Am J Physiol Cell Physiol 303: C806–C814, 2012.
First published July 3, 2012; doi:10.1152/ajpcell.00437.2011.
AMP kinase regulation of sugar transport in brain capillary endothelial cells
during acute metabolic stress
Anthony J. Cura and Anthony Carruthers
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School,
Worcester, Massachusetts
Submitted 12 December 2011; accepted in final form 27 June 2012
blood brain barrier; endothelial cells; glucose transport; membrane;
metabolism
THE ADULT MAMMALIAN BRAIN uses glucose as its primary energy
source. While brain function and development require a constant glucose supply, diffusional exchange of glucose between
blood and brain is severely restricted by the blood-brain barrier. This barrier comprises a nonfenestrated, endothelium
formed by capillary endothelial cells connected by tight junctions. Glucose that enters the brain must, therefore, cross the
endothelium by protein-mediated trans-cellular transport. In
mammalian brain, this is catalyzed by the glucose transport
protein GLUT1, which is expressed in luminal and abluminal
membranes of microvasculature endothelial cells and is essential for brain metabolic homeostasis (13, 15, 30, 36, 43).
Under resting conditions and at physiologic blood glucose
levels, GLUT1-mediated glucose transport across the bloodbrain barrier barely exceeds brain glucose utilization (42).
Under conditions where glucose consumption could exceed
glucose supply, for example during hypoglycemia, hypoxia, or
intense neuronal activation, blood-brain barrier glucose transport capacity is increased by two different mechanisms. During
chronic metabolic stress [e.g., hypoxia (24) and hypoglycemia
(4, 33)], endothelial cell GLUT1 gene and protein expression
increases in vitro (3) and in vivo (5), thereby increasing
Address for reprint requests and other correspondence: A. Carruthers, 364
Plantation St., LRB Rm. 926 Worcester, MA 01605 (e-mail: anthony.carruthers
@umassmed.edu).
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blood-brain barrier sugar transport. In contrast, acute metabolic
stress [e.g., short-lived hypoglycemia, hypoxia, metabolic poisoning (14), or seizures (10, 35)] is without effect on GLUT1
expression but promotes recruitment of intracellular GLUT1 to
the plasma membrane (16), thereby increasing blood-brain
barrier sugar transport (14). The signals that regulate bloodbrain barrier sugar transport during acute metabolic stress are
not known.
Phosphorylated AMP-activated kinase (AMPK) plays a key
role in maintaining energy homeostasis in skeletal muscle (19,
45), heart (12, 32), brain (29, 37, 40), and endothelial cells (17)
by switching cellular metabolism to lowered ATP consumption
and increased ATP production. AMPK regulates glycolysis,
fatty acid oxidation, and glucose transport (11, 22, 23), the
activity of cell surface GLUT1 (1, 2) in cultured rat liver
Clone-9 cells and translocation of the insulin-sensitive glucose
transporter GLUT4 to the plasma membrane in heart and
muscle (25).
Aglycemia, hypoxia, and oxygen glucose deprivation activate AMPK in primary bovine brain microvascular endothelial
cells (44). Several forms of acute metabolic stress, including
glucose starvation, KCN or FCCP treatment induce AMPK
phosphorylation and sugar transport stimulation (14) in the
cultured brain microvascular endothelial cell line bEnd.3 (34).
These observations establish that immortalized and primary
cultures of cerebral microvasculature endothelial cells share a
common response to cellular metabolic stress but do not
directly address the role of AMPK activation in controlling
GLUT1-mediated sugar uptake. In the present study, we employ
AMPK agonists and antagonists and AMPK small interfering
(si)RNAs to perturb AMPK signaling. Our results strongly suggest that AMPK directs the trafficking of GLUT1 between endothelial cell membrane and intracellular pools and thereby regulates
blood-brain barrier endothelial cell sugar transport in response to
altered cellular metabolic status.1
EXPERIMENTAL PROCEDURES
Tissue culture. bEnd.3 cells were obtained from American Type
Culture Collection (Manassas, VA) and maintained in Dulbecco’s
modified Eagle’s medium (DMEM) from Gibco supplemented with
10% fetal bovine serum (FBS) from Hyclone and 1% penicillinstreptomycin (Pen-Strep) solution (Gibco) at 37°C in a humidified 5%
CO2 incubator as described previously (14).
Antibodies. A custom, affinity-purified rabbit polyclonal antibody
raised against a synthetic peptide corresponding to GLUT1 amino
acids 480 – 492 was produced by New England Peptide. Rabbit polyclonal and monoclonal antibodies against AMPK and phosphorylated
1
This article is the topic of an Editorial Focus by Warren L. Lee and Amira
Klip (26a).
0363-6143/12 Copyright © 2012 the American Physiological Society
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Cura AJ, Carruthers A. AMP kinase regulation of sugar transport
in brain capillary endothelial cells during acute metabolic stress. Am
J Physiol Cell Physiol 303: C806 –C814, 2012. First published July 3,
2012; doi:10.1152/ajpcell.00437.2011.—AMP-dependent kinase (AMPK)
and GLUT1-mediated sugar transport in blood-brain barrier endothelial cells are activated during acute cellular metabolic stress. Using
murine brain microvasculature endothelium bEnd.3 cells, we show
that AMPK phosphorylation and stimulation of 3-O-methylglucose
transport by the AMPK agonist AICAR are inhibited in a dosedependent manner by the AMPK antagonist Compound C. AMPK ␣1or AMPK ␣2-knockdown by RNA interference or AMPK inhibition
by Compound C reduces AMPK phosphorylation and 3-O-methylglucose transport stimulation induced by cellular glucose-depletion, by
potassium cyanide (KCN), or by carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Cell surface biotinylation studies
reveal that plasma membrane GLUT1 levels are increased two- to
threefold by cellular glucose depletion, AICAR or KCN treatment,
and that these increases are prevented by Compound C and by AMPK
␣1- or ␣2-knockdown. These results support the hypothesis that
AMPK activation in blood-brain barrier-derived endothelial cells
directs the trafficking of GLUT1 intracellular pools to the plasma
membrane, thereby increasing endothelial sugar transport capacity.
AMPK REGULATION OF SUGAR TRANSPORT IN ENDOTHELIAL CELLS
ment of sugar uptake. Cells were then prepared and processed as
previously described.
Biotinylation of bEnd.3 cells. Biotinylation was carried out as
previously described (14) with the following modifications: 100 mm
plates of confluent bEnd.3 cells were placed in serum-free DMEM
with 2 mM AICAR ⫾ 10 ␮M Compound C for 2 h, 2 mM AICAR ⫾
10 ␮M Compound C for 1.5 h followed by serum-free, glucose-free
DMEM ⫾ 10 ␮M Compound C for 30 min, or 5 mM KCN ⫾ 10 ␮M
Compound C for 10 min at 37°C before proceeding with biotinylation.
For AMPK knockdown experiments, cells were split into 100 mm
dishes before AMPK knockdown, and cells were treated with siRNA
or Dharmafect alone as previously described for 48 h before biotinylation. On the day of the experiment, plates were treated with 5 mM
KCN for 10 min at 37°C, then biotinylated and processed as previously described.
Data analysis. Differences in sugar transport, cell surface GLUT1
biotinylation, or AMPK phosphorylation in control and test conditions
were analyzed by using Student’s t-test.
RESULTS
Compound C inhibits AICAR-dependent AMPK phosphorylation.
AMPK is activated by phosphorylation of AMPK threonine
172 (22) and this phosphorylation is inhibited in bovine brain
microvasculature endothelial cells by Compound C (44). To
analyze Compound C inhibition of AMPK activity in bEnd.3
cells, we treated cells with 2 mM AICAR to activate AMPK in
the absence or presence of increasing concentrations of Compound C. Thr172 phosphorylation was analyzed by using an
AMPK-phospho-Thr172-specific antibody (Fig. 1A). AICARstimulated AMPK phosphorylation decreases with increasing
concentrations of Compound C. AMPK phosphorylation at 2
mM AICAR plus 10 ␮M Compound C is indistinguishable
from AMPK phosphorylation in the absence of AICAR. The
apparent inhibitory constant [Ki(app); 5.5 ⫾ 2.2 ␮M] for Compound C inhibition of AMPK phosphorylation was computed
by nonlinear regression analysis of the concentration dependence of Compound C inhibition of AICAR-dependent AMPK
phosphorylation by assuming simple Michaelis-Menten inhibition (Fig. 1B).
We also examined phosphorylation of ACC, a direct downstream target of AMPK (21). Using an antibody specific to
phosphorylated Ser79 on ACC, we measured AICAR-stimulated ACC phosphorylation in the presence of increasing Compound C concentration ([Compound C]) (Fig. 1, C and D).
Compound C reduces ACC phosphorylation in a dose-dependent manner with Ki(app) ⫽ 2.9 ⫾ 1.2 ␮M. Both AMPK and
ACC are phosphorylated in the absence of AICAR, suggesting
either that untreated bEnd.3 cells experience a low, but measurable level of metabolic stress or that there is a low level of
AMPK and ACC phosphorylation independent of metabolic
stress.
Compound C inhibits AICAR-stimulated 3-OMG uptake. The
facilitative glucose transport protein GLUT1 mediates sugar
transport in control and AICAR-treated bEnd.3 cells where
AICAR increases Vmax for 3-OMG net uptake by twofold (14).
We therefore asked whether AICAR stimulation of transport is
suppressed by Compound C.
bEnd.3 cells were treated with 2 mM AICAR plus increasing
concentrations of Compound C, and uptake of 20 mM 3-OMG
was measured over 30 s at 4°C (Fig. 2). 3-OMG is a nonmetabolizable GLUT1 substrate whose net uptake proceeds until
intracellular [3-OMG] ⫽ extracellular [3-OMG] and unidirec-
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AMPK (P-Thr172), and phosphorylated acetyl-CoA carboxylase
(ACC) (P-Ser79) were obtained from Cell Signaling Technology, and
an antibody against cyclophilin B was obtained from Abcam. Horseradish peroxidase- conjugated goat anti-rabbit secondary antibody
was obtained from Jackson Immunoresearch.
Buffers. Cell lysis buffer consisted of 5 mM HEPES, 5 mM MgCl2,
150 mM NaCl, 50 ␮M EDTA, and 1% SDS. Uptake stop solution
included 10 ␮M cytochalasin B (Sigma) and 100 ␮M phloretin
(Sigma) in Dulbecco’s phosphate-buffered saline (DPBS). TBS was
composed of 20 mM Tris base and 135 mM NaCl, pH 7.6. TBST
comprised TBS buffer with 0.2% Tween 20. Biotin Quench solution
was composed of 250 mM Trizma Base. Biotin lysis buffer contained
TBS with 0.5% Triton X-100.
Western blotting of bEnd.3 cells. Confluent 100 mm dishes of
bEnd.3 cells were treated with 2 mM AICAR (ThermoFisher) or 2
mM AICAR plus 1, 2, 5, 10, or 20 ␮M Compound C (Tocris
Bioscience) for 2 h. Cells were then washed twice with DPBS, lysed,
and analyzed for total protein concentration using a Micro BCA kit
(Pierce). Western blot analyses were performed as previously described (14) using a 1:1,000 dilution of either rabbit AMPK P-Thr172,
rabbit ACC P-Ser79 antibody, or a 1:10,000 dilution of rabbit cyclophilin B antibody. Samples were run in triplicate, and band densities
were quantitated using ImageJ software (National Institutes of Health,
Bethesda, MD).
Knockdown of AMP kinase in bEnd.3 cells. Pools of four separate
siRNA oligonucleotides targeting the mouse AMPK subunits ␣1 and
␣2, as well as cyclophilin B, and a pool of nonsilencing siRNA were
obtained from Dharmacon. The cationic lipid transfection reagent
Dharmafect 4, RNase-free water, and siRNA resuspension buffer
were also obtained from Dharmacon.
To knock down AMPK and cyclophilin B in bEnd.3 cells, confluent 100 mm dishes were split into 6- or 12-well tissue culture dishes
at a ratio of 1:3 the night before each knockdown. After cells were
verified as 70 – 80% confluent, growth media were replaced with
DMEM ⫹ 10% FBS but lacking Pen-Strep. Oligonucleotide-Dharmafect
complexes were prepared in DMEM without FBS or Pen-Strep.
Dharmafect and the siRNA were allowed to equilibrate with the media
for 5 min before being combined and allowed to complex for 20 min.
Following the complexing step, each well of cells was treated with a
total 4 ␮l of Dharmafect 4 alone or 4 ␮l of Dharmafect 4 and 100
nmol total siRNA for 48 h. For AMPK knockdowns, each well
contained 50 nmol each of AMPK ␣1 and ␣2 siRNA for a total of 100
nmol. Knockdown was verified by Western blotting using antibodies
for AMPK and cyclophilin B.
3-OMG uptake measurements in bEnd.3 cells. Uptake was measured for 30 s at 4°C as previously described (14) using 20 mM
3-O-methylglucose (3-OMG) containing 2.5 ␮Ci/ml [3H]3-O-methylglucose ([3H]3-OMG) in DPBS. Sugar uptake measurements in
AICAR-treated cells were performed as described above with the
following modifications: on the day of the assay, cells were placed in
serum-free DMEM or serum-free DMEM containing either 0 or 2 mM
AICAR or 2 mM AICAR plus 1, 2, 10, or 20 ␮M Compound C for 2
h at 37°C. Cells were washed with 1 ml of either glucose-free DPBS,
DPBS containing 2 mM AICAR, or DPBS containing 2 mM AICAR
plus Compound C, at which point uptake was measured.
Sugar uptake in metabolically poisoned and glucose-depleted cells
was measured as described above with the following modifications:
cells were placed in serum-free DMEM ⫾ 10 ␮M Compound C,
serum-free DMEM containing 2 mM AICAR ⫾ 10 ␮M Compound C
for 2 h, or serum-free DMEM ⫾ 10 ␮M Compound C for 1.5 h
followed by serum-free glucose-free DMEM ⫾ 10 ␮M Compound C
for 30 min. Cells were washed with 1 ml of either glucose-free DPBS,
or glucose-free DPBS plus the appropriate poison (AICAR, KCN, or
FCCP) ⫾ 10 ␮M Compound C at which point uptake was measured.
For AMPK knockdown experiments, cells were treated with siRNA
against the ␣1- and ␣2-subunits of AMPK for 48 h before measure-
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AMPK REGULATION OF SUGAR TRANSPORT IN ENDOTHELIAL CELLS
Fig. 1. Inhibition of AMP-activated protein kinase (AMPK) and acetyl-CoA
carboxylase (ACC) phosphorylation by Compound C. A and C: representative
Western blot of whole cell bEnd.3 lysates in the absence or presence of AICAR
and Compound C (concentrations indicated above the blots). Cell lysate (30 ␮g
total protein) was loaded into each lane and probed with AMPK P-Thr172 (A)
or ACC P-Ser79 (C) antibody. The mobility of molecular weight standards is
indicated. B and D: quantitation of Western blot band volumes from A and C,
respectively. Ordinate, AMPK (B) or ACC (D) phosphorylation relative to
control conditions (%); abscissa, concentration of Compound C (␮M). Data are
shown for control cells () and AICAR-treated cells exposed to Compound C
(Œ). Curves drawn though the Compound C data were computed by nonlinear
regression assuming simple competitive inhibition and provide the apparent
inhibitory constant [Ki(app)] for Compound C inhibition of phosphorylation.
Data points represent means ⫾ SE of three separate experiments.
tional 3-OMG uptake and exit are quantitatively identical. Net
uptake at 30 s achieves only 9% and 20% equilibration in
control and FCCP-treated cells, respectively (14), indicating
that transport determinations at 30 sc underestimate steadystate transport rates by ⬍7%. In the present study, AICAR
Fig. 2. Sugar uptake at 4°C in bEnd.3 cells in response to Compound C.
3-O-methylglucose (3-OMG; 20 mM) uptake was measured for 30 s in
untreated cells () or in cells exposed to 2 mM AICAR and increasing
[Compound C] (Œ) for 2 h at 37°C before cooling, glucose depletion, and
transport initiation. Ordinate, 3-OMG uptake (dpm per ␮g total cell protein);
abscissa, [Compound C] (in ␮M). The curve drawn though the AICAR-treated
cell data was computed by nonlinear regression assuming simple saturable
inhibition of uptake by Compound C but that uptake inhibition is incomplete.
This permits calculation of the Ki(app) for transport inhibition. Each point
represents the mean ⫾ SE of three separate experiments.
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stimulates 3-OMG uptake approximately 2.5-fold over control
cells. Addition of Compound C inhibits bEnd.3 sugar uptake in
a dose-dependent manner. Ki(app) for Compound C inhibition of
AICAR-activated sugar uptake is 1.1 ⫾ 0.2 ␮M. The 95%
confidence interval for Ki(app) for transport inhibition (0.1–2.1
␮M) overlaps with the confidence interval for Ki(app) for
Compound C inhibition of AMPK phosphorylation (0 –11 ␮M,
Fig. 1B). Transport in the absence of AICAR is increased by
1.4-fold by Compound C, demonstrating that inhibition of
AICAR-stimulated transport does not result from a direct
inhibition of GLUT1 by Compound C. We address Compound
C stimulation of transport in the DISCUSSION.
Acute metabolic stress-induced AMPK phosphorylation is
reduced with Compound C treatment and AMPK knockdown.
Treatment of bEnd.3 cells with siRNA oligonucleotides targeting mouse AMPK subunits ␣1 plus ␣2 reduces AMPK expression by 2.2-fold. Treatment with cyclophilin B siRNAs and a
pool of nonsilencing siRNAs is without effect on bEnd.3
AMPK expression (Fig. 3, A–D). The use of isoform-specific
AMPK siRNAs indicates that message knockdown by AMPK ␣1
or ␣2 siRNA is specific and is not accompanied by upregulation
of the nontargeted AMPK isoform (Fig. 4, A and B).
Pretreatment of cells with AMPK-targeted siRNAs for 48 h
reduces acute stress-induced AMPK phosphorylation. Cells
were treated with AICAR, 5 mM KCN, 8 ␮g/ml FCCP, 10 ␮M
Compound C, or glucose starved, and assayed for AMPK
phosphorylation by Western blot analysis (Fig. 5A). Quantitation of band densities (Fig. 5B) reveals that basal AMPK
phosphorylation is reduced by 72% following siRNA treatment. The responses to glucose deprivation, AICAR, Compound C, KCN, and FCCP treatments are reduced by 25%,
70%, 71%, 53%, and 46%, respectively, following siRNA
treatment. AMPK knockdown is incomplete (Figs. 3A and 5A)
and the question remains as to whether the residual AMPK,
AMPK REGULATION OF SUGAR TRANSPORT IN ENDOTHELIAL CELLS
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Fig. 3. AMPK small interfering (si)RNA inhibition of AMPK expression and effects
of cyclophilin B siRNAs (CypB siRNA) and a pool of nonsilencing (NS) siRNAs on
AMPK and cyclophilin B expression. A: Western blot of whole cell bEnd.3 lysates
from untreated cells (control), mock-transfected cells (mock), and cells transfected with
AMPK siRNAs, cyclophilin B siRNAs, or nonsilencing siRNAs. Cell lysate (30 ␮g
total protein) was loaded into each lane and probed with AMPK antibody and band
volumes were quantitated. The mobility of molecular weight markers is indicated.
B: ordinate, relative AMPK band volume (%); abscissa, experimental condition. Data
represent the mean of two separate experiments. C: Western blot of whole cell bEnd.3
lysates from untreated cells, mock-transfected cells, and cells transfected with AMPK
siRNAs, cyclophilin B siRNAs, or nonsilencing siRNAs. Cell lysate (30 ␮g total
protein) was loaded into each lane and probed with CypB antibody and band volumes
were quantitated. The mobility of molecular weight markers is indicated. D: ordinate,
relative cyclophilin B band volume (%); abscissa, experimental condition. Data
represent means ⫾ SE of three separate determinations. Two-tailed t-test analysis
indicates as follows: *P ⬍ 0.01, significantly different from control; §P ⬍ 0.01,
significantly different from mock transfected.
which continues to respond to metabolic stress (Fig. 5, A and
B), is sufficient to activate glucose transport.
Compound C inhibits acute metabolic stress-induced AMPK
activation in endothelial cells. We subjected bEnd.3 cells to 2
mM AICAR (10 min), glucose starvation (30 min), 5 mM KCN
(10 min), or 8 ␮g/ml FCCP (10 min) in the absence or presence
of Compound C, and assayed AMPK phosphorylation by
Western blot analysis. An untreated control was measured for
basal AMPK phosphorylation (Fig. 5C). Quantitation of phosphorylation (Fig. 5D) indicates that all stress conditions and
AICAR treatment increase AMPK phosphorylation by 4- to
10-fold. Cells treated with only Compound C show no significant increase in phosphorylation over controls. Compound C
treatment reduces stress-induced AMPK phosphorylation by
36% in glucose-starved cells, 56% in AICAR-treated cells,
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Fig. 4. qPCR analysis of AMPK ␣1 and AMPK ␣2 mRNA expression in cells
treated with AMPK ␣1, AMPK ␣2, or AMPK ␣1 ⫹ ␣2 siRNAs. A: AMPK ␣1
expression. Ordinate, relative AMPK ␣1 message levels (%) in mock-transfected cells and in cells transfected with AMPK ␣1, AMPK ␣2, or AMPK
␣1 ⫹ ␣2 siRNAs. B: AMPK ␣2 expression. Ordinate, relative AMPK ␣2
message levels (%) in mock-transfected cells and in cells transfected with
AMPK ␣1, AMPK ␣2, or AMPK ␣1 ⫹ ␣2 siRNAs. Cells were treated with
or without siRNAs for 48 h before lysis and message extraction. Data represent
means ⫾ SE of three separate experiments. One-tailed t-test analysis indicates
as follows: §P ⬍ 0.005, significantly lower than mock-transfected condition.
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AMPK REGULATION OF SUGAR TRANSPORT IN ENDOTHELIAL CELLS
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Fig. 5. Compound C and AMPK siRNA inhibition of AMPK phosphorylation during
metabolic stress. A: representative Western blot of whole cell bEnd.3 lysates from cells
pretreated for 48 h with or without AMPK siRNAs followed by 15 min pretreatment
at 37°C with or without 5 mM glucose (Glc), 5 mM KCN, 8 ␮g/ml FCCP, 10 ␮M
Compound C (Comp C), or for 2 h with 2 mM AICAR. Cell lysate (30 ␮g total
protein) was loaded into each lane and probed with AMPK P-Thr172 antibody and
band volumes were quantitated. B: ordinate, relative band volume (%); abscissa,
experimental condition. Data represent means ⫾ SE of three separate experiments.
*P ⬍ 0.05, significantly different from control condition. §P ⬍ 0.05, AMPK knockdown significantly reduces AMPK phosphoprotein levels relative to the test condition.
C: representative Western blot of whole cell bEnd.3 lysates in the absence or presence
of glucose, KCN, FCCP, AICAR, and Compound C. Before lysis, cells were treated
for 15 min at 37°C with 5 mM glucose (control), 0 glucose, 5 mM KCN, 8 ␮g/ml
FCCP, or for 2 h with 2 mM AICAR in the absence or presence of 10 ␮M Compound
C. Cell lysate (30 ␮g total protein) was loaded into each lane and probed with AMPK
P-Thr172 antibody and band volumes were quantitated. D: ordinate, relative band
volume (%); abscissa, experimental condition. Data represent means ⫾ SE of three
separate experiments. *P ⬍ 0.05, significantly different from control condition. §P ⬍
0.05, Compound C significantly reduces AMPK phosphoprotein levels relative to the
test condition.
69% in KCN-treated cells, and 61% in FCCP-treated cells.
Although AMPK phosphorylation was reduced in the presence
of Compound C, complete phosphorylation inhibition was not
observed.
Compound C and AMPK knockdown inhibit metabolic stressinduced 3-OMG uptake stimulation. We next asked whether
inhibition of AMPK phosphorylation suppresses sugar uptake
stimulation during metabolic stress. 3-OMG uptake was measured in bEnd.3 cells in the absence and presence of 10 ␮M
Compound C in cells that were glucose starved, treated with 2
mM AICAR, treated with 5 mM KCN, or treated with 8 ␮g/ml
FCCP as described above. 3-OMG uptake (Fig. 6A) is stimulated 2.6-fold in glucose-starved cells, 2.6-fold in AICARtreated cells, 2.9-fold in KCN-treated cells, and 3.0-fold in
FCCP-treated cells. Glucose transport in control cells is also
stimulated 1.9-fold by 10 ␮M Compound C. Compound C
inhibits 3-OMG uptake by 40% in glucose-starved cells, 42%
in AICAR-treated cells, by 54% in KCN-treated cells, and by
64% in FCCP-treated cells (Fig. 6A).
In cells where AMPK is knocked down before measurement
of 3-OMG uptake, stress-induced stimulation of uptake is also
reversed (Fig. 6B). Mock-transfected and AMPK knockdown
cells not subjected to stress show no appreciable increase in
3-OMG uptake over control cells. Nontransfected cells treated
with AICAR, KCN, FCCP, or cells subjected to glucose starvation all show a two- to threefold increase in 3-OMG uptake. This
stimulation of sugar uptake is reversed to near-control levels in
AMPK knockdown cells. AMPK knockdown inhibits 3-OMG
uptake by 48% in glucose-starved cells, by 41% in AICARtreated cells, by 50% in KCN-treated cells, and by 43% in
FCCP-treated cells (Fig. 4B). Compound C stimulates 3-OMG
uptake 1.3-fold, but AMPK knockdown has no effect on this
stimulation. The use of nonsilencing siRNAs or mock transfection protocols are without effect on basal sugar uptake or
KCN-stimulated sugar uptake (Fig. 6, A and B). All subsequent
AMPK siRNA experiments therefore used mock transfection
as the appropriate control.
The use of AMPK ␣1 or ␣2 siRNAs specifically reduces
AMPK ␣1 or ␣2 message expression, respectively (Fig. 4). We
therefore asked which AMPK isoform is required for stressinduced sugar transport stimulation in bEnd.3 cells. Transport
stimulation by glucose deprivation or by KCN treatment is
blocked by AMPK ␣1 knockdown, by AMPK ␣2 knockdown,
and by knockdown of AMPK ␣1 plus ␣2 (Fig. 6C). We
therefore conclude that both AMPK ␣1 and ␣2 are required for
sugar transport stimulation during metabolic stress.
Compound C and AMPK knockdown inhibit GLUT1 recruitment during acute metabolic stress. bEnd.3 sugar transport
stimulation during acute metabolic stress is mediated by recruitment of intracellular GLUT1 to the plasma membrane
(14). The role of AMPK in this process was evaluated by
examining the effects of Compound C on GLUT1 recruitment.
Cells were treated with 2 mM AICAR, glucose starved, or
KCN treated as described above in the absence or presence of
10 ␮M Compound C. Cells were then washed, cooled to 4°C,
and treated with the membrane-impermeant Sulfo-NHS-SSBiotin to label exposed primary amines (lysine side-chains) as
described previously (14). The reaction was quenched, the cells
were lysed in detergent-containing lysis buffer, and biotinylated proteins were precipitated with streptavidin beads. Precipitated proteins were analyzed by Western blotting using anti-
AMPK REGULATION OF SUGAR TRANSPORT IN ENDOTHELIAL CELLS
GLUT1 COOH-terminal IgGs (Fig. 7, A, C, and E) and band
densities were quantitated (Fig. 7, B, D, and F). As recently
reported (14), biotinylated, COOH-terminal-reactive GLUT1
is revealed as a 48-kDa species plus a broadly mobile 55-kDa
species. Occasionally, a 42-kDa species is also observed (Fig.
7, C and E). Treatment of membranes with peptide:N-glycosidase F causes all species to collapse to a 40- to 42-kDa GLUT1
species (14). Cell surface expression of the 48-kDa GLUT1
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DISCUSSION
This study examines the hypothesis that AMPK modulates
GLUT1-mediated sugar uptake in brain microvascular endothelial cells by regulating plasma membrane GLUT1 levels
Fig. 6. A: Compound C inhibition of stimulated 3-OMG uptake. Before cooling
to 4°C, glucose depletion, and transport initiation, cells were treated for 15 min
at 37°C with 5 mM glucose (control), 0 glucose, 5 mM KCN, 8 ␮g/ml FCCP,
or for 2 h with 2 mM AICAR in the absence or presence of 10 ␮M Compound
C. Uptake of 20 mM 3-OMG was then measured for 30 s in each condition.
Ordinate, 3-OMG uptake (dpm per ␮g total cell protein). Abscissa, experimental condition. Data represent means ⫾ SE of three separate experiments. A
t-test analysis indicates as follows: *P ⬍ 0.005, all test conditions (minus
Compound C) show significantly greater uptake than control; §P ⬍ 0.005,
Compound C reduces sugar uptake in all instances except in control cells,
where uptake is significantly increased. B: AMPK siRNA inhibition of modulated 3-OMG uptake. Cells were treated with or without AMPK siRNAs for
48 h. Before cooling to 4°C, glucose depletion, and transport initiation, cells
were treated for 15 min at 37°C with 5 mM glucose (control), 0 glucose, 5 mM
KCN, 8 ␮g/ml FCCP, 10 ␮M compound C, or for 2 h with 2 mM AICAR.
Uptake of 20 mM 3-OMG was then measured for 30 s in each condition.
Ordinate, 3-OMG uptake (dpm per ␮g total cell protein). Abscissa, experimental condition. Data represent means ⫾ SE of three separate experiments. A
t-test analysis indicates as follows: *P ⬍ 0.005, all test conditions (minus
AMPK siRNA) show significantly greater uptake than control; sugar uptake by
mock-transfected control cells, by control cells transfected with nonsilencing
siRNAs and by compound C (CC)-treated cells ⫾ AMPK siRNA is not
significantly different from uptake by control cells (P ⬎ 0.19); §P ⬍ 0.005,
AMPK siRNA transfection reduces sugar uptake in all test conditions. C: effects of AMPK ␣1- and ␣2-specific siRNAs on sugar transport stimulation by
glucose deprivation or KCN treatment. Cells were treated with or without
AMPK ␣1, AMPK ␣2, or AMPK ␣1 ⫹ ␣2 siRNAs for 48 h. Before cooling
to 4°C, glucose depletion, and transport initiation, cells were treated for 15 min
at 37°C with 5 mM glucose (control), 0 glucose, or 5 mM KCN. Uptake of 20
mM 3-OMG was then measured for 30 s in each condition. Ordinate, 3-OMG
uptake (dpm per ␮g total cell protein). Abscissa, experimental condition. Data
represent means ⫾ SE of three separate experiments. A t-test analysis indicates
as follows: *P ⬍ 0.005, all test conditions lacking siRNAs show significantly
greater uptake than control; all conditions with siRNAs are not significantly
different from control.
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COOH-terminal antibody reactive species is unchanged by
AICAR, whereas expression of the 55-kDa species is increased
by 2- to 3-fold (Fig. 7, B and F). Glucose depletion and KCN
appear to enhance cell surface expression of the 48- and
55-kDa species by 2- to 4-fold. AICAR treatment increases
total plasma membrane GLUT1 by approximately 2.1-fold,
glucose depletion by approximately 3.0-fold, and KCN treatment by approximately 1.8- to 2.4-fold over controls. These
results mirror the increases in 3-OMG transport capacity produced by metabolic poisons. In contrast, Compound C-treated
cells show no significant change in plasma membrane GLUT1
(Fig. 7, B, D, and F).
AMPK knockdown before KCN treatment also prevents
GLUT1 recruitment to the plasma membrane. KCN treatment
increases GLUT1 at the plasma membrane 1.8- to 2.4-fold over
mock transfected cells, but this recruitment is blocked in
AMPK knockdown cells treated with KCN. Basal plasma
membrane [GLUT1] is unaffected by AMPK knockdown (Fig.
7, G and H). These data are consistent with the results obtained
by Compound C inhibition of AMPK in Fig. 7, E and F.
Biotinylation of cell surface GLUT1 is increased 2.2-fold
following glucose deprivation of mock-transfected cells but is
unchanged following glucose deprivation of cells previously
transfected with AMPK siRNA (data not shown).
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AMPK REGULATION OF SUGAR TRANSPORT IN ENDOTHELIAL CELLS
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Fig. 7. Inhibition of plasma membrane GLUT1
recruitment by Compound C and by AMPK
siRNA. A, C, E, and G: representative Western
blots of cell surface biotinylated bEnd.3 cell
proteins obtained in the absence and presence
of either 2 mM AICAR and 10 ␮M Compound
C (A), 5 mM glucose and 10 ␮M Compound C
(C), 5 mM KCN and 10 ␮M Compound C (E),
or 48 h treatment with AMPK siRNAs (G).
Three COOH-terminal antibody (C-Ab)-reactive species are observed with Mr(app) 42 kDa,
48 kDa, and 55 kDa. Cells were treated for 2 h
with AICAR, were glucose depleted for 15
min, or were poisoned for 15 min at 37°C
before cooling to 4°C and biotinylation. Total
streptavidin-pull down protein (80 ␮g) was
loaded into each lane and blotted with GLUT1
C-Ab. Band densities were quantitated as total,
48 kDa, and 55 kDa GLUT1 species and are
shown in B (AICAR-treated cells), D (glucosedepleted cells), F (KCN-treated cells), and H
(AMPK siRNA-treated cells ⫾ KCN). B, D, F,
and H: ordinate, relative band volume (%).
Abscissa, experimental condition. Each experiment was repeated in triplicate and the results
of quantitations (B, D, F, and H) are shown as
means ⫾ SE. *P ⬍ 0.01, §P ⬍ 0.05, significantly different from control values.
during acute metabolic stress. We show that endothelial cell
AMPK is phosphorylated during metabolic stress and that this
is inhibited in a dose-dependent manner by the AMPK antagonist, Compound C. AMPK activation by the AMPK agonist
AICAR or by metabolic stress is associated with stimulation of
GLUT1-mediated sugar uptake; but transport stimulation is
inhibited by AMPK knockdown and in a dose-dependent
manner by Compound C. Transport stimulation appears to
result from recruitment of intracellular GLUT1 to the cell
surface because Compound C and AMPK knockdown block
AICAR- and metabolic stress-induced GLUT1 recruitment.
Compound C is a high-affinity ligand that competes with
AMP and ATP for binding to AMPK (47). ATP- and Compound C-liganded AMPK is catalytically inactive, but AMPbinding promotes AMPK phosphorylation, resulting in activation (22, 23). ZMP, an AICAR metabolite, also binds at the
AMP-binding site to activate the kinase (22). Compound C and
ZMP binding are thus mutually exclusive, thereby explaining
AJP-Cell Physiol • doi:10.1152/ajpcell.00437.2011 • www.ajpcell.org
AMPK REGULATION OF SUGAR TRANSPORT IN ENDOTHELIAL CELLS
mediates bEnd.3 cell sugar transport stimulation during metabolic stress.
Chronic metabolic stress causes increased GLUT1 expression and increased sugar transport in brain microvascular
endothelial cells (4, 5, 24, 26, 33, 41). In contrast, acute
metabolic stress is without effect on GLUT1 expression but
increases cellular sugar transport capacity (9, 10, 14). The
present work supports the hypothesis that activation of AMPK
[the primary sensor in cellular energy homeostasis (22, 23)]
stimulates sugar transport by rapidly enhancing GLUT1 trafficking to the plasma membrane. AMPK also regulates GLUT4mediated sugar transport in muscle and adipose by chronic control
of gene expression and by acute regulation of protein trafficking to
the plasma membrane (1, 18, 25, 46). AMPK therefore plays a
dual role in each tissue by initiating short-term responses to acute
changes in metabolic state and long-term responses to chronic
alterations in cellular metabolism.
Glucose is a primary energy source for the brain, and its
transport across the blood-brain barrier is rate-limiting for
cerebral glucose utilization (31, 42). When a region of the brain
becomes acutely activated or when blood glucose levels rapidly fall, local blood flow and glucose uptake are activated to
maintain brain glucose availability (4, 5, 26, 33, 39, 41). The
present study supports the hypothesis that elevated intracellular
AMP promotes endothelial cell AMPK phosphorylation, which
leads to intracellular GLUT1 translocation to luminal and
abluminal membranes. The net effect is increased glucose
transport across the blood-brain barrier and enhanced glucose
delivery to astrocytes and neurons.
GRANTS
This grant was supported by National Institutes of Health Grants DK-36081
and DK-44888.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
A.J.C. and A.C. conception and design of the research; A.J.C. performed
the experiments; A.J.C. analyzed the data; A.J.C. and A.C. interpreted the
results of the experiments; A.J.C. and A.C. prepared the figures; A.J.C. and
A.C. drafted the manuscript; A.J.C. and A.C. edited and revised the manuscript; A.J.C. and A.C. approved the final version of the manuscript.
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