W2476 ameliorates β-cell dysfunction and exerts therapeutic effects in mouse models of diabetes via modulation of the thioredoxin-interacting protein signaling pathway

Article abstract of DOI:10.1038/aps.2017.15

Recent evidence shows that high glucose levels recruit carbohydrate response element-binding protein, which binds the promoter of thioredoxin-interacting protein (txnip), thereby regulating its expression in β-cells. Overexpression of txnip not only induc

Full text of DOI:10.1038/aps.2017.15

Acta Pharmacologica Sinica 2017: 1–14
© 2017 CPS and SIMM All rights reserved 1671-4083/17
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Original Article
W2476 ameliorates β-cell dysfunction and exerts
therapeutic effects in mouse models of diabetes via
modulation of the thioredoxin-interacting protein
signaling pathway
Ting LI1, 2, #, Guang-yao LIN2, 3, #, Li ZHONG1, 2, #, Yan ZHOU1, Jia WANG1, Yue ZHU1,2, Yang FENG1, Xiao-qing CAI1, Qing LIU1
Olivier NOSJEAN4, Jean A BOUTIN4, Pierre RENARD4, De-hua YANG1, 2, Ming-wei WANG1, 2, 3, 5,
*
1The National Center for Drug Screening and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China; 2University of Chinese Academy of Sciences, Beijing 100049, China; 3School
of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China; 4Pôle d’Expertise Biotechnologie, Chimie et
Biologie, Institut de Recherches SERVIER, Croissy-sur-Seine 78290, France; 5School of Pharmacy, Fudan University, Zhangjiang High-
Tech Park, Shanghai 201203, China
Abstract
Recent evidence shows that high glucose levels recruit carbohydrate response element-binding protein, which binds the promoter
of thioredoxin-interacting protein (txnip), thereby regulating its expression in β-cells. Overexpression of txnip not only induces β-cell
apoptosis but also reduces insulin production. Thus, the discovery of compounds that either inhibit TXNIP activity or suppress its
expression was the focus of the present study. INS-1E cells stably transfected with either a txnip proximal glucose response element
connected to a luciferase reporter plasmid (BG73) or full-length txnip promoter connected to a luciferase reporter plasmid (CL108)
were used in primary and secondary high-throughput screening campaigns, respectively. From 256 000 synthetic compounds, a
small molecule compound, W2476 [9-((1-(4-acetyl-phenyloxy)-ethyl)-2-)adenine], was identified as a modulator of the TXNIP-regulated
signaling pathway following the screening and characterized using a battery of bioassays. The preventive and therapeutic properties
of W2476 were further examined in streptozotocin-induced diabetic and diet-induced obese mice. Treatment with W2476 (1, 5, and
15 μmol/L) dose-dependently inhibited high glucose-induced TXNIP expression at the mRNA and protein levels in INS-1E cells and rat
pancreatic islets. Furthermore, W2476 treatment prevented INS-1E cells from apoptosis induced by chronic exposure of high glucose
and enhanced insulin production in vitro. Oral administration of W2476 (200 mg·kg-1·d-1) rescued streptozotocin-induced diabetic
mice by promoting β-cell survival and enhancing insulin secretion. This therapeutic property of W2476 was further demonstrated by
its ability to improve glucose homeostasis and insulin sensitivity in diet-induced obese mice. Thus, chemical intervention of the TXNIP-
regulated signaling pathway might present a viable approach to manage diabetes.
Keywords: diabetes; glucose; TXNIP; β-cells; W2476; high-throughput screening
Acta Pharmacologica Sinica advance online publication, 15 May 2017; doi: 10.1038/aps.2017.15
strated that TXNIP regulates a wide variety of physiological
Thioredoxin-interacting protein (TXNIP), also known as vita- processes, including cell growth, differentiation and death,
min D3 up-regulated protein 1 (VDUP1)[1] or thioredoxin-bind- energy metabolism and immune responses[8–11]
ing protein-2 (TBP-2)[2], is a ubiquitously expressed protein
A recent study showed that high concentrations of glu-
Introduction
.
with a molecular weight of 50 kDa. It was initially identified cose could substantially increase the mRNA and protein
as a negative regulator of thioredoxin[2–4] to mediate cellular levels of TXNIP in multiple β-cell lines and cultured islets of
oxidative stress[3–7]. Subsequent investigations have demon- Langerhans from different mouse models of type 1 or type 2
diabetes[12–14]. One of the underlying mechanisms is that glu-
# These authors contributed equally to this work.
To whom correspondence should be addressed.
E-mail: mwwang@simm.ac.cn
Received 2016-12-26 Accepted 2017-02-09
cose recruits carbohydrate response element-binding protein
(ChREBP), which binds the carbohydrate response element
(ChoRE) located in the promoter of txnip[13–15]. Given that
*

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Li T et al
persistent hyperglycemia is a common manifestation of type d=2.17 g/mL, 24.05 mL, 0.2778 mol, 7.57 eq), potassium car-
2 diabetes mellitus (T2DM) and its expression is rapidly and bonate (Mw = 138.2, 17.87 g, 0.129 mol, 3.5 eq) and triethyl-
vigorously induced by glucose, TXNIP was proposed to be a benzylammonium chloride (TEBA, catalytic amount) in ethyl
key link between hyperglycemia and β-cell dysfunction[13, 16, 17]
insulin production[18] and insulin resistance[19]
,
acetate (100 mL) at 60°C. The reaction was refluxed overnight.
After cooling, water was added and the mixture was extracted
.
It is known that TXNIP can induce cell apoptosis in response with ethyl acetate (3×50 mL). The organic phase was washed
to oxidative stress[20]. β cells have low levels of antioxidant with 1 mol/L NaOH solusion and water, and then dried
enzymes[21] and thus are more susceptible to apoptosis upon over anhydrous sodium sulfate. After filtration, the solvent
TXNIP overexpression[17]. TXNIP deficiency in the pancreas was removed in vacuo, and the residue was separated on the
increases β-cell mass by inhibition of apoptosis[16, 22, 23]. TXNIP Biotage SNAP cartridge KP-Sil 340 g column (Biotage, Shang-
has been found to decrease insulin transcription by directly hai, China) eluting with 30% ethyl acetate/petroleum ether.
targeting or indirectly down-regulating v-maf musculoapo- The product was obtained as yellowish oil, which was solidi-
neurotic fibrosarcoma oncogene family protein A (MafA), fied after cooling (7.99 g, yield: 89.6%). The purity was 97.0%
a known insulin transcription factor[18]
.
TXNIP was also by HPLC analysis. LR-ESI: 264.0 (M+Na)+. HR-MS (TOF) m/z
implicated in modulating insulin sensitivity by disrupting calcd for C10H12BrO2 [M+H]+ 243.0021, found 243.0023. H
insulin-stimulated glucose uptake in the skeletal muscle and NMR (CDCl3, 300 MHz) δ 2.57 (s, 3H), 3.67 (t, J = 6.3 Hz, 2H),
adipocytes[23–25]. Several studies using HcB-19 (txnip nonsense 4.36 (t, J = 6.3 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.7
mutation), β-cell-specific txnip knockout (βTKO) and txnip Hz, 2H). 13C NMR (CDCl3, 75 MHz) δ 26.5, 29.8, 68.0, 114.4
deleted ob/ob mice supported the proposition that the inhibi- (3C), 130.8 (2C), 162.0, 196.8.
1
tion of TXNIP is potentially a novel approach to combat type
1 and type 2 diabetes[10, 16, 23, 26, 27] by promoting β-cell survival 9-((1-(4-Acetylphenyloxy)ethyl)-2-)adenine
and improving insulin sensitivity.
Potassium carbonate (Mw = 138.2, 0.27 g, 1.98 mmol, 2 eq) was
To date, no small molecule that can effectively modulate added into the mixture of adenine (Mw = 135.1, 0.13 g, 0.99
glucose-induced TXNIP overexpression has been reported. mmol, 1 eq) and 1-(4-(2-bromoethoxy)phenyl-ethanone (Mw
Here, we describe a high-throughput screening (HTS) assay = 243.1, 0.30 g, 1.23 mmol, 1.25 eq) in anhydrous DMF (30
that was applied to discover such modulators leading to the mL). Then, the reaction was stirred at 80°C overnight. After
identification of W2476, a synthetic compound capable of cooling, it was filtered and the solution was evaporated in
lowering TXNIP expression induced by high concentrations of vacuo to obtain the crude product, which was further recrys-
glucose. Therefore, the present study was designed to inves- tallized with ethanol to obtain the pure product (0.23 g, yield:
tigate the bioactivities of W2476 in β-cells both in vitro and in 79.3%).The purity was 96.0% by HPLC analysis. LR-ESI: 319.2
vivo.
(M+Na)+. HR-MS (TOF) m/z calcd for C15H16N5O2 [M+H]+
298.1304, found 298.1309. 1H NMR (DMSO-d6, 300 MHz) δ 2.50
(s, 3H), 4.47 (t, J = 5.1 Hz, 2H), 4.57 (t, J = 4.8 Hz, 2H), 7.03 (d,
J = 8.4 Hz, 2H), 7.20 (brs, 2H, NH2), 7.90 (d, J = 8.4 Hz, 2H),
8.16 (s, 1H), 8.19 (s, 1H). 13C NMR (DMSO-d6, 75 MHz) δ 26.4,
Materials and methods
Chemistry
General statement
Reagents were of commercial grade and used as received 42.4, 66.0, 114.4 (2C), 118.6, 130.3, 130.5 (2C), 141.2, 149.6, 152.5,
unless otherwise noted. All new compounds were judged to 156.0, 161.8, 196.3.
be ≥95% pure by HPLC. NMR spectra were recorded on Var-
ian Mercury 300 (Varian, Palo Alto, CA, USA). Chemical shifts Reagents
were reported in parts per million (ppm), with the solvent res- Antibodies against TXNIP (JY2), MafA (F-6) and ChREBP and
onance as the internal standard (CDCl3 7.27 ppm for 1H NMR, those used in Western blotting were purchased from MBL
77.23 ppm for 13C NMR, DMSO-d6 2.50 ppm for 1H NMR, 39.51 International (Woburn, MA), Santa Cruz Biotechnology (Santa
ppm for 13C NMR). Low-resolution mass spectral data (elec- Cruz, CA, USA) and Cell Signaling Technology (Danvers, MA,
trospray ionization) were acquired on a Finnigan LCQ-DECA USA), respectively.
mass spectrometer (Thermo Finnigan, Altrincham, Cheshire,
UK). High-resolution mass spectral data (TOF) were acquired Cell culture
on an Agilent 6224 mass spectrometer (Agilent, Santa Clara, L6 myoblasts were cultured in DMEM containing 10% FBS.
CA, USA). Samples were analyzed for purity on an HP1100 Upon differentiation, the concentration was decreased to
series (Agilent) equipped with a Zorbax SB-C18 column (5 μm, 2%. Culture and differentiation of 3T3-L1 cells were con-
4.6 mm×250 mm). Purities of final compounds were deter- ducted according to the literature[28]. Islets of Langerhans
mined using a 5-μL injection with quantification by AUC at from Sprague-Dawley rats and mice were prepared using a
210 nm and 254 nm (Agilent diode array detector).
collagenase digestion procedure as described previously[29]
.
Mouse hepatocytes were isolated using Selgen’s two-step
1-(4-(2-Bromoethoxy)phenyl)ethanone
perfusion method[30]. INS-1E cells and islets were cultured
4’-Hydroxyacetophenone (Mw = 136.1, 5 g, 0.0367 mol, 1 eq) in RPMI-1640, and hepatocytes were maintained in DMEM.
was added to the mixture of 1,2-dibromoethane (Mw = 187.9, INS-1E cells were stably transfected with either a human txnip
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Li T et al
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proximal glucose response element connected to a luciferase administered, respectively. Verapamil (positive control;
reporter plasmid (BG73) or a full-length human txnip pro- Sigma, St Louis, MO, USA) was given in the drinking water
moter connected to a luciferase reporter plasmid (CL108) and (1 mg/mL, equivalent to an average dose of 100 mg·kg-1·d-1).
maintained in RPMI-1640 in the presence of 50 μg/mL gene- Body weight and food intake were monitored daily, and blood
ticin (Life Technologies, Carlsbad, CA, USA). Information glucose levels from 6-h fasting mice were measured. For the
about the TXNIP construct promoter sequences is available oral glucose tolerance test (OGTT), a bolus of 2 g/kg glucose
upon request.
was delivered to 6-h fasting mice, and blood glucose levels
were determined at designated time points. At the end of the
study, plasma insulin level was assessed using a mouse insu-
RNA extraction and quantitative real-time PCR
Total RNA from INS-1E cells, L6 myoblasts, 3T3-L1 cells, hepa- lin ELISA kit (EZRMI-13K; Millipore, Temecula, CA, USA).
tocytes and rat islets were extracted using TRIzol reagent (Life The pancreas was removed from euthanized mice and subse-
Technologies) according to the manufacturer’s instructions. quently fixed in 4% paraformaldehyde (PFA) for immunohis-
RNA was converted to cDNA using a High-Capacity cDNA tochemistry or for isolating the islets.
Reverse Transcription kit (Life Technologies), and quantitative
Four-week-old male C57BL/6J mice were fed on a high-fat
real-time PCR was performed using SYBR Green Master Mix diet (HFD; D12492, Research Diet, New Brunswick, NJ, USA)
(DRR820A; Takara, Dalian, China) on an ABI ViiA7 system to establish a diet-induced obese (DIO) model or a standard
(Life Technologies). The primers used are shown in Table S1. chow diet (SCD; D12450B, Research Diet) and watered ad libi-
All the samples were analyzed in triplicate, and gapdh was tum. After 4- or 12-week HFD feeding, they were randomly
used as an internal control.
assigned to 4 treatment groups (n=9–12 per group) with
matched body weight: (1) 0.5% w/v methylcellulose (MC), (2)
200 mg·kg-1·d-1 oral W2476, (3) 400 mg·kg-1·d-1 oral W2476, and
Annexin V-FITC/PI double-labeled flow cytometry
INS-1E cells were cultured in RPMI-1640 (2 % FBS) in the pres- (4) 100 mg·kg-1·d-1 verapamil contained in drinking water (1
ence of 5 mmol/L or 33.3 mmol/L glucose with or without dif- mg/mL). Body weight and food intake were recorded daily,
ferent concentrations of W2476 for 48 h. Apoptotic cells were and 6-h fasting blood glucose levels were measured every
identified using the Dead Cell Apoptosis Kit with Annexin three weeks. Following a 4-h fasting period, an insulin toler-
V-FITC and PI (Life Technologies) according to the manufac- ance test (ITT) was performed by intraperitoneal injection of
turer’s instructions. Flow cytometry data were acquired using 2.5 IU/kg insulin to measure blood glucose levels thereafter.
an Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, At the end of 6-week treatment, samples were collected to
USA) and analyzed using Accuri C6 software.
examine insulin levels and the expression of TXNIP. Plasma
biochemical indices were analyzed as described previously[32]
.
Western blot analysis
Protein extracts from W2476-treated INS-1E cells were pre- Immunohistochemistry
pared according to the procedure published previously[17]
Immunofluorescence imaging analysis was carried out as
.
The following antibodies were used: TXNIP (1:400), MafA previously described[32]. Briefly, the pancreas was fixed with
(F-6) (1:500), insulin (1:1000), cleaved caspase-3 (1:500), cas- 4% PFA, embedded in paraffin, and sliced into 4-μm cross
pase-3 (1:1000), cleaved caspase-9 (1:1000), caspase-9 (1:1000), sections. Insulin and glucagon were stained with anti-insulin
ChREBP (1:200), β-actin (1:2000), GAPDH (1:2000), goat anti- (1:200) or anti-glucagon (1:200; Abcam, Cambridge, MA, USA)
mouse IgG (1:8000) and goat anti-rabbit IgG (1:8000). Signals antibodies, respectively. The nuclei were visualized with
were detected by a ChemiDocTM MP Imaging System from 4’,6-diamidino-2-phenylidole (DAPI).
Bio-Rad Laboratories (Richmond, CA, USA).
Statistical analysis
Animal studies
Data are presented as means±SEM and analyzed by Student’s
All animal experimentation was conducted in accordance with t-test or one-way ANOVA. P<0.05 was considered to be statis-
the regulations adopted by the Animal Care and Use Commit- tically significant.
tee, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences (approval number: 2014-SIMM-06).
Results
A detailed flowchart of animal study procedures was shown HTS assay development and compound screening
in Figure S1. Briefly, male C57BL/6 mice (SLAC Laboratory Expression of the luciferase reporter gene can be stimulated
Animal Co, Ltd, Shanghai, China) were kept in a temperature- by various concentrations of glucose in both BG73 and CL108
controlled room (22±2 °C), with a light/dark cycle of 12 h. cells, with EC50 values of 9.74±0.42 mmol/L and 9.48±0.95
Mice of 8 weeks of age were injected with streptozotocin (STZ; mmol/L, respectively. BG73 cells responded to glucose stimu-
40 mg·kg-1·d-1 for 5 d, freshly prepared in 0.1 mmol/L sodium lation more sensitively than did CL108 cells, with a greater
citrate; pH 4.3–4.5) according to the procedure published pre- response window of 7.36 times at 25 mmol/L glucose. The
viously[31]. For studying the preventive or therapeutic effect reporter gene in BG73 cells was fully activated at glucose
of W2476 on STZ-induced diabetic mice, either vehicle (0.5% concentrations equal to or above 16.7 mmol/L (Figure S2A).
methylcellulose, MC) or W2476 (200 mg·kg-1·d-1) was orally Thus, 19.4 mmol/L glucose was used in the HTS campaigns
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Li T et al
with BG73 cells. The BG73 assay system showed coefficient phenyloxy)-ethyl)-2-)adenine, Figure 1A and Figure S3], as
of variation (CV) values of 5.94% for high glucose (HG, 19.4 a model for further characterization. W2476 concentration-
mmol/L) and 7.12% for basal glucose (BG, 3.3 mmol/L), a dependently suppressed reporter gene expression in BG73
signal-to-background ratio of 6.91 and a Z’ factor of 0.72 (Fig- cells with IC50 values of 1.7±0.1 μmol/L at HG (19.4 mmol/L)
ure S2B). These parameters indicate that the assay system was or 3.1±0.6 μmol/L at BG (3.3 mmol/L), respectively (Figures
of high quality and well suited to HTS[33]. Of the 256000 syn- 1B). In CL108 cells, the IC50 of W2476 was 3.3±0.6 mmol/L at
thetic compounds from the collection of the Chinese National HG, with no concentration-dependent response at BG (Figure
Compound Library screened, 892 (0.34%) initial hits were 1C), implying that it did not inhibit TXNIP expression at BG,
identified using 80% inhibition as the cut-off line (Figures S1C, which might allow TXNIP to retain other functions[34–38]
S1D). They subsequently went through a secondary screening To investigate whether W2476 could affect TXNIP expres-
involving both BG73 and CL108 cells that yielded 4 confirmed sion in INS-1E cells, we treated the cells with 5 mmol/L or
.
hits (0.45% or an overall hit rate of 0.02‰).
25 mmol/L glucose for 6, 12, 24 and 48 h in the presence or
absence of W2476 (10 μmol/L). Western blot analysis illus-
trated that 25 mmol/L glucose could significantly increase
W2476 modulates TXNIP expression in INS-1E cells
Following the above confirmation studies, we selected TXNIP expression from 6 h to 48 h, which was completely
one compound, WNN2476-A011 [W2476, 9-((1-(4-acetyl- inhibited by 10 μmol/L W2476 (Figure 1D). W2476 at 1, 5 and
Figure 1. Structure of W2476 and its characterization in BG73, CL108 and INS-1E cells. (A) Chemical structure of W2476 (C15H15N5O2, MW: 297.31).
BG73 or CL108 used for counter screening were treated with either 19.4 mmol/L or 3.3 mmol/L glucose with or without different concentrations of
W2476 for 24 h. The reporter gene activity was evaluated and the modulating activity of W2476 was examined under high glucose (HG, 19.4 mmol/L)
and basal glucose (BG, 3.3 mmol/L) conditions in both BG73 (B) and CL108 (C) cells. INS-1E cells were treated with 10 μmol/L W2476 in the presence
of 5 mmol/L or 25 mmol/L glucose for 6, 12, 24 or 48 h (D), or with different doses of W2476 for 24 h (E, F), followed by immunoblotting of TXNIP.
Values represent means±SEM for three independent experiments. **P<0.01 vs 5 mmol/L glucose. ##P<0.01 vs 25 mmol/L glucose.
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15 μmol/L suppressed TXNIP expression by 25.31% (P<0.01), (d 4 to 8). As shown in Figure 4A, while 6-h fasting blood
40.09% (P<0.01) and 63.54% (P<0.001; Figures 1E, 1F), respec- glucose in the control group reached 10.64±0.88 mmol/L (dia-
tively. These data demonstrate that the modulating effect of betic state) on d 17, that of verapamil- or W2476-treated mice
W2476 on TXNIP was both time- and concentration-depen- remained normal (7.77±0.41 mmol/L and 7.78±0.38 mmol/L,
dent. The repressive property of W2476 on TXNIP expression respectively). This phenomenon was accompanied by a 49%
was also observed in mouse hepatocytes, L6 myotubes and and 37% reduction of TXNIP protein levels in W2476- and
3T3-L1 adipocytes (Figure S4).
verapamil-treated group, respectively (Figures 4B, 4C). To
evaluate islet function, OGTT was performed, and 15.04% and
20.77% reduction of the area under the curve (AUC0–120) for
W2476 inhibits high glucose-induced apoptosis
INS-1E cells were treated with 5 mmol/L or 33.3 mmol/L blood glucose levels were found in mice treated with either
glucose for 48 h and subjected to various concentrations of 100 mg/kg verapamil or 200 mg/kg W2476 compared to vehi-
W2476, followed by detection of TXNIP expression and cell cle-treated control (P<0.05 and P<0.01, respectively; Figures
apoptosis. We found that W2476 could significantly decrease 4D, 4E). In addition, immunohistochemistry revealed that islet
TXNIP expression at 33.3 mmol/L glucose (Figures 2A, 2B). structure was severely disrupted, with poor insulin staining
Cell apoptosis was evaluated, revealing that 33.3 mmol/L glu- in STZ-treated mice, whereas normal insulin-containing islets
cose induced INS-1E cell apoptosis, which could be rescued by were observed in both verapamil- and W2476-treated groups
10 μmol/L and 15 μmol/L W2476 (Figures 2C, 2D).
(Figure 4F).
We also examined activation of caspases, critical biomark-
To further investigate whether W2476 could reverse overtly
ers of cell apoptosis, in INS-1E cells. Western blot analysis STZ-induced diabetes, C57BL/6 mice were given daily STZ
showed that 33.3 mmol/L glucose increased the cleavage of (40 mg·kg-1·d-1) ip injection for 5 d and rested for 10 d. Star-
caspase-3, which could be dose-dependently inhibited by ing from d 15, mice divided into three groups were treated
W2476 (Figures 2E–2G). Moreover, W2476 exerted similar with W2476 (200 mg·kg-1·d-1), verapamil (100 mg·kg-1·d-1) or
effects on the cleavage of caspase-9 (Figures 2H–2J). These vehicle (0.5% MC) for the next 10 d. As depicted in Figure
results indicate that W2476 was able to reverse high glucose- 5A, fasting blood glucose levels in verapamil- and W2476-
induced INS-1E cell apoptosis.
treated groups were both reduced by 27.7% on d 25. The
OGTT study showed that glucose AUC0–120 values in W2476-
and verapamil-treated animals were reduced by 23.8% and
W2476 elevates insulin and MafA expression
Because TXNIP has been proposed to down-regulate the 21.8%, respectively (P<0.01 and P<0.01; Figures 5B, 5C). This
transcription of mafA and insulin, we investigated whether was accompanied by parallel increases in plasma insulin levels
W2476 could reverse the impact of high glucose on MafA and by 34.43% (W2476) and 34.65% (verapamil) compared to STZ
insulin via reduction of TXNIP expression. INS-1E cells were control (Figure 5D). Immunofluorescence staining of pancre-
incubated for 24 h with 5 mmol/L or 25 mmol/L glucose in atic cross-sections exhibited severe islet destruction in the STZ
the presence or absence of W2476, followed by analysis of group, whereas normal morphology and intensive insulin
gene and protein expression. In comparison to 5 mmol/L, 25 staining were observed in W2476- and verapamil-treated ani-
mmol/L glucose significantly attenuated insulin expression at mals (Figure 5E).
both the mRNA and protein levels, which were accompanied
by an increase in TXNIP expression. As TXNIP was dimin- Preventive and therapeutic properties of W2476 in DIO mice
ished by W2476, MafA and insulin gene (Figures 3A–3C) and To study whether chronic administration of W2476 exerts
protein (Figures 3D–3F) expression were increased in a con- protective or therapeutic effect in DIO mice, we fed C57BL/6
centration-dependent manner. Isolated rat islets of Langer- mice with HFD either for 4 weeks to create a pre-obese state,
hans were also applied to verify this effect of W2476, in which or for 12 weeks to present overweight, hyperglycemic, hyper-
islets were exposed to 33.3 mmol/L glucose with or without 15 insulinemic and dyslipidemic features[41]. This was followed
μmol/L W2476 for 48 h. It was shown that W2476 decreased by 6-week daily treatment of either W2476 (200 mg/kg or 400
TXNIP mRNA level by 2-fold, and this reduction was accom- mg/kg) or verapamil (100 mg/kg). While the body weight on
panied by an increase in insulin transcription (Figures 3G–3H). the last day of dosing and the average food intake during the
However, changes in mafA transcription were not observed 6-week treatment period did not significantly alter between
under this condition (Figure 3I).
HFD control and treatment groups in both preventive (Figures
6A, 6B) and therapeutic experiments (Figures 7A, 7B), signifi-
cant reduction in fasting blood glucose and plasma insulin
Therapeutic property of W2476 in STZ-induced diabetic mice
Previous studies suggest that genetic deletion and pharma- levels were observed after the treatment (Figures 6C, 6F, 7C,
cological inhibition of TXNIP were able to protect animals 7D). This was accompanied by improved ITT responses in
against STZ-induced diabetes[16, 39, 40]. In this study, STZ- W2476- but not verapamil-treated mice (Figures 6G, 6H, 7E,
induced diabetic mice were employed to examine whether 7F). To determine if the above beneficial effects are mediated
W2476 exerts a similar effect via modulating TXNIP. After via inhibition of TXNIP and the difference between W2476 and
treatment with W2476 or verapamil for 3 d, mice were intra- verapamil, we examined the level of TXNIP in metabolism-
peritoneally (ip) injected with STZ (40 mg·kg-1·d-1) for 5 d related organs after treatment. W2476, but not verapamil,
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Li T et al
Figure 2. Effect of W2476 on high glucose-induced INS-1E cell apoptosis. INS-1E cells were incubated at 5 mmol/L or 33.3 mmol/L glucose in the
presence or absence of varying concentrations of W2476 with 2% FBS for 48 h. (A, B) Protein expression levels of TXNIP were measured by Western
blotting. (C, D) Cell apoptosis was assessed by flow cytometry using Annexin V/PI staining. Immunoblotting of cleaved caspase-3 (E, F) and caspase-3 (E,
G) as well as cleaved caspase-9 (H, I) and caspase-9 (H, J) after treatment with W2476. Representative immunoblots were shown and protein contents
were separately quantified. All values are presented as mean±SEM of at least three independent experiments; *P<0.05, **P<0.01 vs 5 mmol/L glucose;
#P<0.05, ##P<0.01 vs 33.3 mmol/L glucose.
significantly decreased pancreatic TXNIP expression (Figures muscle (Figure S5). Pharmacokinetic analysis suggests that
6D, 6E). Neither W2476 nor verapamil exhibited any inhibi- this differentiated action of W2476 may have resulted from its
tory effect on TXNIP in the subcutaneous fat, liver and skeletal different tissue distribution (Figure S6, Tables S2, S3).
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Li T et al
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Figure 3. Effects of W2476 on insulin and MafA expression. INS-1E cells were exposed to 5 mmol/L or 25 mmol/L glucose with or without varying
concentrations of W2476 for 24 h, followed by measurement of mRNA levels of txnip (A), mafa (B) and insulin (C) using quantitative real-time PCR.
Protein levels of TXNIP (D), MafA (D, E) and insulin (D, F) were evaluated by Western blotting. Representative immunoblots were shown and protein
contents were separately quantified. Isolated rat islets of Langerhans were incubated for 48 h with 33.3 mmol/L glucose and 15 μmol/L W2476,
and mRNA levels of txnip (G), insulin (H) and mafa (I) were assessed by quantitative real-time PCR. Data shown are mean±SEM of three independent
experiments. *P<0.05, **P<0.01 vs 5 mmol/L glucose. #P<0.05, ##P<0.01 vs 25 mmol/L glucose in A–F. ##P<0.01 vs 33.3 mmol/L glucose in G–I.
At the end of the 6-week therapy, the plasma triacylglycerol
Discussion
level was markedly elevated in the HFD control, which was ChREBP shuttling from the cytoplasm to nucleus binds to the
significantly reversed by W2476; no significant difference was ChoRE in the promoters of many genes[42] involved in gly-
found in plasma cholesterol and nonesterified fatty acid lev- colysis, lipogenesis and gluconeogenesis[42, 43]. Among them,
els among the treatment groups in two experimental settings TXNIP shows prominence[15]. Therefore, a search for small
(Tables S4, S5). We also compared the low-density lipopro- molecules capable of modulating ChREBP and the down-
tein-cholesterol/high-density lipoprotein-cholesterol (LDL-C/ stream signaling pathway is of interest.
HDL-C) ratio in these animals, showing that long-term expo- ChREBP mainly regulates the expression of TXNIP in pancre-
sure to HFD increased the LDL/HDL ratio by 61.5%, which atic β-cells. The link between TXNIP and diabetes has been
was decreased by 9.52% (200 mg/kg, P<0.05) and 23.81% recently strengthened with identification of effective modu-
(400 mg/kg, P<0.01) after two doses of W2476 administration lators of TXNIP. Exenatide, a marketed anti-diabetic drug,
(Table S5). This phenomenon was not observed in mice that was shown to suppress TXNIP expression in INS-1E cells and
received prophylactic W2476 treatment (Table S4).
primary islets, leading to increased β-cell mass[22]. Verapamil,
a calcium channel blocker, was also found to inhibit TXNIP
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Li T et al
Figure 4. Protective effect of W2476 on STZ-induced diabetic mice. Male C57BL/6 mice received either verapamil in drinking water (100 mg·kg-1·d-1) or
an oral dose of W2476 (200 mg·kg-1·d-1) between d 1 and 17. Three days after the treatment, all groups received daily intraperitoneal injection of STZ (40
mg·kg-1·d-1) for 5 d. (A) Blood glucose levels at indicated times minus baseline values measured on d 3 (n=12 per group). (B, C) Effects of W2476 and
verapamil on pancreatic islet TXNIP protein levels in each group. On d 17, OGTT was performed to assess blood glucose levels (D) and the glucose area-
under-the-curve integrated from 0 to 120 min (AUC0–120; E) in all treatment groups. (F) Immunofluorescence staining of pancreatic islets prepared from
three groups after 17-d treatment. Red, glucagon; green, insulin; blue, nuclei/DAPI. Scale bar, 30 μm. Original magnification, 10× (upper panel) and
60× (lower panel). Data shown are mean±SEM. *P<0.05, **P<0.01 vs STZ group.
expression in β-cells and exert a therapeutic effect on several that transcription of txnip is regulated by glucose, and its pro-
diabetic animal models. However, the concentration of vera- moter has a ChoRE site crucial for glucose-induced ChREBP/
pamil that inhibited TXNIP was extremely high (50 mmol/L) Mlx heterodimer binding in β-cells[15]. BG73 cells with proxi-
in vitro, suggesting its poor efficacy[31]. In this paper, we pres- mal elements of ChoRE in the txnip promoter were more
ent a small molecule, W2476, as a modulator of TXNIP expres- sensitive to glucose in our reporter assay used for primary
sion discovered by HTS and characterized using a battery of in screening. Because CL108 cells with an intact txnip promoter
vitro and in vivo bioassays.
responded to glucose in a manner similar to induction of
A human microarray study initially established that TXNIP TXNIP expression under physiological conditions, they were
was powerfully stimulated by glucose[44] and may play an applied to secondary screening. Of note, W2476, identified by
important role in the pathogenesis of T2DM[45]. It is known this cell-based assay system, exerted modulating action only
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Figure 5. Therapeutic effect of W2476 on STZ-induced diabetic mice. Male C57BL/6 mice received daily intraperitoneal injection of STZ (40 mg·kg-1·d-1)
for 5 d and rested for 10 d. On d 15, 6-h fasting blood glucose levels were measured, and those with glucose levels higher or equal to 8 mmol/L were
selected to receive daily treatment of W2476 (200 mg/kg), verapamil (100 mg/kg) or vehicle (control). (A) Blood glucose levels measured on d 1, 15
and 25 (n=6 per group). Blood glucose levels (B) and AUC0-120 (C) during OGTT on d 25. (D) Plasma insulin levels on d 25. (E) Immunofluorescence
staining of pancreatic islets prepared from three groups following 10-d treatment. Red, glucagon; green, insulin; blue, nuclei/DAPI. Scale bar, 30 μm.
Original magnification, 10× (upper panel) and 60× (lower panel). Data shown are mean±SEM. *P<0.05, **P<0.01 vs STZ group.
in the high-glucose environment and had little impact when verapamil could prevent β-cells from apoptosis by decreasing
the glucose level was normal, as observed in CL108 cells. It TXNIP expression[22, 31], we found that W2476 was also capable
is likely that W2476 may affect trans-acting factors bound to of protecting INS-1E cells from apoptosis induced by high
cis-acting elements located in the txnip gene[15]. The effect of glucose, probably through inhibition of the cleavage of cas-
W2476 was subsequently validated in INS-1E cells: it dose- pase-3 and caspase-9. Impaired insulin transcription in dia-
dependently reduced both TXNIP mRNA and protein levels.
betes appears to be involved in excessive β-cell expression of
TXNIP is implicated in glucotoxicity and hence, β-cell apop- TXNIP: it is up-regulated in β-cells from diabetic subjects, and
tosis[17]. Consistent with previous findings that exenatide and its overexpression induces miR-204, leading to impaired MafA
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Li T et al
Figure 6. Protective effect of W2476 on DIO mice. Four-week-old male C57BL/6 mice were fed on SCD or HFD for 4 weeks. At approximately 8 weeks
of age, they were treated with W2476 (200 or 400 mg·kg-1·d-1), verapamil (100 mg·kg-1·d-1) or vehicle for 6 weeks (n=11–12 per group). (A, B) Body
weight on the last day of dosing and the average food intake during the 6-week treatment period. (C) The 6-h fasting blood glucose levels at weeks 0,
3 and 6. (D, E) Effects of W2476 and verapamil on pancreatic TXNIP protein levels in each group. At the end of treatment, plasma insulin levels were
examined (F), and ITT was carried out to measure blood glucose response (G) and associated AUC0–120 (H) in each group. Data shown are mean±SEM.
*P<0.05, **P<0.01 vs SCD group. #P<0.05, ##P<0.01 vs HFD group.
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Figure 7. W2476 improves glucose homeostasis and insulin sensitivity in DIO mice. Male C57BL/6 mice fed on SCD or HFD for 12 weeks were
chronically treated with W2476 (200 or 400 mg·kg-1·d-1), verapamil (100 mg·kg-1·d-1) or vehicle for 6 weeks (n=9 per group). (A) Body weight at the end of
treatment. (B) Average daily food intake during the 6-week treatment period. (C) The 6-h fasting blood glucose levels at weeks 0, 3 and 6. (D) Plasma
insulin levels following 6-week treatment. At the end of the experiment, ITT was carried out to measure blood glucose response (E) and associated
AUC0–120 (F) in each group. Data shown are mean±SEM. *P<0.05, **P<0.01 vs SCD group. #P<0.05, ##P<0.01 vs HFD group.
transcription and insulin production[18]. In this study, we Langerhans. Failure of W2476 to rescue MafA mRNA levels
have demonstrated that: (1) TXNIP was significantly elevated might have resulted from the indifference of this transcription
by high glucose; (2) this phenomenon was accompanied by factor to 48 h stimulation by either 33.3 mmol/L or 5 mmol/L
down-regulation of MafA and insulin expression; (3) W2476 glucose in isolated rat islets (data not shown). These protec-
could reverse this course via modulation of TXNIP; and (4) tive effects on β-cells exerted by W2476, including enhanced
most of these effects were confirmed in isolated rat islets of β-cell mass by inhibiting apoptosis and improved function
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Li T et al
by increasing insulin production, appear to mimic those pro- characterized in vitro and in vivo. W2476 improved β-cell dys-
duced by TXNIP-deficient HcB-19 mice[16, 18]
.
function by protecting the cells against apoptosis and enhanc-
TXNIP nonsense mutation (HcB-19) mice were found to ing MafA and insulin expression. It exerted therapeutic prop-
exhibit lower blood glucose levels, increased β-cell mass and erties in both STZ-induced diabetic and DIO mice. Our find-
resistance to STZ-induced diabetes[46, 47]. bTKO mice also dis- ings warrant further functional studies of ChREBP-regulated
played defense against β-cell apoptosis induced by STZ injec- genes such as txnip to explore their potential application in
tion[16]. In our in vivo experiments, a similar protective effect treating T2DM.
was observed in STZ-treated mice receiving W2476. Because
verapamil was reported to inhibit β-cell TXNIP expression
Acknowledgements
and prevent STZ-induced diabetes, it was applied as a posi- This work was partially supported by the National Health
tive control. Both W2476 and verapamil not only lowered and Family Planning Commission (No 2012ZX09304-011,
blood glucose levels but also relieved diabetic symptoms and 2013ZX09401003-005, 2013ZX09507001 and 2013ZX09507-
rescued β-cell loss in STZ-treated animals by lowering TXNIP. 002), Shanghai Science and Technology Development Fund
Our results are in line with previous reports that TXNIP defi- (No 15DZ2291600 and 14431901200), the Ministry of Science
cient mice were resistant to STZ, and hence, development and and Technology International Cooperation Program (No
progression of diabetes[39]
.
2014DFG32200), Les Laboratories Servier (France) and the
It has been demonstrated that disruption of TXNIP Thousand Talents Program in China. The authors declare no
improved glucose homeostasis and ameliorated insulin sen- conflict of interest in the design, execution and publication of
sitivity through activating insulin receptor substrate-1/Akt this article.
signaling[23]. Obesity is one of the most important factors in
We are indebted to Xu SHEN, Cai-hong ZHOU, Ting XIAO,
the development of insulin resistance. We employed a DIO and Wen-juan HU for valuable discussions and technical assis-
model[41] to further elucidate the therapeutic property of tance.
W2476. Following induction with HFD, C57BL/6J mice exhib-
ited significant increase in body weight and moderate hyper-
Author contribution
glycemia and hyperinsulinemia. After treatment with W2476 Olivier NOSJEAN, Jean A BOUTIN, Pierre R, and Ming-wei
for 6 weeks, the fasting glucose level was markedly decreased WANG conceived the idea. Ming-wei WANG, Olivier NOS-
without obvious changes in body weight and food intake. JEAN, Ting LI, and Li ZHONG designed the study. Ting LI,
This observation is in agreement with the conclusion drawn Guang-yao LIN, Li ZHONG, Yan ZHOU, Jia WANG, Yue
from the investigations in HcB-19, TXNIP-deficient ob/ob and ZHU, Yang FENG, Xiao-qing CAI, and Qing LIU performed
verapamil-treated BTBR ob/ob mice[16, 26, 31, 47]
.
the experiments. Ting LI, Li ZHONG, De-hua YANG, and
DIO is characteristic of hyperinsulinemia and insulin Ming-wei WANG analyzed the data and wrote the manu-
resistance[41]. Following HFD induction, plasma insulin was script.
significantly elevated in obese mice, which was corrected by
6-week treatment with W2476, but not verapamil. ITT results
Supplementary information
also support this phenomenon, ie, verapamil was less effica- Supplementary information is available on the website of Acta
cious (prophylactic setting) or unable to (therapeutic setting) Pharmacologica Sinica.
improve insulin sensitivity. Because verapamil, as a calcium
channel blocker, interrupts intracellular calcium releases
to affect insulin secretion and sensitivity[40], its behavior
described above is expected.
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