Insulin Stimulates Glucose Transport Via Nitric Oxide/Cyclic GMP Pathway in Human Vascular Smooth Muscle Cells
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Insulin Stimulates Glucose Transport Via Nitric
Oxide/Cyclic GMP Pathway in Human Vascular Smooth
Muscle Cells
L. Bergandi, F. Silvagno, I. Russo, C. Riganti, G. Anfossi, E. Aldieri, D. Ghigo, M. Trovati, A. Bosia
Objective—In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric
oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated
NO/cyclic GMP pathway plays a role in the regulation of glucose uptake.
Methods and Results—Glucose transport in human vascular smooth muscle cells was measured as uptake of
2-deoxy-D-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the
plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruit-
ment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited
glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP– dependent protein kinase;
furthermore, glucose transport was stimulated by an analog of cyclic GMP.
Conclusions—Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into
the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which
stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP– dependent protein kinase.
(Arterioscler Thromb Vasc Biol. 2003;23:2215-2221.)
Key Words: insulin signaling 䡲 muscle, smooth, vascular 䡲 glucose transport 䡲 nitric oxide 䡲 cyclic GMP
I nsulin-stimulated glucose uptake is primarily mediated by
the facilitative transporter GLUT4, which after hormonal
stimulation is recruited from intracellular membranes to the
muscle.9 –11 Recent data suggest that NO elicits glucose
uptake in skeletal muscle through the activation of an
AMP-activated protein kinase (AMPK), a mechanism that is
cell surface, resulting in an enhanced glucose uptake (for distinct from both insulin and contraction signaling
review see Foster and Klip1). The precise mechanism by pathways.12,13
which insulin directs exocytosis of GLUT4-containing vesi- It is known that insulin has vasodilator actions in vivo that
cles in different cell types has not been completely clarified. depend on endothelium-derived NO.14,15 Indeed, insulin stim-
Multiple studies have suggested a necessary role of the ulates production of NO in human endothelial cells in vitro,16
insulin receptor– catalyzed tyrosine phosphorylation of insu- and the insulin receptor tyrosine kinase, PI-3K, and Akt all
lin receptor substrate proteins and the subsequent activation play significant roles in insulin-dependent production of NO
of the p85/p110-type phosphatidylinositol 3-kinase (PI-3K).2 in endothelium.17
However, several lines of evidence suggest that insulin must It has been proposed that insulin-induced vasodilation is
generate additional and possibly separate signals to increase mediated, at least in part, by the stimulation of endothelial
glucose transport, such as the activation of the small GTPase NO production, causing inhibition of contraction of the
TC10,3 and the rearrangement of the microtubule- and actin- underlying vascular smooth muscle cells (VSMCs) via acti-
based cytoskeleton.4,5 vation of the VSMC guanylate cyclase (GC). The relaxant
Another cell signaling pathway that appears to markedly effect exerted by insulin in VSMCs is not necessarily medi-
stimulate glucose uptake in skeletal muscle involves the nitric ated by endothelial cells only. Actually, a direct effect of
oxide (NO)/cyclic GMP (cGMP) pathway. NO stimulates insulin on cultured VSMCs has been observed in vitro,
glucose transport in isolated skeletal muscles and mediates in showing that these cells are target of insulin action (for
vivo exercise-induced glucose transport:6 – 8 part of the in vivo review, see Trovati and Anfossi18). Indeed, insulin elicits
mechanism involves the release of NO from the endothelium, antiapoptotic signaling in VSMCs by inducing rapid tyrosine
causing enhanced blood flow and glucose delivery to the phosphorylation of the insulin receptor and IRS-1, followed
Received June 7, 2003; revision accepted July 1, 2003.
From the Department of Genetics, Biology and Biochemistry, University of Turin (L.B., F.S., C.R., E.A., D.G., A.B.), Turin, Italy; and Metabolic
Disease and Diabetes Unit, Department of Clinical and Biological Sciences, University of Turin, San Luigi Gonzaga Hospital (I.R., G.A., M.T.),
Orbassano-Torino, Italy.
Correspondence to Amalia Bosia, Dipartimento di Genetica, Biologia e Biochimica - Sezione di Biochimica, Via Santena, 5/bis - 10126 Torino - Italy.
E-mail amalia.bosia@unito.it
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000107028.20478.8e
2215
Downloaded from http://atvb.ahajournals.org/ by guest on February 4, 20152216 Arterioscler Thromb Vasc Biol. December 2003
by a 3-fold increase of IRS-1– bound PI-3K and a robust Glucose Transport Assay
enhancement of Akt-3 phosphorylation and activity down- At the end of the different incubation periods in the presence of
stream of PI-3K.19,20 In addition, it has been reported that in agonists and/or inhibitors, cells were rinsed once with glucose-free
HEPES-buffered saline solution (mmol/L: 140 NaCl, 20 HEPES/Na,
cultured VSMCs, insulin increases (1) cGMP production via 5 KCl, 2.5 MgSO4, and 1 CaCl2 at pH 7.4) and were subsequently
a NO-dependent mechanism21 and (2) inducible NOS expres- incubated in the same buffer for 8 minutes with 10 mol/L
sion and cGMP generation via the PI-3K pathway.22 2-deoxy-D-glucose containing 0.3 Ci/mL 2-deoxy-D-[3H]glucose.
We recently demonstrated that human VSMCs (hVSMCs) Under these conditions the glucose uptake was linear for at least 20
express an endothelial-type, calcium/calmodulin-dependent minutes (data not shown). Uptake was stopped by rapid removal of
the buffer, followed by 3 washes in ice-cold PBS. Cells were
NO synthase (NOS) and exhibit an increased production of disrupted by adding 50 mmol/L NaOH, and the radioactivity asso-
both NO and cGMP in response to the calcium ionophore ciated with the cells was determined by scintillation counting.
ionomycin.23 Insulin stimulates hVSMC calcium-dependent Protein concentrations were determined by the BCA kit from Pierce,
NOS activity in a few minutes, and insulin-induced NO and and the uptake was expressed in pmol 2-deoxy- D -
glucose 䡠 min⫺1 䡠 mg⫺1 cell protein. Uptake values were corrected for
cGMP production is completely blunted by the NOS inhibitor the noncarrier-mediated transport by measuring hexose uptake in the
NG-nitro-L-arginine methyl ester (L-NAME).23 presence of 10 mol/L cytochalasin B (a potent inhibitor of
The aim of the present study was to determine whether in facilitated glucose transport). The nonspecific association of deoxy-
hVSMC insulin-derived NO plays any role in the regulation glucose with the cells was typically ⬍10% of the total uptake.
of one of the most important effects of insulin, ie, its ability
to increase the rate of cellular glucose transport. We addi-
cGMP Determination
At the end of a 45-minute incubation in 35-mm Petri dishes in the
tionally investigated which signaling events are required for presence of agonists and/or inhibitors, the medium was removed, and
NO to act as a regulator of insulin-mediated glucose uptake. 300 L of absolute ethanol was added to make the cells permeable.
After ethanol was completely evaporated, 300 L of Tris-EDTA
Methods buffer (50 mmol/L Tris-HCl, 4 mmol/L EDTA at pH 7.5) was added
to each dish. After a 10-minute equilibration period, 100 L of
Materials supernatant was tested for the cGMP level with a [3H]cGMP assay
Dulbecco’s modified Eagle’s medium (DMEM), FCS, and other kit provided by Immuno Biological Laboratories, and the results
tissue culture products were obtained from GIBCO BRL; plastic for were expressed as picomoles cGMP per milligram cell proteins. For
cell culture was from Falcon (Becton Dickinson). 2-deoxy-D- the cGMP assay, the discrimination was 0.05 pmol/mL, and the
glucose, cytochalasin B, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1- cross-reactivity with cAMP was ⬍0.001%.
one (ODQ), 8-bromo guanosine-3⬘:5⬘-cyclic 1 monophosphate (8-
Br-cGMP), sodium nitroprusside (SNP), S-nitrosoglutathione (GS- Western Blot Analysis
NO) were obtained from Sigma Chemical. Human recombinant Cells were directly solubilized in boiling Laemmli buffer containing
insulin and 8-bromo guanosine-3⬘:5⬘-cyclic monophosphorothioate, the protease inhibitor cocktail set III (mmol/L: 100 AEBSF, 0.08
Rp-isomer (Rp-8-Br-cGMPS) were from Calbiochem-Novabiochem aprotinin, 5 bestatin, 1.5 E-64, 2 leupeptin, and 1 pepstatin;
Corporation. 2-Deoxy-D-[3H]glucose (6.1 Ci/mmol) was from NEN Calbiochem-Novabiochem Corporation), and proteins, separated by
Dupont. Electrophoresis reagents were obtained from SDS-PAGE (12%), were transferred to polyvinylidene difluoride
Bio-Rad Laboratories. filter membrane (Immobilon P, Millipore) and probed with rabbit
polyclonal antibodies (diluted 1:500 in PBS-BSA 1%) anti-human
Cell Cultures GLUT4 (Santa Cruz Biotechnology; catalog no. sc-7938) and anti-
human cGMP-dependent protein kinase (PKG) I␣ (Calbiochem,
Experiments were done with hVSMCs derived from microarterioles
VWR International; catalog no. 370652), or with the mouse mono-
obtained from male patients, 45 to 55 years old, undergoing
clonal antibody 16C2 (1:2000), specific for vasodilator-stimulated
abdominal surgery because they were affected by gallbladder stones
phosphoprotein (VASP) phosphorylated at serine 239 (Upstate,
without inflammatory processes. Isolation and characterization were
D.B.A.; catalog no. 05-611).26 After an overnight incubation, the
made according to classical procedures24; ie, cells were characterized membrane was washed with PBS-Tween 0.1% and subjected to a
by phenotype and checked for the presence of the smooth muscle peroxidase-conjugated antibody (diluted 1:1000 in PBS-Tween with
isoform of ␣-actin and the absence of factor VIII. Cells retained Blocker nonfat dry milk 5%, Bio-Rad) anti-rabbit or anti-mouse IgG
differentiated properties even after multiple passages. Cells were (donkey; Amersham International; catalog no. NA934V and
cultured in a 5% CO2 atmosphere at 37°C by using DMEM NA931V). The polyvinylidene diflouride membrane was washed
supplemented with 10% FCS, and were buffered with 10 mmol/L again with PBS-Tween, and proteins were detected by enhanced
N-Tris (hydroxymethyl) methyl-2-aminoethane-sulfonic acid and chemiluminescence (Amersham International).
10 mmol/L HEPES. For the experiments, cells were used at the
fourth to sixth passage and cultured in 35- or 100-mm-diameter Petri Immunofluorescence Studies
dishes until 75% confluence was achieved; then the medium with
Cells were grown on polylysine-coated glass coverslips. At the end
serum was removed, and cells were made quiescent by serum
of the different incubation periods in the presence of agonists and/or
starvation and culture in DMEM containing 0.1% (w/v) bovine
inhibitors, cells were fixed in paraformaldehyde/sucrose fixing
serum albumin (DMEM/BSA). After a 24-hour quiescence, the
solution (3% paraformaldehyde, 4% sucrose in PBS at pH 7.4, for 20
medium was removed, and fresh DMEM/BSA was added to the cell
minutes at room temperature), blocked in 1.5% normal goat serum,
layer. Measurements were performed after incubation of hVSMCs in and incubated overnight at 4°C with anti-GLUT4 polyclonal anti-
the presence of different agonists (including the NO donors), with bodies (1:250 dilution) without a permeabilization step, in order to
and without coincubation with different inhibitors. analyze the fluorescence associated with the cell surface only.
Afterward, cells were treated with biotinylated anti-mouse IgG2a
Aerobic Consumption of Glucose (Vector; catalog no. BA2001) followed by fluorescein-avidin D, and
After a 45-minute incubation in the absence or presence of NO mounted. Staining was observed with an Olympus IX-70 inverted
donors, the cells were washed, detached, and checked for the rate of confocal laser scanning microscope equipped with a Crypton-Argon
mitochondrial respiration by measuring 14CO2 evolution from ion laser (488/568 nm). All images were taken with the same
[6-14C]glucose, as previously described.25 scanning setting, and fluorescence intensity average of each image
Downloaded from http://atvb.ahajournals.org/ by guest on February 4, 2015Bergandi et al Glucose Transport in Smooth Muscle 2217
Figure 1. Left, hVSMCs were incubated with insulin at the con-
centrations shown for 45 minutes, and 2-deoxy-D-glucose trans-
port was assayed as described in the Methods section. Right,
hVSMCs were stimulated with 100 nmol/L insulin for various
periods of time before assay of 2-deoxy-D-glucose transport.
Data are presented as mean⫾SEM (n⫽8). *P⬍0.05 vs basal
value (0); **P⬍0.005 vs basal value (0). Figure 2. Role of endogenous and exogenous NO in 2-deoxy-D-
glucose uptake in hVSMC cells. Cells were incubated for 45
minutes in the absence (ctrl) or presence of 100 nmol/L insulin
was quantified by Scion Image Software (Scion Corporation) in 20 (ins), 1 mmol/L L-NAME (L-name), 100 nmol/L insulin⫹1 mmol/L
randomly chosen cellular fields. Fluorescence intensity was ex- L-NAME (ins/L-name), 25 mol/L GS-NO (gs-no), and
pressed as arbitrary units and plotted as a graph. 25 mol/L SNP (snp), and 2-deoxy-D-glucose transport was
assayed as described in the Methods section. Data are present-
ed as mean⫾SEM (n⫽8). *P⬍0.01, **P⬍0.005, and ***P⬍0.001
Statistical Analysis vs control (ctrl). EP⬍0.001 vs insulin (ins).
All data in text and figures are provided as mean⫾SEM. Statistical
analysis has been carried out by means of Student’s t test.
donors (SNP: 5.83⫾1.15 pmol CO2/h/mg cell proteins, n⫽3;
Results GS-NO: 4.93⫾1.05 pmol CO2/h/mg cell proteins, n⫽3).
The immunoblotting with a specific anti-GLUT4 antibody
Insulin Stimulates Glucose Transport and a Rapid demonstrated that the hVSMCs used in the present study
Increase of GLUT4 at the Cell Surface in expressed this glucose transporter isoform (data not shown).
hVSMCs: Both Are Mediated by NO To check whether insulin and NO increase the GLUT4
hVSMCs were incubated with different concentrations of translocation at the plasma membrane, we performed confo-
insulin for 45 minutes before the assay of 2-deoxy-D-glucose cal microscopy analysis of the transporter localization with-
transport (Figure 1, left), and with 100 nmol/L insulin for out permeabilization in order to stain the cell surface only. In
different time periods up to 1 hour (Figure 1, right). Maximal the absence of insulin, few transporter molecules were de-
stimulation of 2-deoxy-D-glucose uptake, corresponding to an tected on the cell surface; after brief exposure to insulin, the
⬇1.7- to 1.8-fold increase over basal, was achieved after a GLUT4 content of the plasma membrane increased (Figure 3)
45-minute exposure to 100 nmol/L insulin: this experimental 3.3-fold. The incubation with insulin in the presence of the
condition was then used in the subsequent experiments. NOS inhibitor L-NAME reverted the insulin effect (Figure 3).
Cytochalasin B decreased basal glucose uptake by ⬎90% The NO donor GS-NO exerted a remarkable insulin-like
(data not shown), which confirmed that glucose uptake effect on GLUT4 subcellular distribution (3.9-fold increase in
resulted from a specific carrier-mediated transport. the plasma membrane), which was not influenced by
To investigate the role of NO in insulin-stimulated glucose L-NAME coincubation (Figure 3).
transport, hVSMCs were incubated with insulin in the pres-
ence of the NOS inhibitor L-NAME, which completely GC and PKG Are Involved in the
abolished the insulin-mediated 2-deoxy-D-glucose uptake Insulin-Stimulated Glucose Transport of hVSMCs
(Figure 2). Moreover, the NO donor GS-NO elicited a 2-fold When the cells were incubated with insulin in the presence of
increase in 2-deoxy-D-glucose uptake (Figure 2). A similar ODQ, a selective inhibitor of soluble GC (sGC), the insulin-
effect was evoked by SNP (Figure 2). As the mitochondrion stimulated 2-deoxy-D-glucose uptake was abolished (Figure
has been proposed to be one of the primary cellular targets of 4). The inhibitory effect of ODQ was reversed by the
the cytotoxic action of NO (for review see Wink and coincubation with the membrane permeable cGMP analog
Mitchell27), after exposure to GS-NO and SNP, the cells were 8-Br-cGMP (Figure 4), which, per se, was a potent inducer of
checked for the rate of mitochondrial respiration by measur- 2-deoxy-D-glucose uptake (Figure 4). Also the GS-NO-
ing 14CO2 evolution from [6-14C]glucose.25 The amount of elicited 2-deoxy-D-glucose uptake was inhibited by ODQ
14
CO2 produced, used as an index of tricarboxylic acid cycle (Figure 4). We had already shown23 that in hVSMCs, insulin
operation under aerobic conditions, did not significantly induces a NO-dependent cGMP increase. The stimulating
change when cells were incubated in the absence (5.04⫾0.92 effect of insulin on the cGMP intracellular concentration
pmol CO2/h/mg cell proteins, n⫽3) or presence of the NO (Figure 5) was mimicked by SNP and GS-NO (Figure 5). In
Downloaded from http://atvb.ahajournals.org/ by guest on February 4, 20152218 Arterioscler Thromb Vasc Biol. December 2003
Figure 4. Role of GC in the insulin- and GS-NO–stimulated
2-deoxy-D-glucose uptake of hVSMCs. Cells were incubated for
45 minutes in the absence (ctrl) or presence of 100 nmol/L insu-
lin (ins), 100 nmol/L insulin⫹50 mol/L ODQ (ins/odq), 100
nmol/L insulin⫹50 mol/L ODQ followed by a further 45 min-
utes incubation with 1 mmol/L 8-Br-cGMP (ins/odq ⬎ br),
1 mmol/L 8-Br-cGMP (br), 25 mol/L GS-NO (gs-no), and
25 mol/L GS-NO⫹50 mol/L ODQ (gs-no/odq), and 2-deoxy-
D-glucose transport was assayed as described in the Methods
section. Data are presented as mean⫾SEM (n⫽8). *P⬍0.002,
**P⬍0.001, and ***P⬍0.0001 vs control (ctrl); EP⬍0.001 versus
insulin (ins); 〫P⬍0.001 vs insulin⫹ODQ (ins/odq); ⽧ P⬍0.001
vs GS-NO (gs/no).
owing to the phosphorylation of specific target proteins,
including VASP, the 46/50-kDa vasodilator-stimulated-
phosphoprotein, which is expressed in VSMCs and phosphor-
ylated at serine 239 by PKG.26,28 VASP phosphorylation at
Figure 3. Confocal microscopy analysis of the GLUT4 trans-
porter concentration at the hVSMC plasma membrane. Cells
serine 239, measured with the monoclonal antibody 16C2,
were incubated for 45 minutes in the absence (ctrl) or presence
of 1 mmol/L L-NAME (L-name), 100 nmol/L insulin (ins), 100
nmol/L insulin⫹1 mmol/L L-NAME (ins/L-name), 25 mol/L
GS-NO (gs-no), and 25 mol/L GS-NO⫹1 mmol/L L-NAME (gs-
no/L-name): GLUT4 was detected as indicated in the Methods
section. The figures in the upper panel are representative of 3
similar experiments, giving superimposable results. In the graph
the fluorescence intensities, expressed as arbitrary units, are
presented as mean⫾SEM (n⫽3). *P⬍0.0001 vs control (ctrl);
EP⬍0.0001 vs insulin (ins).
keeping with the observations on glucose transport (Figure 4),
the insulin-, SNP- and GS-NO–induced cGMP increase was
completely inhibited by ODQ (Figure 5).
When hVSMCs were incubated with insulin in the pres-
ence of Rp-8-Br-cGMPS, a selective inhibitor of PKG, the
insulin-stimulated 2-deoxy-D-glucose uptake was abolished
(Figure 6, top). The inhibitor blocked also the activating
effect of 8-Br-cGMP on 2-deoxy-D-glucose uptake (Figure 6, Figure 5. cGMP synthesis in hVSMCs after addition of insulin
top). The incubation with the PKG inhibitor also resulted in a and NO donors. Cells were incubated for 45 minutes in the
significant reduction of basal 2-deoxy-D-glucose transport absence (ctrl) or presence of 100 nmol/L insulin (ins), 100
nmol/L insulin⫹50 mol/L ODQ (ins/odq), 25 mol/L SNP (snp),
(Figure 6, top). hVSMCs expressed predominantly PKG I␣, 25 mol/L SNP⫹50 mol/L ODQ (snp/odq), 25 mol/L GS-NO
as checked by Western blotting experiments (data not (gs-no), and 25 mol/L GS-NO⫹50 mol/L ODQ (gs-no/odq),
shown). So far, we looked for a direct measurement of PKG and cGMP concentration was determined as described in Meth-
ods. Data are presented as mean⫾SEM (n⫽6). *P⬍0.002 vs
activation elicited by insulin, NO donor, and cGMP analog in control (ctrl); EP⬍0.0001 vs insulin (ins), SNP (snp), and GS-NO
hVSMCs. The functional effects of PKG are thought to be (gs-no).
Downloaded from http://atvb.ahajournals.org/ by guest on February 4, 2015Bergandi et al Glucose Transport in Smooth Muscle 2219
hVSMCs owing to its ability to increase endogenous NO
production via activation of NOS, as the NOS inhibitor
L-NAME completely blocks both insulin-mediated effects. In
addition, different NO donors (GS-NO and SNP) behave as
insulin mimetics and effectively enhance both glucose trans-
port and GLUT4 translocation in hVSMCs, bypassing the
inhibitory effect of L-NAME. These results suggest that in
hVSMCs, NO plays an important role in both insulin-
dependent relaxation/vasodilation and glucose uptake.
VSMCs have metabolic properties that make them peculiarly
sensitive to glucose transporter expression and activity: glu-
cose transport is rate-limiting for glucose metabolism in
VSMCs,34 as GLUT4 is nearly or completely saturated at
most physiological concentrations of glucose.35
To understand further the mechanism whereby NO regu-
lates insulin-mediated glucose transport in hVSMCs, we
examined the contribution of GC and cGMP in the insulin
effect. NO is known to activate sGC, which is the predomi-
nant intracellular NO receptor in VSMCs (for review, see
Figure 6. Top, Effect of a selective inhibitor of PKG on 2-deoxy- Lincoln and Cornwell36), leading to increased levels of
D-glucose uptake in insulin-stimulated hVSMCs. Cells were cGMP. We have already shown23 that in hVSMCs, insulin
incubated for 45 minutes in the absence (ctrl) or presence of induces a significant enhancement of NO production and a
100 nmol/L insulin (ins), 100 nmol/L insulin ⫹50 mol/L Rp-8-
Br-cGMPS (ins/rp), and 100 nmol/L insulin⫹50 mol/L Rp-8-Br- NO-dependent cGMP increase. Our present results show that
cGMPS followed by a further 45-minute incubation with 8-Br- ODQ, which renders sGC insensitive to activation by NO,37
cGMP (ins/rp ⬎ br). Data are presented as mean⫾SEM (n⫽9). abolishes the insulin- and the GS-NO–stimulated glucose
*P⬍0.002 vs control (ctrl); EP⬍0.0001 vs insulin (ins). Bottom,
Enhancement of phosphoserine 239-VASP formation in the
uptake, and the insulin-, SNP- and GS-NO–induced cGMP
presence of insulin, GS-NO, and 8-Br-cGMP, as well as inhibi- increase. So far, both insulin and NO donors require NO-
tion of this phosphorylation by ODQ and Rp-8-Br-cGMPS. Rep- dependent cGMP production to stimulate glucose transport in
resentative original blot. Cells were incubated for 45 minutes in hVSMC. Indeed, the membrane permeable cGMP analog
the absence (ctrl) or presence of 100 nmol/L insulin (ins), 100
nmol/L insulin⫹50 mol/L ODQ (ins/odq), 100 nmol/L insu- 8-Br-cGMP reverses the ODQ inhibitory effect and is a
lin⫹50 mol/L Rp-8-Br-cGMPS (ins/rp), and 100 nmol/L insu- potent inducer of glucose uptake per se.
lin⫹50 mol/L Rp-8-Br-cGMPS followed by a further 45-minute Our results suggest that the role of NO/cGMP signaling in
incubation with 8-Br-cGMP (ins/rp ⬎ br), 25 mol/L GS-NO (gs-
no), or 1 mmol/L 8-Br-cGMP (br). ODQ and Rp-8-Br-cGMPS
glucose transport is different between smooth muscle and
alone gave results superimposable to ctrl (data not shown). skeletal muscle. Indeed, in rat skeletal muscle and mouse
Western blot analysis was performed as described in the Meth- H-2K b muscle cells,12 the increase in glucose transport is
ods section. completely blocked by inhibition of NOS only under those
conditions that lead to increased AMPK activity, and inhib-
was increased in hVSMCs by insulin, GS-NO, and 8-Br- itors of NOS have no effect on the increase in glucose
cGMP (Figure 6, bottom). Inhibition of sGC (by ODQ) and of transport caused by stimuli, such as insulin, that do not
PKG (by Rp-8-Br-cGMPS) almost completely abolished the activate AMPK. Our data show that in human vascular
phosphorylation of VASP at serine 239 in response to insulin. smooth muscle, the increase in glucose transport is promoted
by insulin via a NOS- and GC-dependent mechanism.
Discussion We next examined the contribution of PKG in the insulin-
VSMCs have been shown to express the insulin-responsive and cGMP-stimulated glucose transport of hVSMCs. PKG
glucose transporter GLUT429 –31 and exhibit a significant regulates smooth muscle relaxation, platelet aggregation, cell
insulin-responsive glucose uptake,32,33 similar to that of growth, and differentiation (for review, see Pfeifer et al38).
skeletal muscle and adipose tissue. In most GLUT4- The expression of the type I PKG, and particularly the I␣
expressing tissues (eg, heart muscle, skeletal muscle, fat), the isoform, predominates in VSMCs39 and decreases as VSMCs
great majority of GLUT4 molecules reside within an intra- are passaged in vitro.40 Our results show that in cultured
cellular vesicular compartment until the cells are stimulated hVSMCs, which express predominantly PKG I␣ (data not
by insulin, at which time they are translocated and inserted shown), the stimulating effects of insulin and cGMP on
into the plasma membrane, where they mediate glucose glucose transport require the cGMP-mediated PKG activa-
uptake.1 Because insulin is known to act as a potent vasodi- tion, as the pretreatment of hVSMCs with a selective inhibitor
lator14 –17 and to increase NO production in VSMCs,21,23 we of PKG completely blocks the glucose uptake elicited by both
examined the contribution of the NO signaling pathway in insulin and 8-Br-cGMP. VASP, a protein expressed in vas-
insulin-mediated glucose transport and GLUT4 translocation cular smooth muscle, is specifically phosphorylated at serine
in hVSMCs. 239 by PKG.28 Insulin elicits the NO/cGMP-dependent acti-
The results of the present study indicate that insulin vation of PKG, as VASP phosphorylation at serine 239 is
stimulates glucose transport and GLUT4 translocation in significantly increased by insulin, and this effect is almost
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This work was supported by grants from the University of Torino
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Downloaded from http://atvb.ahajournals.org/ by guest on February 4, 2015Insulin Stimulates Glucose Transport Via Nitric Oxide/Cyclic GMP Pathway in Human
Vascular Smooth Muscle Cells
L. Bergandi, F. Silvagno, I. Russo, C. Riganti, G. Anfossi, E. Aldieri, D. Ghigo, M. Trovati and
A. Bosia
Arterioscler Thromb Vasc Biol. 2003;23:2215-2221; originally published online November 13,
2003;
doi: 10.1161/01.ATV.0000107028.20478.8e
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