPK), phospho-IRS-1 (p-IRS-
1), Akt, GSK-3b, AMPK and IRS-1 were obtained from Cell Signaling
Technology (Danvers, MA, USA). A specic antibody to PTP1B was
purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and the
secondary antibodies were obtained from GE Healthcare Life
Sciences (Piscataway, NJ, USA). Wortmannin (Wo) was supplied by
Cell Signaling Technology. Human recombinant insulin was
obtained from Sigma Chemical Corp. (St. Louis, MO, USA).
Electrophoresis reagents, including Bis-Tris gels, running buffer,
and poly (vinylidene uoride) (PVDF) membrane were obtained
from Invitrogen (Carlsbad, CA, USA).
2.2. Cell cultures
L6 myoblasts were maintained in DMEM supplemented with
10% (v/v) FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin
at 37  C in a humidied 5% CO2 atmosphere. When the cells
reached 8090% conuence, the growth medium was changed to
differentiation medium (DMEM supplemented with 2% horse
serum, 50 U/ml penicillin, and 50 mg/ml streptomycin), until the
cells were fully differentiated.
2.3. Glucose consumption and lactic acid assays
Glucose consumption was measured as described previously
[20]. L6 skeletal muscle cells were cultured in 96-well tissue
culture plates for 8 h at 37  C. Culture medium was replaced by the
serum-free DMEM supplemented with 0.25% BSA for 6 h before the
treatment with compounds AC (up to 50 mM) and/or insulin
(100 nM) for 1 h. The glucose concentration in the culture medium
was determined using Glucose Oxidase Assay Kit of Applygen
Technologies Inc (Beijing, PR China) according to the manufactur-
er's instructions. The glucose of the wells with cells was subtracted
from the glucose of the blank wells to calculate the glucose
consumption. To measure lactic acid production, cells were treated
using the same culturing procedure as described above, and lactate
concentration was detected using a commercial Lactic Acid Assay
Kit of Jiancheng Bioengineering Institute (Nanjing, PR China)
according to the manufacturer's instructions.
2.4. Glycogen content and hexokinase activity assays
Glycogen content was tested in L6 cells in the presence or
absence of insulin in 6 well plates using Muscle Glycogen Assay Kit
made by Jiancheng Bioengineering Institute (Nanjing, PR China).
Results were normalized by protein concentration measured using
Thermo Scientic Pierce BCA Protein Assay Kit (Rockford, USA), and
glycogen content was presented as micrograms glucose per well
and expressed as mg/g of protein. Hexokinase activity was detected
using a Hexokinase Assay Kit of Nanjing Jiancheng Bioengineering
Institute according to the manufacturer's instructions.
2.5. Western blotting analysis
Whole cell lysate was made with lysis buffer (25 mM HEPES pH
7.8, 50 mM KCl, 1% NP-40, 10 mg/ml leupeptin, 20 mg/ml
aprotinin,125 mM DTT, 1 mM Na3VO4, and 1 mM PMSF). Protein
concentration was measured using the Thermo Scientic Pierce
BCA Protein Assay Kit (Rockford, USA). Equal amounts of pre-
denatured protein (50 mg/lane) were separated by SDS-PAGE on
10% polyacrylamide gels and electrotransferred onto PVDF
membranes. After incubation with a blocking buffer (5% BSA in
Tris-buffered saline with 0.1% Tween-20) for 1 h, the membranes
were incubated with the primary antibodies (1:1000 dilution)
overnight at 4  C. After washed three times with TBST buffer, the
membranes were incubated with the secondary antibody conju-
gated with horseradish peroxidase (HRP) for 1 h at room
temperature. The immunoreactive bands were detected with
ECL Western Blotting Detection Reagent of GE Healthcare.
2.6. Statistical analysis
Experimental data are represented as meanSD. All experi-
ments were performed independently in cells at least three times.
Data were analyzed using one-way ANOVA (SPSS 12.0). Bands from
western blots were quantied using the Image J software. *P < 0.05
and **P < 0.01 were considered statistically signicant.
3. Results
3.1. Identication of compounds A, B and C
Nigelladines (A-C), were isolated from the seeds of N.
glandulifera, and were identied by extensive analysis of 1D, 2D
NMR and HRESIMS as described previously [19]. They shared the
same molecular formula of C19H25NO. The purity of nigelladines
(A-C) was higher than 95% as observed from 13C NMR (See
Supplementary data), and was higher than 98% as observed from
HPLC under 365 nm wavelength (Fig. 1 A-C).
3.2. Compounds A-C increase glucose consumption and lactic acid
production
L6 cells were treated with compounds A-C (050 mM) for 1 h to
investigate their impact on glucose metabolism. Glucose con-
sumption was measured using glucose oxidase method (GOD).
When the concentrations of compounds AC was less than
12.5 mM, glucose consumption increased linearly with the
concentration, but it failed to increase when the concentration
is greater than 12.5 mM (Fig. 2A). After insulin (100 nM) and
compounds AC treatment, glucose consumptions were increased
by 1.5-fold (**P < 0.01), 1-fold (**P < 0.01) and 0.5-fold (**P < 0.01)
as compared with insulin control group. As shown in Fig. 2B, lactic
acid release in L6 cells was enhanced by 79% (**P < 0.01), 76%
(**P < 0.01) and 49% (**P < 0.01) after co-treatment with 12.5 mM
compounds A-C and insulin. However, there was no obvious
alteration in lactic acid production by any of the three compounds
in the absence of insulin.
3.3. Compounds A-C increase glycogen synthesis and hexokinase
activity
Glycogen content and hexokinase activity of L6 cells treated
with three compounds were tested. In the absence of insulin, the
glycogen content was not altered by compounds A-C although a
slight increase was observed for the compound C. In the presence
of insulin, the glycogen was increased obviously by compound B
and C for 72% (*P < 0.05) and 57% (**P < 0.01) (Fig. 2C). No
remarkable effect was observed for compound A in the assay. The
compound A, B and C increased hexokinase activity by 42%
(*P < 0.01), 15% and 36% (*P < 0.05), respectively in the presence of
 D. Tang et al. / Biomedicine & Pharmacotherapy 87 (2017) 145152 149

insulin, whereas this positive effect was not observed in the
absence of insulin (Fig. 2D).
3.4. Effect compounds A-C on PTP1 B protein and Akt insulin
pathway
We sought to determine the effects of compounds AC on
PTP1 B protein levels in L6 cells. In the absence of insulin,
compounds AC had no obvious effect on PTP1 B protein level
(Fig. 3A). In the presence of insulin, a signicant reduction was
observed in PTP1 B levels by compound A (20%), B (43%, *P < 0.05)
and C (70%, **P < 0.01) compared with the insulin control (Fig. 3B).
However, PTP1 B protein level signicantly increased in L6 cells
after pretreated with 1 mM wortmannin for 1 h (Fig. 3B). There is
no difference between wortmannin and wortmannin combine
with compounds AC.
The proteins (IRS, Akt and GSK3b) that may interact with PTP1 B
involved in insulin transduction signal pathways were explored
examined. In the absence of insulin, compounds AC did not
exhibit any activity in the regulation of IRS-1 phosphorylation
(Fig. 3C). In the presence of insulin, all three compounds
signicantly increased the phosphorylation of IRS-1 by 47%, 68%

Fig. 3. Effects of compounds AC on PTP1B expression and phosphorylation of IRS-1 and Akt. (A) Western blot of PTP1B protein in response to treatment of 12.5 mM
compounds AC for 1 h in the absence or presence of insulin (100 nM). (B) Expression analysis of PTP1B in L6 myotubes treated with or without 1 mM Wo and 12.5 mM
compounds AC. (C) Expression analysis of p-IRS-1 in response to treatment of 12.5 mM compounds A-C for 1 h in the absence or presence of insulin (100 nM). (D) Western
blot of p-IRS-1 in L6 myotubes treated with or without 1 mM Wo and 12.5 mM compounds AC. (E) Western blot of p-Akt in response to treatment of 12.5 mM compounds A-C
for 1 h in the absence or presence of insulin (100 nM). (F) Expression analysis of p-Akt in L6 myotubes treated for 1 h with or without 1 mM Wo and 12.5 mM AC. Protein
expression was determined by Western blot and expressed as densitometry of PTP1B/b-actin, p-IRS-1/IRS-1 and p-Akt/Akt. b-Actin was used as an internal reference. Typical
results from three independent experiments were shown. Each bar is a mean  SD of three experiments.* P < 0.05, ** P < 0.01 vs the control.
150 D. Tang et al. / Biomedicine & Pharmacotherapy 87 (2017) 145152

(*P < 0.05) and 88% (**P < 0.01), respectively (Fig. 3D). The up-
regulation by compounds AC were blocked after pretreatment
with Wortmannin (Fig. 3D). In the presence of insulin, Akt
phosphorylation was enhanced by compounds A (1-fold increase,
*P < 0.05), B (1.5-fold increase, **P < 0.01) and C (1.2-fold increase,
**P < 0.01), respectively (Fig. 3E). But the positive effect was
abolished by Wortmannin (Fig. 3F). Compounds A-C on phosphor-
ylation of GSK-3b and AMPK
Thereafter, we examined phosphorylation levels of GSK-3b and
AMPK involved in insulin signaling. In the absence of insulin, the
phosphorylation of GSK-3b was not alterated by any of the three
compounds in L6 cells. In the presence of insulin, the phosphory-
lation was obviously increased by compound A (40%), B (50%,
**P < 0.01) and C (42%, *P < 0.05), respectively (Fig. 4A), but the
activities were blocked by pre-treatment of cells with Wortmannin
(Fig. 4B). AMPK activity was also examined by its phosphorylation
status in this study. In the absence of insulin, AMPK phosphoryla-
tion was not changed by compounds AC. In the presence of
insulin, phosphorylated AMPK was enhanced by 57% (compound B,
*P < 0.05) and 55% (compound C, *P < 0.05) (Fig. 4C). These effects
were blocked by Wortmannin as well (Fig. 4D).
4. Discussion
In the present study, the three alkaloids (compounds AC) of N.
glandulifera were found to improve insulin-dependent glucose
consumption and promote glycogen synthesis in L6 myotubes. To
observe the effect of compounds AC on glucose consumption of
L6 cell, the differentiated myoblast cells were treated with AC,
and glucose consumption was measured using glucose oxidase
method (GOD). The results indicate that compounds AC promote
glucose consumption in a concentration-dependent manner. The
optimal dose of compounds AC in glucose consumption was
12.5 mM, and this concentration was used in the following
experiment.
Glycolysis, a simple pathway of glucose metabolism, critically
regulates insulin secretion and metabolic functions of various cells.
Glycolysis is an important step in the process of glucose
metabolism, which leads to production of lactic acid [21].
Hexokinase catalyzes the rst step in glucose metabolism and
involves in glycolysis and glucose conversion. The compounds AC
enhanced the lactic acid production and hexokinase activity in an
insulin-dependent manner, resulting in the increases of glucose
consumption in L6 myotubes. These ndings indicate that the
increase in glucose consumption induced by compounds AC is
related with activation of glycolysis pathway.
The positive effect of compound A on lactic acid production and
hexokinase activity were stronger than compounds B and C. The
difference of bioactivity among the three compounds is likely a
consequence of different spatial structure. Although compound A,
B and C have the same molecular formula (C19H25NO), there is
some different in their skeleton structure, which inuence their
effects on biological activities and signaling pathway. The result
suggest compound A with special structure mainly act on

Fig. 4. Effect of compounds AC on phosphorylation of GSK-3b (Ser9) and AMPK (Thr172). (A) Western blot of p-GSK-3b (Ser9) in response to treatment of 12.5 mM
compounds AC for 1 h in the absence or presence of insulin (100 nM). (B) Expression analysis of p-GSK-3b (Ser9) in L6 myotubes treated with or without 1 mM Wo and
12.5 mM compounds AC. (C) Expression analysis of p-AMPK in response to treatment of 12.5 mM compounds AC for 1 h in the absence or presence of insulin (100 nM). (D)
Western blot of p-AMPK in L6 myotubes treated with or without 1 mM Wo and 12.5 mM compounds AC. Data are expressed as fold change of phosphorylated protein relative
to total protein (p-GSK-3b/GSK-3b and p-AMPK/AMPK). Typical results from three independent experiments were shown. Each bar is a mean  SD of three experiments.
*P < 0.05, ** P < 0.01 vs the control.
 D. Tang et al. / Biomedicine & Pharmacotherapy 87 (2017) 145152
glycolysis processes. Thus the effect of compound A on glycogene-
sis pathway (Akt/GSK) is weaker than compounds B and C.
Glucose is used in synthesis of glycogen upon insulin
stimulation to store glucose in cells in glucose metabolism [22].
Liver and muscle glycogen level directly reect the metabolism of
the organism. Therefore, glycogen content of L6 cells treated with
compounds AC was examined. The results indicate that
compounds AC promote muscle glycogen synthesis in an
insulin-dependent manner. The activities of compound B and C
on glycogen content were stronger than compound A. The results
indicate that compound B and C, possessing 6/6/5-tricyclic
heterocyclic skeleton, mainly work on glycogenesis pathway
(IRS/Akt/GSK).
PTP1B is an important molecule in the negative feedback
regulation of insulin signaling pathway [23]. PTP1B plays a critical
role in the metabolic processes of muscle cells. PTP1B content is
associated with insulin resistance [24]. It was observed that
compounds AC were sufcient to reduce the overexpression of
PTP1B in L6 myotubes, indicating that compounds AC is likely to
improve glucose metabolism by inhibiting PTP1B. It is a very
important result since PTP1B has been emerged as a potential drug
target in the treatment of T2DM [25].
Insulin action is initiated by hormone binding to cell-surface
insulin receptors (IR and IRS-1), which activates the protein kinase
associated with the insulin signaling [26]. PTP1B negatively
regulates insulin signaling by directly dephosphorylating the IR
and IRS-1 [27]. We hypothesized that inhibition of PTP1B by
compounds AC might enhance IRS-1 (Tyr895) phosphorylation in
L6 cells. In fact, the western blot results are consistent with our
hypothesis. The observations suggest that inhibition of PTP1B by
compounds AC facilitate Tyr895 phosphorylation of IRS-1, and
activate insulin-induced Akt signaling pathway.
Akt and GSK-3b is the downstream proteins of IRS in the insulin
signaling pathway [28,29]. We further investigate the effects of
compounds AC on Akt and GSK-3b, which facilitate glucose
synthesis and utilization in fully differentiated L6 myotubes [30].
We observed a signicant positive effect of compounds AC on the
insulin-activated Akt phosphorylation. Glycogen synthase kinase-
3 (GSK-3) is inhibited by phosphorylation upon Akt, consequently
promotes glycogen synthesis through insulin signaling pathway
[31,32]. The phosphorylation occurs at Ser9 in GSK-3b protein
following Akt activation [33]. Compounds AC caused an increase
of GSK-3b (Ser9) phosphorylation, suggesting that compounds A
C increased glycogen synthesis by the induction of Akt and GSK-3b
phosphorylation. The up-regulated effects of compound B and C on
phosphorylated levels of IRS, Akt and GSK-3b proteins were more
signicant than compound A. It is because that compound B and C
with 6/6/5-tricyclic heterocyclic skeleton primarily act on Akt/GSK
pathway.
AMP-activated protein kinase (AMPK) is an energy sensor in
cells including muscle cells. AMPK activation leads to promotion of
glycolysis and lactic acid production [34]. Compounds A-C resulted
in an increase of lactic acid production and AMPK phosphorylation,
which suggest that they possibly improve glycolysis through AMPK
activation.
In conclusion, Compounds AC play efcient role against
hyperglycemia through promoting glucose consumption and
glycogen synthesis, and the anti-diabetic effect is realized by
inhibition of PTP1B expression, induction of phosphorylation of
IRS-1, Akt, GSK-3b and AMPK in the stimulation of insulin.
However, the positive effect of compounds A-C was blocked by
wortmannin (PI3-kinase/Akt inhibitors). The results suggest that
PI3-kinase/Akt is required for IRS-1 tyrosine phosphorylation and
GSK-3b phosphorylation, and the activities of compounds AC are
dependent on PI3K/Akt signaling pathway.
151
Conict of interest
All authors declare that there are no conicts of interest.
Acknowledgments
This research was supported by the Joint Funds of the National
Natural Science Foundation of China (No. U1203203), the National
Science Foundation of China (No. 81173658), and Natural Science
Foundation of Xinjiang, China (2014211B45).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.
biopha.2016.12.058.
References
[1] J.E. Shaw, R.A. Sicree, P.Z. Zimmet, Global estimates of the prevalence of
diabetes for 2010 and 2030, Diabetes Res. Clin. Pract. 87 (2010) 414.
[2] J. Ye, Mechanisms of insulin resistance in obesity, Front. Med. 7 (2013) 1424.
[3] Y.K. Jarvinen, H. Baillieres, Action of insulin on glucose metabolism in vivo,
Clin. Endocrinol. Metab. 7 (1993) 903927.
[4] J.E. Pessin, A.R. Saltiel, Signaling pathways in insulin action: molecular targets
of insulin resistance, J. Clin. Invest. 106 (2000) 165169.
[5] L.D. Klaman, O. Boss, O.D. Petoni, J.K. Kim, J.L. Martino, J.M. Zabolotny, N.
Moghal, M. Lubkin, Y.B. Kim, A.H. Sharpe, Increased energy expenditure,
decreased adiposity and tissue-specic insulin sensitivity in protein-tyrosine
phosphatase 1B-decient mice, Mol. Cell. Biol. 20 (2000) 54795489.
[6] T.O. Johnson, J. Ermolieff, M.R. Jirousek, Protein tyrosine phosphatase 1B
inhibitors for diabetes, Nat. Rev. Drug Discov. 1 (2002) 696709.
[7] X.J. Sun, P. Rothenberg, C.R. Kahn, J.M. Backer, E. Araki, P.A. Wilden, D.A. Cahill,
B.J. Goldstein, M.F. White, Stucture of the insulin receptor substrate IRS-1
denes a unique signal transduction protein, Nature 352 (1991) 7377.
[8] B. Simone, A.J. Bradley, X. Linhua, I. Flurin, T. Thomas, B. Nicola, A.S. Giatgen, N.
Markus, From signal transduction to signal interpretation: an alternative
model for the molecular function of insulin receptor substrates, Arch. Physiol.
Biochem. 118 (2012) 148155.
[9] H. Kubota, R. Noguchi, Y. Toyoshima, Y. Ozaki, S. Uda, K. Watanabe, W. Ogawa, S.
Kuroda, Temporal coding of insulin action through multiplexing of the AKT
pathway, Mol. Cell. 46 (2012) 820832.
[10] Y. Katsura, H. Kubota, K. Kunida, A. Kanno, S. Kuroda, T. Ozawa, An optogenetic
system for interrogating the temporal dynamics of Akt, Sci. Rep. 5 (2015)
1458914599.
[11] N. Tian, T. Kanno, Y. Jin, T. Nishizaki, Lithium potentiates GSK-3b activity by
inhibiting phosphoinositide 3-kinase-mediated Akt phosphorylation,
Biochem. Biophys. Res. Commun. 450 (2014) 746749.
[12] J. Ye, Challenges in drug discovery for thiazolidinedione substitute, Acta
Pharm. Sin. B 1 (2011) 137142.
[13] J. Yin, H. Zhang, J. Ye, Traditional chinese medicine in treatment of metabolic
syndrome, Endocr. Metab. Immune. Disord. Drug Targets 8 (2008) 99111.
[14] R.A. Khalaf, Exploring natural products as a source for antidiabetic lead
compounds and possible lead optimization, Curr. Top. Med. Chem. 16 (2016)
25492561.
[15] A. Aikemu, X. Xiaerfuding, C. Shiwenhui, M. Abudureyimu, D. Maimaitiyiming,
Immunomodulatory and anti-tumor effects of Nigella glandulifera Freyn and
sint seeds on ehrlich ascites carcinoma in mouse model, Pharmacogn. Mag. 9
(2013) 87191.
[16] B.H. Ali, G. Blunden, Pharmacological and toxicological properties of Nigella
sativa, Phytother. Res. 17 (2003) 299305.
[17] X.L. Xin, Q. Wang, H.A. Aisa, H.Q. Wang, Anti-diabetes properties of n-hexane
and petroleum ether extracts from seeds of Nigella glandulifera Freyn, J. Food
Biochem. 34 (2010) 811824.
[18] X.L. Xin, H.Q. Wang, H.A. Aisa, Chemical constituents from the seed of Nigella
glandulifera freyn, Nat. Prod. Res. Dev. 24 (2012) 892899.
[19] Q.B. Chen, X.L. Xin, Y. Yang, S.S. Lee, H.A. Aisa, Highly conjugated
norditerpenoid and pyrroloquinoline alkaloids with potent PTP1B inhibitory
activity from Nigella glandulifera, J. Nat. Prod. 77 (2014) 807812.
[20] J. Yin, R. Hu, M. Chen, J. Tang, F. Li, Y. Yang, J. Chen, Effects of berberine on
glucose metabolism in vitro, Metabolism 51 (2002) 14391443.
[21] N.Y. Hong, Z.G. Cui, H.K. Kang, D.H. Lee, Y.K. Lee, D.B. Park, p-synephrine
stimulates glucose consumption via AMPK in L6 skeletal muscle cells,
Biochem. Biophys. Res. Commun. 418 (2012) 720724.
[22] M. Ragano-Caracciolo, W.K. Berlin, M.W. Miller, J.A. Hanover, Nuclear glycogen
and glycogen synthase kinas 3, Biochem. Biophys. Res. Commun. 249 (1998)
422427.
[23] M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A.L. Loy, M.J.
Gresser, M.L. Tremblay, B.P. Kennedy, Increased insulin sensitivity and obesity
152 D. Tang et al. / Biomedicine & Pharmacotherapy 87 (2017) 145152
resistance in mice lacking the protein tyrosine phosphatase-1 B gene, Science
283 (1999) 15441548.
[24] A. Bourdeau, N. Dube, M.L. Tremblay, Cytoplasmic protein tyrosine
phosphatases: regulation and function: the roles of PTP1B and TC-PTP, Curr.
Opin. Cell Biol. 17 (2005) 203209.
[25] O. Ukkola, M.J. Santaniemi, Potein tyrosine phosphatase 1B: a new target for
the treatment of obesity and associated comorbidities, Intern. Med. 251 (2002)
467475.
[26] S.E. Kahn, The relative contributions of insulin resistance and beta-cell
dysfunction to the pathophysiology of Type 2 diabetes, Diabetologia 46 (2003)
319.
[27] B.L. Seely, P.A. Staubs, D.R. Reichart, P. Berhanu, K.L. Milarski, A.R. Saltiel, et al.,
Protein tyrosine phosphatase 1B interacts with the activated insulin receptor,
Diabetes 45 (1996) 13791385.
[28] Z. Gao, D. Hwang, F. Bataille, M. Lefevre, D. York, M.J. Quon, Serine
phosphorylation of insulin receptor substrate 1 by inhibitor kappa B kinase
complex, J. Biol. Chem. 277 (2002) 4811548121.
[29] V. Aguirre, T. Uchida, L. Yenush, R. Davis, M.F. White, The c-Jun NH (2)-terminal
kinase promotes insulin resistance during association with insulin receptor
substrate-1 and phosphorylation of Ser (307), J. Biol. Chem. 275 (2000) 9047
9050.
[30] A.M. Marteli, I. Faenza, A.M. Billi, L. Manzoli, C. Evangelisti, F. Fala, L. Cocco,
Intramuscular 30 -phosphoinositide metabolism and Akt signaling: new
mechanisms for tumorigenesis and protection against apoptosis, Cell Signal.
18 (2006) 11011107.
[31] G.I. Welsh, C. Wilson, C.G. Proud, GSK3: a SHAGGY frog story, Trends Cell. Biol.
6 (1996) 274279.
[32] A.K. Srivastava, S.K. Pandey, Potential mechanism(s) involved in the regulation
of glycogen synthesis by insulin, Mol. Cell. Biochem. 182 (1998) 135141.
[33] D.A. Cross, D.R. Alessi, P. Cohen, M. Andjelkovich, B.A. Hem-mings, Inhibition of
glycogen synthase kinase-3 by insulin mediated by protein kinase B, Nature
378 (1995) 785789.
[34] D.G. Hardie, Minireview: the AMP-activated protein kinase cascade: the key
sensor of cellular energy status, Endocrinology 144 (2003) 51795183.