ORIGINAL
RESEARCH
New Insight Into Metformin Action: Regulation of
ChREBP and FOXO1 Activities in Endothelial Cells
Xiaoyu Li, Karen L. Kover, Daniel P. Heruth, Dara J. Watkins, Wayne V. Moore,
Kathyrin Jackson, Mengwei Zang, Mark A. Clements, and Yun Yan
Division of Endocrinology (X.L., K.L.K., D.J.W., W.V.M., K.J., M.A.C., Y.Y.), Department of Pediatrics,
and Division of Experimental and Translational Genetics (D.P.H.), Department of Pediatrics, Children’s
Mercy Hospital and University of Missouri-Kansas City, Kansas City, Missouri 64108; and Department of
Medicine (M.Z.), Vascular Biology Section, Whitaker Cardiovascular Institute, Boston University School of
Medicine, Boston, Massachusetts 02481
Metformin has been considered a potential adjunctive therapy in treating poorly controlled type
1 diabetes with obesity and insulin resistance, owing to its potent effects on improving insulin
sensitivity. However, the underlying mechanism of metformin’s vascular protective effects remains obscure. Thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox state
induced by high-glucose concentration, decreases thioredoxin reductase activity and mediates
apoptosis induced by oxidative stress. Here we report that high glucose-induced endothelial
dysfunction is associated with induction of TXNIP expression in primary human aortic endothelial
cells exposed to high-glucose conditions, whereas the metformin treatment suppresses highglucose-induced TXNIP expression at mRNA and protein levels. We further show that metformin
decreases the high-glucose-stimulated nuclear entry rate of two transcription factors, carbohydrate response element-binding protein (ChREBP) and forkhead box O1 (FOXO1), as well as their
recruitment on the TXNIP promoter. An AMP-activated protein kinase inhibitor partially compromised these metformin effects. Our data suggest that endothelial dysfunction resulting from
high-glucose concentrations is associated with TXNIP expression. Metformin down-regulates
high-glucose-induced TXNIP transcription by inactivating ChREBP and FOXO1 in endothelial cells,
partially through AMP-activated protein kinase activation. (Molecular Endocrinology 29:
1184 –1194, 2015)
M
etformin, a first-line oral antidiabetic drug for adult
and pediatric type 2 diabetes has long been known
to promote its lipid-lowering and insulin sensitivity-improving actions in liver and pancreatic cells (1–5). It inhibits the mitochondrial respiratory chain complex I,
which increases the AMP to ATP ratio, thus leading to the
activation of AMP-activated protein kinase (AMPK) (6),
an important cellular energy sensor that may reflect glucose homeostasis. Activated AMPK is phosphorylated at
Thr-172 and subsequently phosphorylates multiple
downstream effectors to dictate cellular metabolism for
the restoration of energy homeostasis (7), thereby confer-
ring improved insulin sensitivity and lower hepatic lipid
content on cells. These properties of metformin make it an
attractive candidate for a potential adjunctive therapy for
treating type 1 diabetes (T1D). Recent clinical trial data
have shown that metformin treatment has a positive impact on improving insulin sensitivity in the overweight
youth with T1D (8).
Poor glycemic control increases the incidence of cardiovascular complications that are associated with endothelial dysfunction. Although multiple studies suggest
that metformin reduces cardiovascular disease risk in patients with type 2 diabetes (9 –11), the underlying mech-
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in USA
Copyright © 2015 by the Endocrine Society
Received March 27, 2015. Accepted June 30, 2015.
First Published Online July 6, 2015
Abbreviations: ACC, acetyl-CoA carboxylase; AMPK, AMP-activated protein kinase; ChIP,
chromatin immunoprecipitation; ChoRE, carbohydrate response element; ChREBP,
ChoRE-binding protein; DHE, dihydroethidium; FOXO1, forkhead box O1; FXBS, FOXO
binding site; HAEC, human aortic endothelial cell; ICAM-1, intercellular adhesion molecule 1; MAEC, mouse aortic endothelial cell; Mlx, max-like factor X; PARP, poly(ADPribose)polymerase; qRT-PCR, quantitative RT-PCR; ROS, reactive oxygen species; siRNA,
small interfering RNA; T1D, type 1 diabetes; TXNIP, thioredoxin-interacting protein.
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Mol Endocrinol, August 2015, 29(8):1184 –1194
doi: 10.1210/ME.2015-1090
doi: 10.1210/ME.2015-1090
anism by which metformin improves endothelial cell
functions remains poorly understood. Understanding the
mechanisms of the vascular-protective effects of metformin during hyperglycemia is necessary before clinicians can evaluate whether metformin might be beneficial
for patients with T1D by conferring protection against
the micro- and macrovascular complications of T1D.
Thioredoxin-interacting protein (TXNIP), a protein
acutely induced by high glucose concentrations, has
emerged as a key factor in maintaining glucose homeostasis (12). It binds to, and thus inactivates, the antioxidant protein thioredoxin, which is required for reducing
cellular oxidative stress. In addition, it inhibits peripheral
glucose uptake, which is independent of thioredoxin
binding (12, 13). TXNIP transcription has been shown
to be controlled by the transcription complexes carbohydrate response element (ChoRe)-binding protein
(ChREBP)-max-like factor X (Mlx), which is predominantly expressed in liver, and by MondoA-Mlx, which
mainly exists in skeletal muscle (14). ChREBP-Mlx and
MondoA-Mlx bind to the ChoRE in the TXNIP promoter to activate its expression in response to increased
glucose influx into cells (14). Many factors, including
metformin, have already been found to regulate TXNIP
mRNA levels by affecting this binding reaction (15–27).
Although metformin has been previously found to repress
TXNIP expression in pancreatic ␤-cells (21), metformin’s
effects on TXNIP in endothelial cells remain unknown.
Of note, previous studies indicate that the regulation of
TXNIP transcription may be in a tissue-specific pattern
(26, 28 –30). Transcription factor forkhead box O1
(FOXO1) up-regulates TXNIP expression in neurons
(29) and endothelial cells (30) but inhibits it in liver (28)
and pancreatic ␤-cells (26). Therefore, our study was directed to investigating metformin effects on endothelial
TXNIP to elucidate the mechanism that underpins this
vascular-protective function of metformin. We demonstrated that metformin benefits endothelial cells by ameliorating high-glucose effects partially via AMPK activation-dependent inactivation of ChREBP and FOXO1 in
TXNIP transcriptional initiation, suggesting that metformin may be a potential adjunctive therapy for protection against the micro- and macrovascular complications
of T1D.
Materials and Methods
Materials
Metformin (1,1-dimethylbiguanide hydrochloride), mannitol, and anti-␤-actin were purchased from Sigma. Compound C
was purchased from Millipore. Anti-AMPK␣, anti-phospho-
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AMPK␣, anti-phospho-acetyl-CoA carboxylase (ACC), antiACC, anti-FOXO1, and FOXO1 small interfering RNA
(siRNA) were from Cell Signaling Technology. Anti-TXNIP
was from Invitrogen. Anti-ChREBP, siRNAs of AMPK,
ChREBP, and the negative control were from Santa Cruz Biotechnology. Protein lysis buffer consisted of 20 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na3VO4, 1 ␮g/mL leupeptin; the mouse
aortic endothelial cell (MAEC) line growth media consisted of
Medium 199 supplemented with 5 ng/mL vascular endothelial
growth factor, 10 mM HEPES, 0.12 mU/mL heparin sodium,
5% fetal bovine serum, and 1% penicillin/streptomycin; and the
human aortic endothelial cell (HAEC) growth media consisted
of endothelial cell basal medium supplemented with EGM
SingleQuots from Lonza.
Cell culture and treatments
MAEC was obtained from Dr Ichiro (Tsurumi University,
Yokohama, Japan) (31). MAECs were maintained in MAEC
growth media at 37°C and 5% CO2. Unless otherwise indicated,
the glucose concentration of the normal growth medium for
MAEC was 5.5 mM. MAECs were serum starved overnight in
normal growth medium prior to any treatments. Primary
HAECs were obtained from Lonza and cultured according to
the manufacturer’s instructions. Unless otherwise indicated, the
glucose concentration of the normal growth medium for
HAECs was 5.5 mM, and only the cells at passage 5– 6 were
used. For metformin treatment, cells were exposed to 5.5 mM
D-glucose (normal glucose), 30 mM D-glucose (high glucose),
25 mM mannitol (osmotic control), or high-glucose growth medium with 2 mM metformin in the absence or presence of 10 ␮M
compound C for the times indicated.
Rats and treatments
Male Sprague Dawley rats (weight 250 –300 g, age 6 – 8
weeks) were purchased from Harlan Laboratories. The rats
were randomly divided into three groups: nondiabetic, diabetic,
and diabetic with metformin treatment. Diabetes was induced
by giving a one-time ip injection of streptozotocin (Sigma), 70
mg/kg, as we previously reported (32) and defined as random
blood glucose levels of greater than 250 mg/dL for 3 consecutive
days. One week after diabetes induction, the streptozotocininjected rats received metformin in their drinking water for 4
weeks, starting at 50 mg/kg䡠d body weight and increasing the
dose by 50 mg/kg every day to the final dose of 150 mg/kg䡠d.
Water was changed daily. At the end of the treatment period, the
rats were humanely euthanized. The aortas were collected then
saved snap frozen in liquid nitrogen for further studies.
The rats were housed in a 12-hour light, 12-hour dark cycle
with free access to water and regular food. This study was carried out in strict accordance with the recommendations in the
Guide for Care and Use of Laboratory Animals of the National
Institutes of Health. The protocol was approved by the University of Missouri-Kansas City Institutional Animal Care Use
Committee protocol 1229.
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Assessment of intracellular reactive oxygen
species generation
Confluent HAECs in six-well plates were exposed to normal
glucose (5.5 mM), high glucose (30 mM), or high glucose plus
metformin (2 mM) for 24 hours. For measurement of intracellular reactive oxygen species (ROS) generation, treated cells
were then stained with 10 ␮mol/L dihydroethidium (DHE) for
20 minutes and subsequently photographed by fluorescence microscopy (Fluoview-300; Olympus). Single-cell fluorescence intensities were determined for 60 cells from six randomly selected
fields (⫻200) per group per experiment, using ImageJ analysis
software [National Institutes of Health (NIH), Bethesda, Maryland]. Three independent experiments were selected for quantification for each group.
Mol Endocrinol, August 2015, 29(8):1184 –1194
PCR system (Eppendorf) using iQ SYBR Green Supermix (BioRad Laboratories). Primers used are listed in Supplemental Table 1. All the data were normalized with the expression of ␤-actin run as an internal standard.
Construction of TXNIP promoter reporter plasmids
The DNA fragment that covers 1 kb Txnip promoter and the
first exon of Txnip cDNA was amplified from mouse genomic
DNA by PCR and then cloned into a pGL3-basic vector (Promega) to generate a functional Txnip promoter-driven reporter
construct named as pTXP-1000. The sequences of the Txnip
promoter region in all the constructs were verified by DNA
sequencing. All the primers used here are listed in Supplemental
Table 1.
Transient transfection
MAECs or HAECs were grown in six-well plates and transfected with mouse-ChREBP-specific siRNA oligonucleotide (mChREBPsi) (100 nM), mouse FOXO1-specific siRNA oligonucleotide (m-FOXO1si), human-ChREBP-specific siRNA
oligonucleotide (h-ChREBPsi) (100 nM), human-FOXO1-specific siRNA oligonucleotide (h-FOXO1si) (100 nM), humanAMPK␣1/2-specific siRNA oligonucleotide (h-AMPKsi) (100
nM), scrambled oligonucleotides (100 nM), or FLAG-FOXO1
(Addgene) (0.4 ␮g/well) using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s instructions (33). All the treatments were performed after an overnight starvation at 24 hours
after the transfection.
Luciferase reporter assay
MAECs were seeded in 96-well plates and transfected with a
variety of DNA constructs or with an Simian virus 40 driven
pGL3 control vector (0.16 ␮g/well), using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s instructions. The
transfected cells were treated after an overnight starvation at 24
hours after the transfection. The relative firefly luciferase activities were measured in a TriStar LB 941 multimode microplate
reader (Berthold Technologies GmbH & Co KG) using the dual
luciferase reporter assay kit from Promega, following the manufacturer’s instructions (33). Data were normalized with the
Renilla luciferase activity.
Quantitative RT-PCR
A quantitative RT-PCR (qRT-PCR) analysis was performed
as described previously (33). Total RNA was extracted using an
RNeasy Plus minikit (QIAGEN) according to the manufacturer’s instructions. Reverse transcription reactions were performed using a SuperScript II first-strand kit (Invitrogen). qRTPCR was performed on a Mastercycler ep realplex real-time
Figure 1. Metformin potently suppresses high glucose-induced TXNIP
overexpression in endothelial cells. A, Time-dependent effects of
metformin on Txnip expression in MAECs. MAECs were harvested after
6 and 24 hours of exposure to 5.5 mM glucose (NG), 30 mM glucose
(HG), or metformin treatments indicated in the figure and then
subjected to qRT-PCR analysis. B, Metformin suppressed the Txnip
promoter activity in MAECs. MAECs were transiently transfected with
a full-length Txnip promoter-driven luciferase reporter-containing
construct and then at 24 hours after transfection treated for another
24 hours as indicated. C, Metformin suppressed TXNIP mRNA level in
primary HAECs. HAECs were harvested and then subjected to qRT-PCR
analysis after the 48 hours of treatments as indicated. Data were
corrected for ␤-actin and quantified. D, Metformin suppressed
hyperglycemia-induced TXNIP expression in rat aortas. Aortas were
collected from saline-injected control (NG; n ⫽ 3), streptozotocininduced diabetic (HG; n ⫽ 3), and diabetic plus 150 mg/kg metformin
(HG⫹Met; n ⫽ 3) groups of rats after 4-week treatments. TXNIP
mRNA (upper panel) and protein (lower panel) levels in rat aortas were
analyzed by qRT-PCR and immunoblotting. Data were corrected for
␤-actin and quantified. In panels A–D, the data are represented as the
mean ⫾ SEM (n ⫽ 3). *, P ⬍ .001 vs normal glucose; #, P ⬍ .001 vs
high glucose.
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Western blot analysis
Coimmunoprecipitation assay
Western blot analysis was performed as described previously
(33). Whole-cell lysates were prepared on ice using lysis buffer
(Cell Signaling Technology) with protease inhibitor cocktail
(Roche) and phosphatase inhibitor cocktail (Roche) after two
ice-cold rinses with PBS. Immunoreactive bands were visualized
by enhanced chemiluminescence Western blotting detection reagent (GE Healthcare Life Sciences) and quantified by the NIH
ImageJ software.
For immunoprecipitations, MAEC cell lysates prepared in protein
lysis buffer were incubated with an anti-FLAG antibody for 4 hours at
4°C and then incubated with protein-G Sepharose beads (GE Healthcare Life Sciences) overnight at 4°C. After three washes in protein lysis
buffer, the beads were boiled in Laemmli sample buffer.
Chromatin immunoprecipitation assay
Chromatin
immunoprecipitation
(ChIP) assays were performed as described elsewhere (33). Briefly, the
TXNIP-promoter-ChREBP complex or
the TXNIP-promoter-FOXO1 complex
was immunoprecipitated with antibodies against ChREBP or FOXO1 from the
cell lysates of the MAECs and HAECs
treated with 5.5 mM D-glucose, 30 mM
D-glucose, or 30 mM D-glucose plus 2
mM metformin. A normal rabbit IgG
was used as a negative control. The
DNA extracts were then subjected to
qRT-PCR analysis. The primers used for
ChoRE and the FOXO binding site
(FXBS) in the TXNIP promoter are
listed in Supplemental Table 1.
Immunostaining and confocal
microscopy
HAECs were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and then stained with a rabbit anti-ChREBP antibody (1:100; Novus Biologicals) or a rabbit anti-FOXO1 antibody (1:100; Cell Signaling), followed by
Alexa Fluor 488 goat antirabbit IgG (1:
1000; Invitrogen). Cell imaging was performed on a Zeiss LSM 510 Meta confocal microscope fitted with a PlanApo ⫻63
oil immersion objective.
Statistical analysis
Results represent means ⫾ SEM. P
values were calculated by 1-way
ANOVA followed by post hoc analysis
for data sets from triplicate experiments
using GraphPad Prism 6. Statistical significance was set at P ⬍ .05.
Results
Figure 2. Metformin (Met) ameliorates high glucose-induced increase of ROS production,
proinflammatory state, and early apoptosis in endothelial cells. Confluent primary HAECs were
treated as indicated for 24 hours. For transient knockdown of TXNIP, 100 nM control siRNA and
human-specific TXNIP siRNA were applied to HAECs for 24 hours before the treatments. NG, normal
glucose, 5.5 mM glucose; HG, high glucose, 30 mM glucose. A, Measurement of intracellular ROS
production using DHE. Scale bar, 100 ␮m. Fluorescence intensity quantified using NIH ImageJ software is
also shown. B, ICAM-1 level and PARP cleavage assessed by Western blot analysis. Transient TXNIP
knockdown was also confirmed by Western blot. The protein levels corrected for ␤-actin and quantified
using ImageJ software (NIH) are shown in the right panel. Representative fluorescence images and
Western blots from three independent experiments are shown, and the data are represented as the
mean ⫾ SEM (n ⫽ 3). *, P ⬍ .001 vs normal glucose; #, P ⬍ .001 vs high glucose.
Metformin relieves increased
intracellular ROS production,
proinflammatory state, and
early apoptosis in high-glucosetreated endothelial cells by
blunting TXNIP overexpression
To investigate the possible vascular-protective function of metformin
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using TXNIP as a reporter, we first investigated the TXNIP expression profile in high glucose- vs normal glucose-treated MAECs and primary HAECs, by RT-qPCR
analysis. We discovered that high glucose exposure significantly increased Txnip mRNA levels and that metformin suppressed the Txnip mRNA level in MAECs in a
time-dependent manner (Figure 1A), with no change in 25
mM mannitol treatment (data not shown). We further
tested the Txnip promoter activities in MAECs by luciferase reporter assay using a luciferase reporter-contain-
Mol Endocrinol, August 2015, 29(8):1184 –1194
ing construct driven by the full-length Txnip promoter.
As shown in Figure 1B, metformin (2 mM) effectively
inhibited the induction of Txnip promoter activities by
high glucose, which is also consistent with its effects on
Txnip mRNA expression. We also observed this mRNA
trend in HAECs (Figure 1C), indicating that metformin
suppresses the high-glucose-induced TXNIP overexpression in endothelial cells. Moreover, metformin also expressed this effect in rat aortas (Figure 1D).
Next we determined whether this
metformin-suppressed TXNIP overexpression was associated with the
possible vascular-protective effects
of metformin on endothelial cells.
As shown in Figure 2, exposing
HAECs to high glucose (30 mM) led
to a significant increase in ROS
production, intercellular adhesion
molecule 1 (ICAM-1; an inflammatory marker) expression, and cleavage of poly(ADP-ribose)polymerase
(PARP), an early apoptosis marker,
whereas metformin prevented these effects. Transient knockdown of TXNIP
mimicked the metformin effects in
HAECs (Figure 2, A and B), suggesting
that TXNIP mediates at least the highglucose-induced changes that are consistent with endothelial dysfunction.
Figure 3. Metformin suppresses high glucose-induced TXNIP overexpression at least partially
through activation of AMPK. A and B, Metformin-suppressed TXNIP overexpression is partially
reversed by an AMPK inhibitor, compound C, in primary HAECs (A) and MAECs (B). For Western
blot analysis of TXNIP, HAECs were harvested after the 48-hour exposure to the treatments
indicated; MAECs were harvested after the 24-hour treatments as indicated. For Western blot
analysis of phosphorylated AMPK, cells were serum starved overnight and then treated for 1
hour as indicated. C, Metformin-suppressed Txnip promoter activities is partially blocked by
compound C. MAECs were transfected with a Simian virus 40 -driven pGL3 control vector (data
not shown) or a full-length TXNIP promoter-driven luciferase reporter and then treated 24 hours
after transfection for additional 24-hour treatments as indicated. In panels A–C, the data are
represented as the mean ⫾ SEM (n ⫽ 3). NG, normal glucose, 5.5 mmol/L glucose; HG, high
glucose, 30 mmol/L glucose; HG⫹Met, 30 mmol/L glucose plus 2 mmol/L metformin;
HG⫹Met⫹C, 30 mmol/L glucose plus 2 mmol/L metformin and 10 ␮mol/L compound C. *, P ⬍
.05 vs normal glucose; #, P ⬍ .05 vs high glucose; $, P ⬍ .05 vs high glucose plus metformin. In
panels A and B, representative Western blots from three independent experiments are shown.
Metformin impairs highglucose-induced TXNIP
overexpression partially via
AMPK pathway
Given that metformin is well
known as an AMPK activator (34),
we applied an AMPK inhibitor,
compound C, along with metformin
to endothelial cells under high glucose to inspect the possible involvement of the AMPK pathway in
metformin-down-regulated TXNIP
expression. We first assessed the
phosphorylated AMPK level, which
represents the status of AMPK activation. As seen in Figure 3, A and B,
without any change in the total
amount of AMPK protein, high glucose severely abated AMPK activation, whereas metformin dramatically rescued the cells from this
negative effect. Compound C, how-
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ever, partly reversed the metformin effects on AMPK phosphorylation (Figure 3, A and B). We further observed
that compound C abrogated the metformin regulation at both the promoter
and protein levels to some extent (Figure 3, A–C). Therefore, AMPK activation at least partially mediates the metformin-dependent down-regulation of
TXNIP expression.
Figure 4. Metformin impairs the high-glucose-enhanced binding capacities of ChREBP and FOXO1
to the TXNIP promoter. A, Promoter analysis of the TXNIP promoter. The conserved glucoseresponsible E-Box and putative FXBS are shown. B, ChREBP and FOXO1 elevate the Txnip promoter
activity under high glucose. MAECs were cotransfected with the full Txnip promoter-driven luciferase
reporter plasmid and scrambled oligonucleotide (control siRNA), siChREBP or siFOXO1 and 24 hours
after transfection subjected to the treatments indicated for an additional 24 hours. Bars indicate
relative firefly luciferase activities, and the data are represented as the mean ⫾ SEM (n ⫽ 3). *, P ⬍
.001 vs normal glucose; #, P ⬍ .05 vs high glucose. Successful FOXO1 and ChREBP knockdown was
confirmed by immunoblotting (left panel). C, MAECs (left panel) and primary HAECs (right panel)
were treated for 24 hours (MAECs) or 48 hours (HAECs) prior to ChIP assays. Ctrl IgG, control IgG.
The data are represented as the mean ⫾ SEM (n ⫽ 3). *, P ⬍ .05 vs normal glucose; #, P ⬍ .05 vs
high glucose. D, MAEC cells were transiently transfected with a FLAG-tagged FOXO1 and then
treated for 24 hours as indicated. FLAG or control IgG (C) immunoprecipitates were immunoblotted
with anti-ChREBP and then stripped and reprobed with anti-FOXO1 to control for levels. The wholecell lysates were immunoblotted with anti-FLAG and anti-ChREBP to detect levels of FLAG-tagged
FOXO1 and ChREBP, respectively. Representative Western blots from three independent experiments
are shown. B to D, NG, normal glucose, 5.5 mM glucose; HG, high glucose, 30 mM glucose;
HG⫹Met, 30 mM glucose plus 2 mM metformin; HG⫹Met⫹C, 30 mmol/L glucose plus 2 mmol/L
metformin and 10 ␮mol/L compound C.
ChREBP and FOXO1 bind to the
TXNIP promoter and thus elevate
TXNIP transcription in endothelial
cells
Based on the in silico analysis of
the TXNIP promoter, we hypothesized that the down-regulation of
TXNIP transcription by metformin
is likely mediated by two highly conserved regulatory elements, FXBS
and a ChoRE composed of two Eboxes, within its promoter region
(Figure 4A). ChoRE and FXBS are
known to be recognized by two transcription factors, ChREBP and
FOXO1, respectively, in liver and
pancreatic ␤-cells (26, 28, 35) but
have not been well studied in endothelial cells. To establish the correlation between metformin signaling
and ChREBP- and FOXO1-involved transcriptional regulation of
TXNIP in endothelial cells, we first
performed a ChIP assay to determine whether endothelial ChREBP
and FOXO1 bind to ChoRE and
FXBS, respectively. As expected, a
significant enrichment of endogenous ChREBP and FOXO1 at
ChoRE and FXBS was observed in
both MAECs and HAECs, respectively (Supplemental Figure 1). No
enrichment was indicated in the control IgG immunoprecipitates or at
the GAPDH internal control (Supplemental Figure 1). Next, to examine the effects of ChREBP and
FOXO1 on TXNIP transcription,
we measured the Txnip promoter
activities by luciferase reporter assay
in transient ChREBP and FOXO1
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knockdown MAECs, respectively. We found that the induction of Txnip promoter activities by high glucose was
blunted by the mouse-specific ChREBP or FOXO1
siRNA but not by scrambled oligonucleotides (Figure
4B). Meanwhile, the Simian virus 40 control promoter
activities were unaffected by both the ChREBP and
FOXO1 siRNAs as a negative control (data not shown).
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Metformin suppresses the recruitment of ChREBP
and FOXO1 on the TXNIP promoter
We next performed ChIP assays to determine whether
metformin affects the recruitment of ChREBP and
FOXO1 on the TXNIP promoter. In both MAECs and
primary HAECs, high glucose potently induced the binding of ChREBP and FOXO1 to ChoRE and FXBS, respec-
Figure 5. Metformin hinders nuclear translocations of ChREBP and FOXO1 via the AMPK pathway. A, HAECs were treated for 48 hours as
indicated. Western blot analysis of the nuclear fractions under different conditions is also shown in the bottom panel. The protein levels were
corrected for Lamin-A and quantified using ImageJ software (NIH). B, AMPK knockdown diminishes the metformin effect on nuclear translocations
of ChREBP and FOXO1 under high-glucose conditions. HAECs were transiently transfected with the human-specific AMPK␣ (␣1 and ␣2) siRNA or
scrambled siRNA and then treated 24 hours after transfection for an additional 24 hours as indicated. Transient AMPK knockdown was confirmed
by the Western blots shown in the bottom panel. In panels A and B, representative images from two independent experiments are shown.
Immunocytochemistry and confocal imaging were performed as described in Materials and Methods. Scale bars, 5 ␮m. NG, normal glucose, 5.5
mM glucose; HG, high glucose, 30 mM glucose; HG⫹Met, 30 mM glucose plus 2 mM metformin; HG⫹Met⫹C, 30 mM glucose plus 2 mM
metformin and 10 ␮M compound C. The data are represented as the mean ⫾ SEM (n ⫽ 3).
doi: 10.1210/ME.2015-1090
tively, whereas metformin significantly aborted this highglucose effect (Figure 4C). By contrast, no significant
change was detected among all the treatments when control IgG was used in the ChIP assays (Figure 4C). Similarly, the GAPDH promoter failed to show any enrichment in the immunoprecipitates as a negative internal
control (data not shown), confirming the specificity of the
ChIP assays performed. This metformin-disturbed coregulation of TXNIP transcription by ChREBP and
FOXO1 prompted us to explore the possible protein-protein interaction between ChREBP and FOXO1 as well as
metformin actions on this interaction. By coimmunoprecipitation analysis using MAECs transiently transfected
with a FLAG-tagged mouse FOXO1, we revealed that
the interaction of ChREBP with FOXO1 existed at a
moderate level even under normal glucose conditions.
This interaction was significantly increased upon high
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1191
glucose yet was compromised by the metformin treatment
(Figure 4D), conforming to the observed metformin-impaired enrichment of ChREBP and FOXO1 on the
promoter.
Metformin inactivates intracellular ChREBP and
FOXO1 via the AMPK pathway
Given that ChREBP and FOXO1 require nuclear
translocation for their activation, we further investigated
metformin’s effect on the nuclear entry of endothelial
ChREBP and FOXO1 and the role of AMPK activation in
this process. Immunofluorescence staining and subcellular fractionation analysis (Figure 5A) indicated that metformin caused a notable decrease in high-glucose-stimulated nuclear accumulation of ChREBP and FOXO1,
whereas compound C dramatically reversed this effect.
We further created AMPK␣ (␣1 and ␣2) knockdown
Figure 6. Ablation of ChREBP and FOXO1 ameliorates high-glucose-induced endothelial dysfunction in HAECs. HAECs were transiently
transfected with the human-specific siRNAs of ChREBP and FOXO1 or scrambled siRNA and then treated 24 hours after transfection for an
additional 24 hours as indicated. NG, normal glucose, 5.5 mM glucose; HG, high glucose, 30 mM glucose. A, Measurement of intracellular ROS
production by DHE staining. Scale bar, 100 ␮m. Fluorescence intensity quantified using NIH ImageJ software is also shown. B, Protein level analysis
of TXNIP and PARP cleavage. Transient ChREBP and FOXO1 knockdown were also confirmed by Western blot. C, Protein level analysis of ICAM-1.
The protein levels corrected for ␤-actin and quantified using NIH ImageJ software are shown in panel D. Representative fluorescence images and
Western blots from three independent experiments are shown. The data are represented as the mean ⫾ SEM (n ⫽ 3). *, P ⬍ .001 vs normal
glucose; #, P ⬍ .001 vs high glucose.
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Metformin Depresses Endothelial ChREBP and FOXO1
HAECs to confirm this observation. As expected, the
transient knockdown of AMPK␣ exactly reproduced the
effect of compound C, which was further confirmed by
the concomitant decrease in the phosphorylation of ACC,
an AMPK downstream target (Figure 5B), supporting the
concept that in endothelial cells, metformin represses the
activities of ChREBP and FOXO1 by reducing their nuclear entry rate in a manner dependent on AMPK
activation.
All these findings taken together indicate that the metformin-mediated regulation of the TXNIP promoter activity lies in the recruitment of ChREBP and FOXO1 on
the TXNIP promoter. This inhibitory effect of metformin
in the nucleus is supported, at least partially, by the metformin-suppressed nuclear translocations of ChREBP
and FOXO1.
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ChREBP and FOXO1 activities in endothelial cells, partly
through AMPK activation.
Discussion
In this study, we discovered an indispensable role of the
metformin-AMPK-TXNIP pathway, which contributes
to the potential vascular-protective function of metformin by regulating the activities of two transcription
factors, ChREBP and FOXO1.
ChREBP (or MondoA) is a central glucose-sensing
transcription factor that is responsible for 75% of intracellular glucose-induced transcriptional response (14).
TXNIP, which fundamentally mediates glucose homeostasis, is one of its direct targets (36). Recently several
publications have reported that another transcription facMetformin mitigates high-glucose-induced TXNIP
tor, FOXO1, may also regulate TXNIP transcription (26,
overexpression by suppressing ChREBP and FOXO1
28 –30). Moreover, FOXO1 has been found to compete
Given that metformin impairs the transcriptional ac- with ChREBP to inhibit TXNIP transcription in pancretivities of ChREBP and FOXO1, we examined the possi- atic ␤-cells (26). In the present study, we have identified a
bility that the ablation of endothelial ChREBP and FOXO1 cross talk between endothelial ChREBP and FOXO1,
will ameliorate endothelial dysfunction caused by high glu- which is distinct from the one reported in pancreatic
cose. Transient knockdown of ChREBP and FOXO1 de- ␤-cells. Our data show that both ChREBP and FOXO1
creased ROS production and alleviated the proinflamma- are dramatically recruited to the TXNIP promoter to
tory state and early apoptosis in primary HAECs under transactivate strong expression of TXNIP upon high gluhigh-glucose conditions, resembling the effects of the met- cose, similar to the previously revealed FOXO1-upreguformin treatment (Figure 6, A–D). This finding indicates lated TXNIP expression in neurons (29). However, in
that metformin imposes its effects on TXNIP transcription liver and pancreatic ␤-cells, FOXO1 was observed to invia the regulation by ChREBP and FOXO1.
hibit TXNIP expression (26, 28) and was further conAltogether, metformin attenuates high-glucose-in- firmed in pancreatic ␤-cells to down-regulate TXNIP
duced TXNIP transcription by depressing intracellular transcription by decreasing ChREBP binding to the TXNIP promoter (26). All this evidence, considered alongside our results, indicate that the FOXO1
actions are tissue specific, at least in
the context of high-glucose-regulated transcription.
The antidiabetic drug, metformin, has been demonstrated to
reduce TXNIP expression in pancreatic ␤-cells (21) and HeLa cells (22)
by preventing some transcription
factors, including ChREBP (21),
MLX, and nuclear transcription factor Y subunit-␣ (22), from binding
to the TXNIP promoter. In our
study, this metformin down-reguFigure 7. Model for metformin action on TXNIP transcription in endothelial cells under highlated TXNIP expression is also conglucose conditions. Metformin prevents the nuclear entry of ChREBP and FOXO1 from cytosol
firmed in endothelial cells at mRNA
and further inhibits their binding capacity to the TXNIP promoter and thus suppresses TXNIP
and protein levels. Furthermore, our
transcription at last. The inhibitory effect of metformin on nuclear translocation is AMPK
ChIP data show that not only
phosphorylation dependent.
doi: 10.1210/ME.2015-1090
ChREBP, but also FOXO1, is inhibited by metformin
from binding to the TXNIP promoter in endothelial cells.
By coimmunoprecipitation analysis, we further observed
the physical interaction that existed between ChREBP
and FOXO1. We speculate that this interaction might
facilitate more stable binding of ChREBP and FOXO1 to
the promoter, owing to the enrichment of ChREBP and
FOXO1 on the TXNIP promoter in response to increased
glucose influx into cells. Nonetheless, further evidence is
needed to verify whether this interaction has any functional consequence in transcriptional regulation.
The AMPK mediation of metformin actions has long
been reported. Recently AMPK activation by metformin
has been linked to metformin-mediated suppression of
TXNIP expression in mouse colon and ileum (37) and in
mouse embryonic fibroblast cells (22), consistent with
our observations in endothelial cells. Despite these prior
observations, the mechanism of the metformin-AMPKTXNIP pathway remains ill defined. In this study we further delineated another important role of AMPK activation in the regulation of the intracellular ChREBP and
FOXO1 activities by metformin. As our data demonstrate, metformin abrogates the high-glucose-stimulated
nuclear translocation of ChREBP and FOXO1 via AMPK
activation. It has already been found that phosphorylation of ChREBP and FOXO1 facilitates their cytoplasmic
retention and that ChREBP and FOXO1 can each be
phosphorylated by AMPK (7, 38). These data strongly
support our notion that metformin-suppressed nuclear
entry of ChREBP and FOXO1 is AMPK dependent. The
only existing data supporting FOXO1 phosphorylation
by AMPK come from an in vitro kinase assay (38). Our
data presented here provide the functional consequence
of this phosphorylation. Despite this decisive role, AMPK
does not play a central role in terms of metformin-suppressed transcriptional initiation of TXNIP, which was
confirmed by our qRT-PCR and ChIP assays in the compound C-treated HAECs and MAECs (Supplemental Figure 2 and Figure 4C). We deduce that an as-yet-unknown
pathway within the nucleus mediates the metformin-induced compromised binding of ChREBP and FOXO1 to
the TXNIP promoter.
Overall, metformin negatively regulates TXNIP transcription in endothelial cells by impairing the transcriptional activities of ChREBP and FOXO1 on multiple steps
that occur both in cytosol and in the nucleus. First, metformin prevents the nuclear entry of ChREBP and
FOXO1 from cytosol. Next, for those escaped or already
translocated ChREBP and FOXO1, metformin further
inhibits their binding capacity to the promoter, thus eventually ensuring potent and effective suppression of TXNIP transcription (Figure 7). The inhibitory effects of
press.endocrine.org/journal/mend
1193
metformin on nuclear translocation require AMPK-mediated phosphorylation (Figure 7). Because our data are
based on ex vivo cell studies, a more detailed underlying
mechanism of metformin actions on endothelial TXNIP
still needs to be defined and confirmed in animal models.
Acknowledgments
We thank Peiying Tong and Chaoying Zhang for their help on
some of the animal experiments, We also thank Dr Shui-Qing
Ye (Children’s Mercy Hospital and University of Missouri-Kansas City, Kansas City, Missouri) for a critical reading of this
manuscript. The authors also thank the Medical Writing Center
at Children’s Mercy Hospital for editing this manuscript.
Address all correspondence and requests for reprints to: Yun
Yan, MD, and Mark A. Clements, MD, PhD, Division of Endocrinology, Department of Pediatrics, Children’s Mercy Hospital and University of Missouri-Kansas City, Kansas City, MO
64108. E-mail: [email protected].
This work was supported by Children’s Mercy Hospital Physician Scientist Award (to Y.Y. and M.A.C.), partially supported by Diabetes Action Research and Education Foundation
Grant 399 (to Y.Y. and M.A.C.), and partially supported by The
Endocrine Society Helmsley Charitable Trust Abstract Award
(to X.L.).
Disclosure Summary: The authors have nothing to disclose.
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