Antidiabetic drug metformin (GlucophageR) increases
biogenesis of Alzheimer’s amyloid peptides via
up-regulating BACE1 transcription
Yaomin Chena,b, Kun Zhoua, Ruishan Wanga, Yun Liua, Young-Don Kwaka, Tao Maa,b, Robert C. Thompsona,
Yongbo Zhaob, Layton Smithc, Laura Gasparinid, Zhijun Luoe, Huaxi Xua, and Francesca-Fang Liaoa,1,2
aNeurodegenerative Disease Research Program, Burnham Institute for Medical Research, La Jolla, CA 92037;bDepartment of Neurology, Shanghai First
People’s Hospital, Shanghai Jiao Tong University, Shanghai 200080, China;cDepartment of Pharmacology, Burnham Institute for Medical Research
at Lake Nona, Orlando, FL 32819;dDepartment of Neuroscience and Brain Technologies, Italian Institute of Technology, Via Morego 30,
16163 Genova, Italy andeDepartment of Genetics and Genomics, Boston University School of Medicine, Boston, MA 02118
Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved December 19, 2008 (received for review August 13, 2008)
Epidemiological, clinical and experimental evidence suggests a link
between type 2 diabetes and Alzheimer’s disease (AD). Insulin
modulates metabolism of ?-amyloid precursor protein (APP) in
neurons, decreasing the intracellular accumulation of ?-amyloid
(A?) peptides, which are pivotal in AD pathogenesis. The present
study investigates whether the widely prescribed insulin-sensitiz-
ing drug, metformin (GlucophageR), affects APP metabolism and
A? generation in various cell models. We demonstrate that met-
formin, at doses that lead to activation of the AMP-activated
protein kinase (AMPK), significantly increases the generation of
both intracellular and extracellular A? species. Furthermore, the
effect of metformin on A? generation is mediated by transcrip-
tional up-regulation of ?-secretase (BACE1), which results in an
elevated protein level and increased enzymatic activity. Unlike
insulin, metformin exerts no effect on A? degradation. In addition,
we found that glucose deprivation and various tyrphostins, known
inhibitors of insulin-like growth factors/insulin receptor tyrosine
kinases, do not modulate the effect of metformin on A?. Finally,
inhibition of AMP-activated protein kinase (AMPK) by the phar-
macological inhibitor Compound C largely suppresses metformin’s
effect on A? generation and BACE1 transcription, suggesting an
AMPK-dependent mechanism. Although insulin and metformin
display opposing effects on A? generation, in combined use,
metformin enhances insulin’s effect in reducing A? levels. Our
findings suggest a potentially harmful consequence of this widely
prescribed antidiabetic drug when used as a monotherapy in
elderly diabetic patients.
contributing to its development and progression. AD is not only
characterized by pathological deposition of A? peptides and
neurofibrillary tangles but is also associated with microglia-
mediated inflammation and dysregulated lipid homeostasis and
glucose metabolism. Amyloid peptides are derived from sequen-
(APP) by ?-secretase (BACE1) and ?-secretase. Full-length
APP can undergo alternative processing by ?-secretase, releas-
ing a soluble fragment (sAPP?) extracellularly, which precludes
A? formation. Compelling evidence indicates that A?, especially
the oligomers, are toxic to neurons; excessive generation and
pathological cascade in AD (1–3).
Epidemiological studies strongly suggest that metabolic de-
fects correlate with the functional alterations associated with
aging of the brain and with AD pathogenesis (4–11). The vast
majority of AD cases are late onset and sporadic in origin with
aging being the most profound risk factor. Insulin signaling is
known to be involved in the process of brain aging (12–20).
Insulin dysfunction/resistance in diabetes mellitus (DM) is not
lzheimer’s disease (AD) is a devastating neurodegenerative
disorder, with aging, genetic, and environmental factors
factor for AD, especially for vascular dementia (21, 22). The link
between DM and AD, plus the high prevalence of both diseases
in the elderly population, prompted us to search for desirable
concomitant pharmacotherapy based on the FDA-approved
drugs. Clinical findings indicated that insulin has beneficial
effects on cognition in patients with dementia (23, 24). More-
over, clinical trials on the PPAR? agonist rosiglitazone, one of
the FDA-approved thiazolidinediones (TZDs) for treating type
2 diabetes, showed improved cognition and memory in patients
with mild to moderate AD (25–28). In addition, we have shown
that insulin regulates APP processing/trafficking in neuronal
cultures, reducing intracellular levels of A? (29). In this context,
it would be of interest to learn whether another FDA-approved
insulin-sensitizing drug, metformin, which likely acts indepen-
dently of the PPAR pathways, has a similar effect on APP/A?
metabolism. Metformin (GlucophageR, 1, 2-dimethylbiguanide
hydrochloride; ?36 million U.S. prescriptions in 2003) (30), is a
biguanide that has pleiotropic effects on metabolism, including
insulin-sensitization, increased glucose uptake, decreased he-
patic glucose synthesis, activation of AMP activated protein
kinase (AMPK, an enzyme involved in glucose and fatty acid
metabolism), and mitochondria inhibition (31, 32).
Metformin Increases A? Generation. To examine the effects of
metformin on APP metabolism, we used 2 cellular models
including primary cortical neurons and N2a neuroblastoma cells
stably expressing human APP. We treated N2a695 cells with
metformin and found that metformin increased levels of both
extracellular (Fig. 1A) and intracellular (data not shown)
A?40/42 in dose-dependent manners, with the maximum effect
(?3-fold) seen after 24 h at 10 mM. Similar effects were seen in
primary neurons at a much lower concentration of metformin
(10 ?M) (Fig. 1B). To ascertain that the intracellular A?
measured from cell lysates did not include the secreted A? that
is often associated with cell membranes, we briefly treated cells
with trypsin and then with trypsin inhibitors before lysis and
found no significant difference in the intracellular A? levels with
or without trypsin cleavage (Fig. S1A).
Author contributions: F.-F.L. designed research; Y.C., K.Z., R.W., Y.L., Y.-D.K., T.M., R.C.T.,
and L.S. performed research; Y.C., Y.Z., L.G., Z.L., H.X., and F.-F.L. analyzed data; and F.-F.L.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com or
2Present address: Department of Pharmacology, University of Tennessee Health Science
Center, Memphis, TN 38163.
This article contains supporting information online at www.pnas.org/cgi/content/full/
www.pnas.org?cgi?doi?10.1073?pnas.0807991106 PNAS ?
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no. 10 ?
We then analyzed the levels of various APP metabolites
including the cleavage products of ?- and ?-secretases (Fig. 1C).
Metformin reduced ?-cleavage and promoted ?-cleavage, as
evidenced by decreased sAPP? and increased APP C-terminal
fragment, CTF-? (the upper CTF band that resulted from
cleavage by BACE1). No change in the levels of full-length PS1
(presenilin 1, the core component of ?-secretase) or its N-
terminal fragment was detected from total cell lysates.
trafficking. The surface levels of both APP and LRP1 (low-
density lipoprotein receptor-related protein 1), which are known
to comigrate during trafficking, were dramatically reduced after
metformin treatment as detected by biotinylation assays,
whereas their total protein levels remained unchanged (Fig. 1D).
However, the surface and the total BACE1 were markedly
sucrose gradients, we showed that metformin treatment caused
changes in the compartmentalization of APP, as evidenced by
increased distribution in trans-Golgi network (TGN) vesicles
(fraction 2), including those trafficks en route to early endo-
somes and TGN (fraction 3), and decreased distribution in
membranes (fraction 5) (Fig. S1 B and C). BACE1 protein levels
were found to be elevated in all 3 fractions: 2, 3, and 5, with an
?2-fold increase of the total protein. The increased distribution
of both APP and BACE1 in fractions 2 and 3 are expected to
favor A? generation within TGN and/or endocytic compart-
ments, the two compartments with mild acidic pH optimal for
BACE1 activity (33, 34). Indeed, we detected increased immu-
nofluorescent A?40 signals after metformin treatment in TGN
of 2 enzymes known to degrade A?, neprilysin and insulin-
degrading enzyme (IDE). Metformin had no effect on both
enzymes, including protein levels and their activities (Fig. S2 A
and B). Moreover, metformin had no effect on A? degradation
as measured by pulse–chase assay (Fig. S2C). We also found that
the increased A? production caused by metformin was not due
to increased APP expression, because the total APP level was
unaltered (Fig. 1D).
Metformin Up-Regulates BACE1 Promoter Activity. As correlated
with its increased protein level (Fig. 1D), metformin increased
the total BACE1 enzymatic activity by 2-fold (Fig. 2A). BACE1
mRNA was also increased by metformin in a time-dependent
manner in both N2a695 and primary cortical neurons (Fig. 2B),
of APP, LRP and BACE1 upon metformin treatment. (E) Immunocytochemistry showing increased A?40 species in the trans-Golgi network colocalized with the
Effects of metformin on APP/A? metabolism. (A) Dose-dependent effects on extracellular A?40/42 in N2a695 cells as measured by ELISAs. Data
in’s effect on BACE 1 enzymatic activity as measured by Sigma’s activity assay
kit. Data therefore represent the relative total BACE1 activity per cell. (B)
Effect of metformin on BACE1 mRNA level. The BACE1 transcript levels were
(C) Metformin up-regulates BACE1 promoter activity. Data represents the
luciferase activity of a 1.5-kb BACE1 promoter-luciferase construct after tran-
(10 mM) or insulin (1 ?M) for 24 h. The BACE1 promoter activity is presented
with respect to the activity of a control plasmid. The gray bars represent the
relative luciferase activity with full-length BACE1 promoter (BACE?F1),
whereas the black bars represent the activity of the truncated promoter
(BACE1?F2) lacking the first 3 PPAR/RXR binding elements as illustrated in the
scheme above the bar graph. In both settings, metformin up-regulates the
promoter activity of BACE1?F1 and BACE1?F2 to a similar degree, suggesting
a PPAR?-independent mechanism. n ? 5.
Effects of metformin on BACE 1 expression and activity. Data pre-
www.pnas.org?cgi?doi?10.1073?pnas.0807991106Chen et al.
as measured by semiquantitative RT-PCR. A luciferase reporter
assay on a 1.5-kb BACE1 promoter (35) showed that metformin
increased promoter activity by ?5-fold whereas insulin had no
effect (Fig. 2C).
Recently, BACE1 promoter activity was reported to be mod-
ulated by PPAR?-dependent transactivation. In addition to the
PPAR?-responsive element (PPRE) identified (36), 3 additional
binding sites for RXR heterodimers were predicted within the
1.5-kb BACE1 promoter based on their consensus motifs (Table
1). We therefore examined whether metformin up-regulates
BACE1 transcription through a PPAR?-RXR-mediated path-
way using a luciferase reporter construct containing a 5? trun-
cated fragment of the rat BACE1 promoter (?1, ?753). This
truncated promoter lacked the first 3 PPRE/RXR elements but
still exhibited 5-fold-increased activity upon metformin treat-
ment (Fig. 2C), suggesting an up-regulation of BACE1 transcrip-
tion independently of PPAR?.
Metformin’s Effect Is Independent of Glucose Metabolism and Insulin
Signaling. To investigate whether the A?-increasing effect of
metformin depends on insulin levels and glucose metabolism,
metformin-treated N2a695 cells were cultured in low-glucose
3A). However, metformin still increased A? production to a
similar degree as normal conditions (25 mM glucose). In con-
trast, cells cultured under serum-free conditions for 24 h, where
the basal intracellular A? level was reduced by 30%, became
that the effect of metformin depends on the presence of growth
factors in the serum, possibly including insulin and insulin-like
growth factors (IGFs).
To determine whether insulin signaling is involved in medi-
ating metformin’s action, we tested several inhibitors to tyrosine
receptor protein kinase, including 2 potent pan-tyrphostins
(A25, AG126) and the insulin/IGF-1 pathway-selective AG538.
Interestingly, all 3 tyrphostins did not show any significant effect
at the concentrations tested (1–25 ?M; Fig. 3B). Taken together,
these results indicate that metformin likely augments A? pro-
duction through mechanisms independent of insulin signaling
and glucose metabolism.
Metformin’s Effect Is Mediated by Activation of the AMP Kinase
(AMPK) in Vitro and in Vivo. We examined whether metformin’s
A?-increasing effect depended on activation of AMPK, a known
molecular target of metformin. Phosphorylation of AMPK at
Thr-172 and its substrate, acetyl CoA carboxylase (ACC), were
found to be both induced by metformin in a dose-dependent
manner (Fig. 4 A and B). We also observed a significant
inhibition of metformin-stimulated A? production by compound
C, a specific AMPK inhibitor, in a dose-dependent manner.
Compound C inhibited metformin’s effect by 50% when used at
for AMPK (32) (Fig. 4C). These results indicate an AMPK-
dependent mechanism for metformin’s effect on A?. Signifi-
cantly, the antagonizing effect of compound C was largely
Table 1. Predicted RXR/PPAR binding elements in BACE1 promoter region
The putative PPAR/RXR heterodimer binding sites in the rat BACE1 promoter predicted by the online program
MatInspector (www.genomatix.de). V$ represents the vertebrate family. The capital letters in the sequence
represent core sequence, and the underlined regions represent ci-value ? 60, according to matrix family
assignment with the RXR consensus sequences. The adenine?1 represents the translational start site.
signaling. (A) For low glucose conditions, cells were cultured in low-glucose
DMEM (5 mM) overnight and then metformin was added for an additional
24 h in this condition compared with normal glucose (25 mM). For serum-free
conditions, cells were cultured in DMEM/Opti-MEM for 24 h. Intracellular A?
production was measured by ELISA of lysates (diluted 50-fold) collected from
cells after the last 4 h incubation in serum-free media. (B) Effects of various
tyrphostins on metformin’s modulation of A? levels. Cells were pretreated
with metformin for 24 h and inhibitors were added at 10 ?M concentrations
the second day after switching to serum-free media. A? ELISAs were performed
using cell lysates collected 4 h after incubation with various inhibitors. n ? 4.
Metformin’s effect is independent of glucose levels and insulin
activates AMPK in N2a695 cells. Western blot analysis shows a marked eleva-
tion of the Thr-172 phosphorylated AMPK. (B) Dose-dependent effect of
metformin on activating ACC, the AMPK downstream substrate, as measured
by Western blot analysis of the phosphorylated ACC (Ser-79). (C) Effect of the
AMPK inhibitor Compound C (Comp. C) on abolishing metformin’s effect on
A? levels. Comp. C completely abolishes the effect of metformin on intracel-
lular A? production as measured by ELISA of cell lysates (diluted 50-fold). (D)
Combinatory effects of metformin and Comp. C on BACE1 transcription as
determined by semiquantitative RT-PCR.
Metformin’s effect depends on AMPK activation. (A) Metformin
Chen et al.PNAS ?
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attributed to suppression of BACE1 transcription because the
mRNA was greatly reduced after treatment with the two drugs
To validate our observations obtained from neuronal culture
systems, we treated C57B6 mice with metformin (2–5 mg/mL in
drinking water) for 6 days and found that its activating effects on
AMPK/ACC and on BACE1 were valid in mouse brains (frontal
region) (Fig. 5), indicating that the drug exerts a similar effect
in vitro and in vivo. Pharmacological analysis, using LC-MS,
indicated that metformin treatment at 2 mg/mL for 6 days in
mice resulted in accumulation of the drug to ?1 ?M concen-
tration in the brain (Table S1), suggesting that metformin likely
exerts a direct effect on APP processing in neurons via activating
the AMPK pathway, as was suggested by neuronal models.
Indeed, we also observed a similar augmenting effect of met-
formin on A? levels in a transgenic mouse line after drinking 2
mg/mL drug for 3 months (Fig. S3).
Antagonizing Effect on Intracellular A? Generation. Because met-
formin is known to sensitize insulin’s effects, one would expect
that the two drugs may exert similar or even synergistic effects
on APP/A? metabolism. Nevertheless, we reported that 1 ?M
insulin reduces intraneuronal A? by accelerating APP traffick-
ing and inhibiting A? degradation (29). Consistent with our
previous finding, we observed a significant reduction of intra-
cellular A? in the presence of 1 ?M, but not 0.25 ?M, insulin.
Interestingly, metformin not only failed to increase intracellular
A?, but also potentiated insulin’s A?-lowering effect, with a
both insulin and metformin (10 mM) were present (Fig. 6).
BACE1 is the predominant neuronal ?-secretase, catalyzing
?-cleavage of APP. Both its protein level and enzymatic activity
are elevated in AD brains, suggesting that abnormal BACE1
regulation may contribute significantly to AD pathogenesis.
Several transcriptional factors have been identified that modu-
late BACE1 transcription and some are involved in the inflam-
matory and chronic stress responses in the brain that are
compromised during aging. For instance, BACE1 is up-regulated
in neurons by (i) oxidative stress, (ii) chronic models of gliosis,
(ii) experimental traumatic brain injury, and (iv) hypoxia con-
ditions as recently demonstrated by our group and others (see
ref. 2 for review).
BACE1 transcription has recently been reported to be regu-
lated by the PPAR? pathway (36). We now demonstrate that the
diabetes drug metformin can also modulate BACE1 transcrip-
tion, likely independently of the PPAR? pathway despite the
presence of several PPAR/RXR binding sites in the promoter
(31, 32). Metformin-mediated transcriptional activation of
BACE1 appears to depended on a pathway involving AMPK.
Recently, the AMP-activated protein kinase (AMPK) has been
identified as one of the molecular targets of metformin, account-
ing for the majority of its pleiotropic effects. AMPK, which acts
as a fuel-sensing enzyme in glucose and fatty acid metabolism,
is ubiquitously expressed and highly conserved in the eukaryotic
kingdom (37, 38). Because AMPK is activated by metformin in
parallel with the up-regulation of A? generation in our exper-
imental systems, and is antagonized by the AMPK inhibitor
compound C (Fig. 5), it is likely that metformin modulates
BACE1 transcription through activating a signaling pathway, at
least in part, involving AMPK.
Our finding that metformin increases A? generation and
secretion raises the concern of potential side-effects, of accel-
erating AD clinical manifestation in patients with type 2 diabe-
tes, especially in the aged population. This concern needs to be
addressed by direct testing of the drug in animal models, in
conjunction with learning, memory and behavioral tests. Al-
remains largely unknown, recent studies (39, 40), together with
ours, suggest that metformin crosses the blood brain barrier and
exerts specific pharmacological effects in rodent brains upon
chronic administration. Indeed, our study using a comparable
dose (2 mg/mL is equivalent to a clinical dosage of 300 mg/kg/
day) indicates that systemically administrated metformin for 6
(Fig. 5). A direct measurement of the metformin concentration
in these mice showed that it reaches 2 ?M and 1 ?M in the
plasma and forebrain region, respectively (Table S1); Consider-
ing that the steady-state plasma level of metformin in patients is
reported to be from 10 ?M to as high as 40 ?M (32) and we
achieved the maximum effect of metformin on BACE1 levels
and AMPK activation in primary cultured neurons at 1–10 ?M,
the potential side-effects of metformin on accelerating AD
pathogenesis must be taken seriously.
Although the study was performed in neuronal models, met-
formin appears to be able to sensitize/enhance insulin’s anti-A?
effect as it can lower the effective concentration of insulin (Fig.
6), which agrees with its insulin-sensitizing effect in other
settings (37, 38). It remains as an interesting question how
metformin specifically sensitizes insulin’s effect on reducing A?
generation while diminishing its own stimulatory effect, when
used together. We believe that the combinatory effect of met-
formin and insulin, as shown in Fig. 6, involves the interplay of
their antagonizing effects on BACE1 transcription and on APP
processing/trafficking. Additional mechanisms may also be in-
volved because insulin signaling has multiple complex effects in
the CNS. The levels of insulin, insulin receptors and IDE are
analysis of phosphorylated AMPK, phosphorylated ACC and BACE1 protein
levels in mouse brain lysates (frontal region) after receiving metformin in
drinking water for 6 days. n ? 4 animals in each groups. (B) The bar graph
shows quantitative data of A.
Metformin activates AMPK/BACE1 in WT C57B6 mice. (A) Western
insulin was added at the 2 concentrations (0.25 ?M or 1 ?M) in serum-free
media for an additional 4 h in combination with metformin. Intracellular A?
levels were measured by IP-Western analysis. To measure insulin’s effect, it
was added directly to the cultures in serum-free media for 4 h before the A?
assays. (B) The bar graph shows quantitative data of the representative
IP-Western blot of A. n ? 3.
Combination of insulin and metformin reduces A? generation. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0807991106Chen et al.
reduced in AD brains (41, 42). Furthermore, a specific inhibitory
effect of soluble A? on insulin signaling has also been reported
(43, 44). Because the neurotoxicity of intraneuronal soluble
forms of oligomeric A? species to synaptic functions has been
increasingly reported, it is important to determine the specific
A? species that are regulated by metformin/insulin. We have
conducted preliminary studies on the brains taken from triple
transgenic mice (45) after a 3-month metformin treatment (4–7
months) and found a significant increase in BACE1 levels and
soluble A? (Fig. S3).
Our data suggest that the potentially deleterious effects of
metformin to AD patients may be avoided by using it in
combination with insulin; the combination may result in a
beneficial effect in treating both type 2 DM and in mitigating AD
progression. Despite the strong link between DM and AD, the
association between DM and the neuropathology of AD is less
clear, based on a few conflicting reports on limited patient
cohorts/populations (46–49). Studying the interaction between
medications for DM and AD neuropathology may clarify the
relationships between diabetes, diabetes medications, and AD.
In particular, comprehensive studies that include a large patient
cohort that take various diabetes medications (monotherapy
with insulin or oral drug versus combination therapy) are
needed. Indeed, two recent reports found significantly fewer
neuritic plaques (NPs) in the brains of diabetics taking a
combination of insulin and oral drugs, compared with those
taking a single drug (insulin or oral medicine) (50, 51), which
supports our observations (Fig. 6), although those patients
taking metformin alone should be further evaluated for AD
neuropathology and A? content in CSF.
Likewise, it is probably beneficial to combine metformin with
certain TZDs based on the known suppressive role of certain
PPAR? agonists in BACE1 transcription (36). The distinct but
complementary mechanisms of action of these two drug types in
insulin-sensitization, the reduction of inflammation and in low-
TZD/metformin combination therapy in patients with type 2
diabetes. In fact, the use of a single pill containing metformin
and rosiglitazone (Avandamet) was approved by the FDA in
reported beneficial effects of certain TZD in attenuating learn-
ing and memory deficits in AD mouse models (53, 54), it remains
to be determined whether a complementary effect for met-
Materials and Methods
Chemicals and Antibodies. Metformin was obtained from 2 sources (Sigma–
Aldrich and Calbiochem). Insulin and tyrphostins AG538 and AG126 were
obtained from Sigma–Aldrich. The monoclonal antibody 6E10 and the ELISA
kits for A? 40 and A? 42 were obtained from Signet Laboratories. The
polyclonal antibodies against the C-terminal portion of APP (369) and CT-
antibodies were obtained from Cell Signaling. Anti-IDE 28H1 clone was from
Santa Cruz Biotechnology; anti-neprilysin and 22C11 monoclonal antibody
were from Millipore. Compound C, anti-?-amyloid 40 (FCA3340) and anti-
?-amyloid 42 (FCA3542) were obtained from Calbiochem.
Cell Lines and Culture Treatment. N2a695 cells were maintained in DMEM.
Mouse P0 primary cortical cultures were prepared as described in ref. 29. For
drug treatments, cells were pretreated with metformin for 24 h and then
switched to serum-free media for an additional 4 h before the cultured
supernatants were collected for A? ELISAs. For the inhibitors, compound C or
tyrphostins were added alone in serum-free conditions for 4 h or after pre-
treatment of cells with metformin for 24 h.
as described to detect intracellular A? (29). Double fluorescent staining was
performed as described in ref. 33, using specific rabbit anti-A? 40 (FCA3340)
Cell Surface Biotinylation. Biotinylation was performed using sulfo-NHS-LC-
biotin (Pierce), which was added to cultures at 0.5 mg/mL for 1 h at 4 °C. After
washes, cells were lysed with Nonidet P-40 lysis buffer. Biotinylated cell
surface proteins were immunoprecipitated by anti-streptavidine-beads and
the surface APP (or BACE1) was detected with specific antibodies (22C11 or
CT-BACE1) by Western blot analysis.
RT-PCR on BACE1 Messages. Total RNA was extracted using TRIzol reagent
(Invitrogen). SuperScript First-Strand kit (Invitrogen) was used to synthesize
the first strand cDNA from samples with an equal amount of RNA, according
to the manufacturer’s instructions. Synthesized cDNAs were amplified using
IQ SYBR green supermix (Biopioneer) and ICycler from Bio-Rad; data were
analyzed using Bio-Rad MyIQ 2.0. Three pairs of primers used for BACE1
amplification are listed below. The pair of mouse specific primers were:
forward, 5?-GATGGTGGACAACCTGAG-3?, and reverse, 5?-CTGGTAGTAGC-
GATGCAG-3?. The rattus primers used were: forward, 5? TTGCCCAAGAAAG-
TATTTGAAG 3?, and reverse, 5? CGGAAGGACTGATTGGTG 3?. Primers used for
GAPDH amplification were: GAPDH-5, 5?-CGTGGAGTCTACTGGTGTC-3? and
GAPDH-3, 5?-ATCATACTTGGCAGGTTTCTC-3?. BACE1 mRNA levels were nor-
malized to levels of GAPDH.
Promoter Activity by Luciferase Assays. We cloned the 1.5-kb segment of the
rat BACE1 promoter into pGL3-Basic vector containing the firefly luciferase
the primers as described in ref. 31. The sequence-confirmed constructs were
transfected into N2a695 cells, using Lipofectamine 2000 (Invitrogen) and
pRL-CMV containing the Renilla luciferase gene (Promega) was cotransfected
as an internal control. The transfected cells were treated with or without 10
mM metformin or 1 ?M insulin for 24 h. The luciferase reporter assay was
performed according to the manufacturer’s instructions (Promega).
Statistical Evaluation. All statistical analysis was performed by ANOVA, fol-
lowed by Dunnett or Tukey–Kramer post hoc tests. Data are means ? SD. The
asterisks indicate significant difference versus control as follows:*, P ? 0.05
and**, P ? 0.01.
For more information, please see SI Methods.
ACKNOWLEDGMENTS. We thank Michael Vicchiarelli for excellent technical
assistance, Dr. Guojun Bu (Washington University School of Medicine, St.
Louis) for LRP antibody, and Drs. Geng-Sheng Feng (Burnham Institute for
Medical Research, La Jolla, CA) and Dale Ludwig (ImClone Systems, Inc., New
York) for constructive discussions. This work was supported by the National
Institutes of Health Grants R01 NS054880 (to F.-F.L.), R01 NS046673 (to H.X.),
and R01 AG030197 (to H.X. and F.-F.L.); Alzheimer’s Association Investigator
Initiated Research Award IIRG-06–26070 (to F.-F.L.); and a Zenith Award
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