Serotonin (5-Hydroxytryptamine), a Novel Regulator of Glucose
Transport in Rat Skeletal Muscle*
Eric
Hajduch,
Franck
Rencurel,
Anudharan
Balendran
,
Ian H.
Batty§,
C. Peter
Downes§, and
Harinder S.
Hundal¶
From the Departments of Anatomy and Physiology and
§ Biochemistry and the
Medical Research
Council Protein Phosphorylation Unit, The University of
Dundee, Dundee DD1 4HN, Scotland
 |
ABSTRACT |
In this study we show that serotonin
(5-hydroxytryptamine (5-HT)) causes a rapid stimulation in glucose
uptake by ~50% in both L6 myotubes and isolated rat skeletal muscle.
This activation is mediated via the 5-HT2A receptor,
which is expressed in L6, rat, and human skeletal muscle. In L6 cells,
expression of the 5-HT2A receptor is developmentally
regulated based on the finding that receptor abundance increases by
over 3-fold during differentiation from myoblasts to myotubes.
Stimulation of the 5-HT2A receptor using methylserotonin
(m-HT), a selective 5-HT2A agonist, increased muscle
glucose uptake in a manner similar to that seen in response to 5-HT.
The agonist-mediated stimulation in glucose uptake was attributable to
an increase in the plasma membrane content of GLUT1, GLUT3, and GLUT4.
The stimulatory effects of 5-HT and m-HT were suppressed in the
presence of submicromolar concentrations of ketanserin (a selective
5-HT2A antagonist) providing further evidence that the
increase in glucose uptake was specifically mediated via the
5-HT2A receptor. Treatment of L6 cells with insulin resulted in tyrosine phosphorylation of IRS1, increased cellular production of phosphatidylinositol 3,4,5-phosphate and a 41-fold activation in protein kinase B (PKB/Akt) activity. In contrast, m-HT
did not modulate IRS1, phosphoinositide 3-kinase, or PKB activity. The
present results indicate that rat and human skeletal muscle both
express the 5-HT2A receptor and that 5-HT and specific 5-HT2A agonists can rapidly stimulate glucose uptake in
skeletal muscle by a mechanism which does not depend upon components
that participate in the insulin signaling pathway.
 |
INTRODUCTION |
Serotonin, also known as 5-hydroxytryptamine
(5-HT),1 is a
neurotransmitter that has been implicated in the regulation of diverse physiological processes, including cellular growth and differentiation (1), neuronal development (2), and regulation of blood glucose concentration (3, 4). This functional diversity stems from the ability
of the neurotransmitter to interact with multiple 5-HT receptors
(currently classified as 5-HT1 through to 5-HT7 with further subtypes within each receptor class) that can trigger and
activate distinct intracellular signaling systems (5). For example,
with the exception of the 5-HT3 receptor which operates as
a ligand-gated ion channel, all known 5-HT receptors belong to the
superfamily of G-protein coupled receptors which can, depending on
receptor class and subtype, couple negatively or positively to adenylyl
cyclase (6), modulate ion channel activity (7), promote hydrolysis of
phosphatidylinositol bisphosphate through activation of phospholipase
C-
(8, 9), and stimulate the mitogen-activated protein kinase
pathway (10).
Of major interest to us, however, has been the observation that
administration of 5-HT, 5-HT precursors, or specific 5-HT receptor
agonists and antagonists can modulate circulating levels of blood
glucose in rodents. While some investigators have reported that 5-HT
promotes hyperglycemia by a mechanism that may involve increased renal
catecholamine release (11), there is a prevailing view that blood
glucose is lowered by 5-HT and that this response can be suppressed by
5-HT receptor antagonists (3, 12-14). The precise nature by which 5-HT
promotes hypoglycemia remains poorly understood, but it is unlikely to
be related to changes in plasma insulin, since circulating levels of
the hormone do not increase significantly following intraperitoneal
administration of tryptophan, a 5-HT precursor (3, 14). This finding is
consistent with the view that biogenic amines normally suppress, rather
than enhance, pancreatic insulin release (14-17). An alternative
possibility is that 5-HT may stimulate glucose transport by acting
directly upon tissues such as skeletal muscle which, by virtue of its
total body mass, could make a significant contribution toward a
reduction in circulating levels of blood glucose. This proposition is
based on recent work showing that rat fetal myoblasts express the
5-HT2A receptor (18) and that activation of this receptor
enhances the expression of genes associated with myogenic
differentiation and that of the fetal glucose transporter, GLUT3 (18).
Increased expression of muscle glucose transporters may play a role in
the hypoglycemic action of 5-HT, but changes in transporter expression are generally slow in onset (18), and previous work has shown that 5-HT
precursors (e.g. 5-hydoroxytryptophan) can reduce blood glucose in fed animals within 1 h of administration (3). In an
attempt to assess whether 5-HT can stimulate skeletal muscle glucose
transport, we have specifically addressed the following questions. (i)
Do L6 muscle cells and skeletal muscle from rats and humans express the
5-HT2A receptor? (ii) Do 5-HT and specific 5-HT2A receptor agonists acutely regulate skeletal muscle
glucose transport, and if so (iii) can the effects be explained on the basis of changes in the subcellular distribution of glucose
transporters? (iv) Does the response involve cellular components (such
as IRS1, phosphoinositide 3-kinase, and protein kinase B), which have
been implicated in the insulin-induced activation of glucose transport in skeletal muscle?
 |
EXPERIMENTAL PROCEDURES |
Materials--
L6 rat skeletal muscle cells were provided by Dr.
Amira Klip (Toronto). D-Glucose and all other reagent grade
chemicals for buffers were obtained from BDH (Poole, Dorset, UK).
Sterile trypsin solution, 2-deoxy-D-glucose (2DG),
cytochalasin B, and human insulin were obtained from Sigma (Poole,
Dorset, UK). 2-[1,2-3H]deoxy-D-glucose was
purchased from New England Nuclear (Dreiech, Germany).
Cell Culture and Incubations--
Monolayers of L6 muscle cells
were grown to the stage of myotubes as described previously (19) in
-minimum essential medium containing 2% fetal calf serum and 1%
antimicotic/antibiotic solution (final concentration 100 units/ml
penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin B) at
37 °C in an atmosphere of 5% CO2, 95% air. Muscle
cells were grown in six-well multidishes for transport measurements and
in 15-cm culture dishes for subcellular fractionation studies.
Rat and Human Skeletal Muscle Procurement--
Human soleus
muscle was obtained from patients at Dundee Royal Infirmary while
undergoing elective limb amputation surgery for peripheral vascular
complications. Upon surgical excision, ~5 g of soleus muscle was
rapidly frozen in liquid nitrogen and stored at
80 °C until
required for study. For isolation of crude rat muscle membranes we used
male Sprague-Dawley rats (200-250 g, Bantin & Kingman, Hull, UK) that
were killed by cervical dislocation. Hindlimb skeletal muscle was
excised and frozen in liquid nitrogen and stored at
80 °C until
required. Rat and human skeletal muscles were homogenized and subjected
to differential centrifugation for isolation of crude muscle membranes
as described previously (20, 21). For glucose uptake studies in
skeletal muscle, smaller rats were used as described below.
Glucose Transport in L6 Muscle Cells and Isolated Rat Soleus
Muscle--
L6 myotubes were exposed to insulin, 5-HT, or to a
specific 5-HT2A receptor agonist
(
-methylhydroxytryptamine (m-HT), Tocris, Bristol, UK), antagonist
(ketanserin tartarate, Tocris, Bristol, UK) or wortmannin at
concentrations and for periods indicated in the figure legends.
Following the appropriate treatments 2DG uptake was assayed as
described previously (22, 23). For glucose uptake in isolated rat
muscle, male Sprague-Dawley rats (50 g, Bantin & Kingman) were killed
by cervical dislocation and soleus muscle from both hindlimbs removed.
Each isolated soleus muscle was cut into two strips, subsequently
weighed (~15 mg), and pinned at the tendon ends onto an inert resin
base in a well of six-well multiculture dish. Each well contained 3 ml
of Krebs Henseleit buffer (KCl, 4.7 mM; CaCl2,
2.5 mM; K2PO4, 1.2 mM,
MgSO4, 1.2 mM; NaHCO3, 25 mM: glucose, 25 mM; bovine serum albumin, 0.1% (w/v), pH 7.4) pregassed with O2/CO2 (95%/5%)
at 37 °C. Muscle strips were allowed to recover for 15 min at
37 °C with continuous oxygenation and gentle rotation on a platform
shaker and then incubated for a further 30 min in the absence or
presence of 1 milliunit/ml insulin (Novo, Denmark) or 50 µM m-HT. At the end of this period, muscle strips were
rapidly washed three times with glucose-free KH buffer (at 37 °C)
and then incubated for 10 min at 37 °C in uptake buffer (KH buffer
containing 10 µM
2-[1,2-3H]deoxy-D-glucose (1 µCi/ml) and
[14C]mannitol (0.2 µCi/ml, used as an extracellular
reference marker). Following this incubation period muscles were washed
three times with ice-cold saline and then maintained in cold saline for
40 min before blotting on filter paper and solubilization in 1 ml of
0.5 N NaOH at 60 °C for 45 min. Solubilized muscle
extracts were then processed for liquid scintillation counting.
Subcellular Fractionation of L6 Muscle Cells--
Total L6 cell
membranes, plasma, and intracellular membranes were prepared from
muscle cells as described previously (24). The protein content of each
of the isolated membrane fractions was determined using the Bradford
assay with BSA as standard (25).
SDS-PAGE and Immunoblotting--
Isolated membrane fractions
from L6 cells and rat and human skeletal muscle were subjected to
SDS-PAGE on 10% resolving gels and immunoblotted as previously
reported (24). IRS1 immunoprecipitates were run on a 7% resolving gel.
Nitrocellulose membranes were probed with antisera against the
1
subunit of the Na,K-ATPase (Mck1, 1:100 generously provided by Dr. K. Sweadner, Harvard University (26)), GLUT4 (1:500, East Acres
Biologicals, Southbridge, MA), GLUT1 (1:500, kindly provided by Dr.
S. A. Baldwin, University of Leeds, Leeds, UK), mouse GLUT3
(1:500, kindly provided by Professor G. W. Gould, University of
Glasgow), 5-HT2A receptor (1:700, PharMingen, San Diego,
CA), anti-p85 (1:100, Upstate Biotechnology Inc., Lake Placid, NY) and
anti-PY (1:3000, Upstate Biotechnology Inc.), anti-PKB and anti-phospho
PKB Ser473 (New England Biolabs, Herts, UK). Primary
antibody detection was performed using either horseradish
peroxidase-conjugated anti-rabbit IgG (1:2000, SAPU, Scotland) or
anti-mouse (1:2000, Scottish Antibody Production Unit) for 1 h and
visualized using enhanced chemiluminescence (Amersham Life Sciences,
Bucks, UK). Immunoreactive signals on autoradiographs were quantitated
using a Bio-Rad 670 densitometer.
IRS1 Immunoprecipitation from L6 Lysates--
L6 cells were
extracted on 10-cm plates in the lysis buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 1 mM
Na3VO4, 10 mM sodium
-glycerophosphate, 50 mM NaF, 5 mM
Na4P2O7, 1 µM
microcystin-LR, 0.27 M sucrose, 0.2 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µg/ml
leupeptin, and 0.1% (v/v) 2-mercaptoethanol. 500 µg of cell lysate
protein was centrifuged at 13,000 × g and IRS1
immunoprecipitated from lysates using a C-terminal IRS1 antibody
(Upstate Biotechnology Inc.). Immunocomplexes were captured by
incubation with protein A-agarose beads and solubilized in Laemmli
sample buffer prior to SDS-PAGE and immunoblotting as described above.
Analysis of Phosphatidylinositol 3,4,5-Phosphate (PIP3) and
PKB
Activity in L6 Cells--
PIP3 was measured using a sensitive
ligand binding displacement assay as reported previously (27). For
analysis of PKB
activity, L6 cells were extracted on 10-cm plates in
lysis buffer (composition as described above). PKB
was
immunoprecipitated from lysates using a C-terminal PKB
antibody (28)
and kinase activity assayed using a synthetic peptide substrate
"crosstide" (GRPRTSSFAEG) corresponding to the sequence in GSK3
surrounding the Ser residue phosphorylated by MAPKAPK1 and
p70S6 kinase as described previously (23, 28). One unit of
activity was defined as that amount which catalyzed the phosphorylation of 1 nmol of substrate in 1 min. Protein concentrations were determined using the Bradford method (25).
Statistical Analysis--
Statistical analysis was carried out
using a two-tailed Student's t test. Data were considered
statistically significant at p values
0.05.
 |
RESULTS AND DISCUSSION |
Previous work has shown that administration of 5-hydroxytryptophan
and pargyline (a monoamine oxidase inhibitor that prevents breakdown of
5-HT) to rodents induces a profound lowering in blood glucose (3, 14).
In these studies the resulting hypoglycemia could not be explained by
an increase in insulin secretion, and the effect could be abolished
when animals were pretreated with an inhibitor of aromatic amino acid
decarboxylation, which prevents the conversion of 5-hydroxytryptophan
to 5-HT (14). These observations collectively suggested that 5-HT was
the active hypoglycemic agent. We entertained the possibility that one
potential mechanism by which 5-HT may promote a lowering in blood
glucose was by directly stimulating glucose uptake in skeletal muscle;
a notion based on recent work showing that rat fetal myoblasts express
the 5-HT2A receptor (18). To test this hypothesis we
initially carried out SDS-PAGE and immunoblotting to determine whether
the 5-HT2A receptor was expressed in total membranes
prepared from L6 muscle cells and crude membranes from mature rat and
human skeletal muscle. Rat brain and human liver microsomes were used
as positive and negative controls, respectively. Using a monoclonal
antibody that specifically recognizes the 5-HT2A receptor
subtype, a single immunoreactive band of ~55 kDa was observed in all
three muscle samples, which migrated alongside that seen in the rat
brain sample (Fig. 1A).
Quantitative analyses of immunoblot data from three separate
experiments revealed that 5-HT2A receptor expression was
higher by 3.2 ± 0.6-fold in fully differentiated L6 myotubes (day
8) compared with that in L6 myoblasts (day 3). The apparent difference
in 5-HT2A receptor expression between myoblasts and myotubes could not be attributed to the aberrant loading of membrane protein on SDS gels, as no differences were observed in the abundance of the
1-Na,K-ATPase subunit in the same L6 membranes (Fig.
1B).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
5-HT2A receptor expression in rat
and human skeletal muscle. A, total membranes (20 µg)
isolated from L6 myotubes, rat skeletal muscle, and human
(Hu.) skeletal muscle were subjected to SDS-PAGE and
immunoblotted with a 5-HT2A receptor antibody as described
under "Experimental Procedures." Microsomes from rat brain and
human liver were run in adjacent lanes as a positive and negative
controls, respectively. B, total membranes (20 µg) from L6
myoblasts (Mb) (day 3) and myotubes (Mt) (day 8)
were subjected to SDS-PAGE and immunoblotted using antibodies against
the subunit of the 1-Na,K-ATPase and 5-HT2A.
|
|
Having established that L6 cells and rat skeletal muscle express the
5-HT2A receptor, we investigated whether 5-HT acutely regulated 2DG uptake in L6 myotubes and isolated rat soleus muscle. When muscle cells were exposed to increasing concentrations of 5-HT
(between 1 nM and 100 µM), there was a
dose-dependent increase in 2DG uptake (Fig.
2). Measurements of circulating 5-HT
levels indicate that whole blood 5-HT is approximately 1 µg/ml
(equating to ~5 µM), whereas the platelet-free plasma
circulating concentration is ~10 nM (29). Thus, the
observation that skeletal muscle expresses a 5-HT2A
receptor and that glucose uptake can be stimulated within the
physiological range of blood 5-HT implies that signaling via this
receptor may represent a novel mechanism regulating muscle glucose
uptake in vivo. In order to gain further insights into the
mechanism by which 5-HT stimulates glucose uptake, all subsequent experiments were performed using maximally effective concentrations (50 µM) of either 5-HT or the 5-HT2A agonist
methylserotonin (m-HT). Both agents increased 2DG uptake by ~50%
(Fig. 3A), and identical results were obtained when using 50 µM quipazine (another
5-HT2 agonist, data not shown). The 5-HT- and m-HT-induced
increase in 2DG uptake was lower than that seen in response to insulin (Fig. 3A), but neither 5-HT nor m-HT could elicit a
significant additive stimulation when simultaneously presented to
muscle cells with insulin (Fig. 3A). Very similar
observations were made in isolated rat soleus strips in which insulin
and m-HT caused a 2.7-fold and 50% increase in 2DG uptake,
respectively (Fig. 3B). As with L6 cells, we found that
exposing soleus strips simultaneously to insulin and m-HT did not
result in any additive stimulation in glucose uptake (Fig.
3B). The finding that m-HT increases glucose uptake in both
cultured muscle cells and isolated rat muscle in a manner similar to
that seen in response to 5-HT is therefore consistent with the
suggestion that the effects of the latter are also mediated via the
5-HT2A receptor. This view is further strengthened by the
observation that the stimulatory effects of m-HT (and 5-HT, data not
shown) were suppressed in the presence of submicromolar concentrations
of the 5-HT2A antagonist, ketanserin (Fig. 3C).
The observed antagonism takes place within the expected Kd range reported for the antagonist in the
literature (i.e. <10 nM (6)) and is, moreover,
in line with findings from other groups reporting that low nanomolar
concentrations of ketanserin block 5-HT action via the
5-HT2 receptor subtype (30).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Dose-dependent activation of 2DG
uptake in L6 myotubes by serotonin (5-HT). L6 myotubes were
incubated with different 5-HT concentrations (between 1 nM
and 100 µM) and
2-[1,2-3H]deoxy-D-glucose (2DG)
assayed as described under "Experimental Procedures." Results are
expressed as a percentage increase in 2DG uptake above that seen in
cells not exposed to 5-HT. Values represent means ± S.E. from at
least three separate experiments and were all found to be significantly
elevated (p < 0.05) compared with the value obtained
with unstimulated cells.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Effects of serotonin (5-HT),
methylserotonin (m-HT), and insulin
(Ins) on 2DG uptake in L6 muscle cells and incubated
rat soleus muscle strips. A, L6 myotubes were incubated
with insulin (100 nM) for 30 min, with 5-HT (50 µM) or m-HT (50 µM) for 10 min, or with
insulin and m-HT (Ins, 30 min/m-HT, added during the last 10 min of insulin incubation).
2-[1,2-3H]deoxy-D-Glucose was assayed as
described under "Experimental Procedures." Values represent
means ± S.E. from at least three separate experiments.
B, rat soleus strips were incubated for 30 min with m-HT (50 µM), insulin (1 milliunit/ml), or with insulin and m-HT
(Ins/m-HT). Results are means ± S.E. of seven
different experiments. C, L6 myotubes were incubated with
m-HT (50 µM) for 10 min in the absence or the presence of
various concentrations (1 nM to 1 µM) of
ketanserin tartarate (KT). Results are the means ± S.E. of three different experiments. The asterisk indicates
a statistically significant change (p < 0.05) compared
with the respective basal value, the annotation a indicates
a significant difference compared with the 5-HT or m-HT values
(p < 0.05).
|
|
In order to determine whether the acute stimulation in 2DG uptake
observed in the presence of m-HT was due to changes in the subcellular
distribution of glucose transporters, we immunoblotted plasma
(PM) and intracellular (IM) membrane fractions
from L6 myotubes with antibodies against GLUT1, GLUT3, and GLUT4. Fig. 4 shows representative immunublots from
three separate experiments showing that m-HT induces an increase in the
plasma membrane abundance of all three transporters by between 40 and
60%. The increase in surface GLUT content takes place as a result of
their recruitment from the intracellular compartment, which showed a
corresponding loss in each of the three GLUT proteins. The increase in
plasma membrane GLUT content following exposure of L6 cells to m-HT was very rapid and highly reminiscent of that seen in response to treatment
of muscle cells with insulin (23, 31). Nevertheless, the observed
translocation of all three transporters seen in response to m-HT was
less than that evoked by insulin (Fig. 4). Given that m-HT and insulin
do not cause any additive increase in glucose uptake, it is conceivable
that both stimuli signal via their respective membrane receptors onto
the same intracellular pool of glucose transporters by either distinct
or convergent signaling pathways.

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of methylserotonin
(m-HT) and insulin on the abundance of GLUT1, GLUT3,
and GLUT4 in subcellular membrane fractions from L6 myotubes. L6
myotubes were incubated with 50 µM m-HT for 10 min or 100 nM insulin for 30 min prior to cell harvesting and
subcellular fractionation as described under "Experimental
Procedures." 20 µg of plasma membrane (PM) or internal
membrane (IM) protein was applied to polyacrylamide gels and
analyzed by SDS-PAGE. Immunoblotting was performed using
isoform-specific antibodies to the three glucose transporters as
described under "Experimental Procedures."
|
|
To gain some insight into whether components of the insulin-signaling
pathway may participate in 5-HT2A receptor signaling we
first investigated the effects of the phosphoinositide 3-kinase (PI3K)
inhibitor, wortmannin, on the agonist induced stimulation in glucose
uptake. In line with previous work from our group, Fig.
5A shows that wortmannin
induced a 50% reduction in basal glucose uptake and completely blocked
insulin-stimulated glucose transport in L6 myotubes (22, 23, 31).
However, exposure of muscle cells to 50 µM m-HT following
pretreatment with 100 nM wortmannin resulted in a modest,
but significant, increase in 2DG uptake by ~25% (similar results
were also obtained when using the structurally unrelated PI3K inhibitor
LY294002, data not shown). Since wortmannin is known to suppress the
externalization of glucose transporters that recycle between the cell
surface and endosomal compartment (32, 33), we believe that this is likely to contribute to the reduced activation in glucose uptake by
m-HT. The finding that m-HT is still capable of causing a significant stimulation in glucose uptake in the presence of wortmannin, whereas insulin fails to elicit any increase, is consistent with the idea that
PI3K participates in insulin signaling but not in 5-HT2A mediated signaling. In an attempt to resolve this issue further, we
investigated whether 5-HT2A receptor stimulation modulated the phosphorylation status of IRS1 and the activities of PI3K and
protein kinase B
.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of m-HT on wortmannin
(WM)-treated L6 myotubes and IRS1 tyrosine
phosphorylation and association with PI3K. A, L6
myotubes were incubated in the presence of 100 nM
wortmannin for 40 min and exposed to 100 nM insulin for the
last 30 min of this incubation or with 50 µM of m-HT for
the last 10 min of the wortmannin incubation. At the end of this
treatment period 2DG uptake was assayed as described under
"Experimental Procedures." Values are mean ± S.E. from up to
four experiments. Data are expressed as a percent change from basal
ascribed a value of 100%. All values were significantly different from
the basal uptake (p < 0.05). The asterisk
indicates a significant difference from the uptake value obtained in
the presence of wortmannin alone (p < 0.05).
B, L6 myotubes were stimulated with insulin (Ins)
(100 nM) or m-HT (50 µM) for 10 min, and
cells were lysed as described under "Experimental Procedures."
Lysates were immunoprecipitated with anti-IRS1 antibody.
Immunoprecipitated proteins were separated by SDS-PAGE and blotted with
either anti-phosphotyrosine (left panel) or anti-P85
(right panel) antibodies. C, the right
panel shows an immunoblot of native PKB expression in L6 lysates
prepared from control and insulin (Ins)- and m-HT-stimulated
cells. The left panel shows the same lysates blotted with an
antibody that recognizes the phosphorylated Ser473 residue
of PKB.
|
|
Fig. 5B shows an anti-phosphotyrosine blot of IRS1
immunoprecipitates prepared from control and insulin- and m-HT-treated muscle cells. Insulin, but not m-HT, induced tyrosine phosphorylation of IRS1, suggesting that the latter was not a downstream target for the
5-HT2A receptor. Moreover, analyses of IRS1 precipitates with an antibody against the regulatory 85-kDa PI3K subunit revealed that p85 was only associated with IRS1 in insulin-treated cells. However, since 5-HT2A belongs to the family of
G-protein-coupled receptors and heterotrimeric G-protein-regulated
forms of PI3K have been identified (34), it is plausible that 5-HT may
stimulate PI3K independently of IRS1. It is also noteworthy that
G-protein-coupled receptors have been shown to activate PKB in human
phagocytes and COS-7 cells ectopically expressing muscarinic
acetylcholine receptors that couple to Gq and
Gi (35, 36). Since PKB lies downstream of PI3K, and we and
others have implicated it in the insulin-mediated translocation of
GLUT4 (23, 37, 38), activation of PI3K by a G-protein-coupled receptor
may represent one potential mechanism of stimulating PKB and hence
glucose uptake in muscle. However, the data shown in Table
I and Fig. 5C does not support such a mechanism. L6 myotubes exposed to 50 µM 5-HT or
m-HT did not display any detectable increase in cellular PIP3 or PKB
phosphorylation and activity. In contrast, insulin provoked a 2.2-fold
increase in PIP3 production and a 41-fold stimulation in PKB
activity and phosphorylation of its Ser473 residue (Table I
and Fig. 5C). The insulin-induced increase in PIP3 and
PKB
activity was abolished when muscle cells were pretreated with
wortmannin prior to stimulation with insulin (Table I). Thus while we
cannot exclude the possibility that 5-HT2A receptor
signaling may converge at some point downstream of PKB with the insulin
signaling pathway, our data rules out any involvement of PI3K or PKB in
the 5-HT2A-mediated increase in muscle glucose uptake. An
alternative signaling mechanism may involve the phospholipase C/protein
kinase C pathway, which the 5-HT2A receptor is thought to
activate via a G-protein-mediated interaction (8, 9). A role for
phospholipase C in the translocation of GLUT4 has been proposed
recently (39), but it currently remains unknown whether phospholipase C
is activated by 5-HT2A receptor agonists in L6 cells and,
if so, whether this plays any part in modulating the number of cell
surface glucose transporters in skeletal muscle. Addressing this issue
remains an interesting topic for future study.
View this table:
[in this window]
[in a new window]
|
Table I
Effects of insulin, serotonin (5-HT), and methylserotonin (m-HT) on
cellular PIP3 levels and protein kinase B activity in L6 muscle cells
L6 myotubes were incubated for 10 min with insulin (100 nM), 5-HT (50 µM), m-HT (50 µM), or wortmannin/insulin (wortmannin, 100 nM, 15 min; insulin 100 nM, 10 min) and
prepared for analyses of PIP3 and PKB activity as described under
"Experimental Procedures." Values are from at least three
experiments for PKB (mean ± S.E.) and from two separate
experiments each conducted in triplicate for PIP3 (mean ± S.D.).
|
|
In summary, our results show that the 5-HT2A receptor is
expressed in rat and human skeletal muscle. Stimulation of this
receptor with 5-HT or a specific 5-HT2A agonist causes a
rapid stimulation in glucose transport that occurs as a result of the
increased recruitment of glucose transporters from an intracellular
pool to the cell surface. The post-receptor signaling events involved in eliciting this stimulation currently remain unknown, but they do not
involve signaling molecules that participate in early events of insulin
signaling (i.e. IRS1, PI3K, or PKB
). The finding that the
5-HT2A receptor can modulate glucose transport is likely to be physiologically significant given that plasma 5-HT levels are known
to increase during muscle exercise (40) and fall during diabetes (29);
conditions during which utilization of glucose is significantly
modulated in skeletal muscle. Understanding how the 5-HT2A
receptor signals an increase in muscle glucose uptake may prove
potentially valuable in developing new strategies aimed at improving
glucose utilization in skeletal muscle during circumstances when this
tissue may be profoundly resistant to insulin action.
 |
ACKNOWLEDGEMENTS |
We are grateful to Froogh Darakhshan and Anne
Blair who participated in some of the studies reported and to Dario
Alessi and Armelle Leturque for useful discussions. We also thank A. Jain and the staff of Orthopedic theater at Dundee Royal Infirmary for
providing us with access to human muscle tissue.
 |
FOOTNOTES |
*
This work was supported by the Medical Research Council,
Biotechnology and Biological Sciences Research Council, Association Langue Francaise Etude Diabete et Metabolisme, British Diabetic Association, and The Wellcome Trust.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Anatomy & Physiology, The University of Dundee, Dundee, DD1 4HN, Scotland. E-mail: h.s.hundal{at}dundee.ac.uk.
 |
ABBREVIATIONS |
The abbreviations used are:
5-HT, 5-hydroxytryptamine;
IRS, insulin receptor substrate;
2DG, 2-deoxy-D-glucose;
m-HT, methylserotonin;
PAGE, polyacrylamide gel electrophoresis;
PIP3, phosphatidylinositol
3,4,5-phosphate;
PI3K, phosphoinositide 3-kinase;
PKB, protein kinase
B.
 |
REFERENCES |
-
Tecott, L.,
Shtrom, S.,
and Julius, D.
(1995)
Mol. Cell. Neurosci.
6,
43-55[CrossRef][Medline]
[Order article via Infotrieve]
-
Eaton, M. J.,
Staley, J. K.,
Globus, M. Y. T.,
and Whittemore, S. R.
(1995)
Dev. Biol.
170,
169-182[CrossRef][Medline]
[Order article via Infotrieve]
-
Smith, S. A.,
and Pogson, C. I.
(1977)
Biochem. J.
168,
495-506[Medline]
[Order article via Infotrieve]
-
Fischer, Y.,
Thomas, J.,
Kamp, J.,
Jungling, E.,
Rose, H.,
Carpene, C.,
and Kammermeier, H.
(1995)
Biochem. J.
311,
575-583[Medline]
[Order article via Infotrieve]
-
Peroutka, S. J.
(1995)
Trends Neurosci.
18,
68-69[CrossRef][Medline]
[Order article via Infotrieve]
-
Hoyer, D.,
Clarke, D. E.,
Fozard, J. R.,
Hartig, P. R.,
Martin, G. R.,
Mylecharane, E. J.,
Saxena, P. R.,
and Humphrey, P. P. A.
(1994)
Pharmacol. Rev.
46,
157-203[Abstract]
-
Nicoll, R. A.
(1988)
Science
241,
545-551[Medline]
[Order article via Infotrieve]
-
Conn, P. J.,
Sandersbush, E.,
Hoffman, B. J.,
and Hartig, P. R.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
4086-4088[Abstract]
-
Julius, D.,
Huang, K. N.,
Livelli, T. J.,
Axel, R.,
and Jessell, T. M.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
928-932[Abstract]
-
Launay, J. M.,
Birraux, G.,
Bondoux, D.,
Callebert, J.,
Choi, D. S.,
Loric, S.,
and Maroteaux, L.
(1996)
J. Biol. Chem.
271,
3141-3147[Abstract/Free Full Text]
-
Wozniak, K. M.,
and Linnoila, M.
(1991)
Life Sci.
49,
101-109[CrossRef][Medline]
[Order article via Infotrieve]
-
Furman, B. L.,
and Wilson, G. A.
(1980)
Diabetologia
19,
386-390[Medline]
[Order article via Infotrieve]
-
Yamada, J.,
Sugimoto, Y.,
Kimura, I.,
Takeuchi, N.,
and Horisaka, K.
(1989)
Life Sci.
45,
1931-1936[Medline]
[Order article via Infotrieve]
-
Lundquist, I.,
Ekholm, R.,
and Ericson, L. E.
(1971)
Diabetologia
7,
414-422[Medline]
[Order article via Infotrieve]
-
Wilson, J. P.,
Downs, R. W., Jr.,
Feldman, J. M.,
and Lebovitz, H. E.
(1974)
Am. J. Physiol.
227,
305-312[Free Full Text]
-
Coulie, B.,
Tack, J.,
Bouillon, R.,
Peeters, T.,
and Janssens, J.
(1998)
Am. J. Physiol.
274,
E317-E320[Abstract/Free Full Text]
-
Uvnasmoberg, K.,
Ahlenius, S.,
Alster, P.,
and Hillegaart, V.
(1996)
Neuroendocrinology
63,
269-274[Medline]
[Order article via Infotrieve]
-
Guillet-Deniau, I.,
Burnol, A. F.,
and Girard, J.
(1997)
J. Biol. Chem.
272,
14825-14829[Abstract/Free Full Text]
-
Mitsumoto, Y.,
and Klip, A.
(1992)
J. Biol. Chem.
267,
4957-4962[Abstract/Free Full Text]
-
Aledo, J. C.,
Lavoie, L.,
Volchuk, A.,
Keller, S. R.,
Klip, A.,
and Hundal, H. S.
(1997)
Biochem. J.
325,
727-732[Medline]
[Order article via Infotrieve]
-
Hundal, H. S.,
Maxwell, D. L.,
Ahmed, A.,
Darakhshan, F.,
Mitsumoto, Y.,
and Klip, A.
(1994)
Mol. Membr. Biol.
11,
255-262[Medline]
[Order article via Infotrieve]
-
McDowell, H. E.,
Walker, T.,
Hajduch, E.,
Christie, G.,
Batty, I. H.,
Downes, C. P.,
and Hundal, H. S.
(1997)
Eur. J. Biochem.
247,
306-313[Abstract]
-
Hajduch, E.,
Alessi, D. R.,
Hemmings, B. A.,
and Hundal, H. S.
(1998)
Diabetes
47,
1006-1013[Abstract]
-
Bilan, P. J.,
Mitsumoto, Y.,
Ramlal, T.,
and Klip, A.
(1992)
FEBS Lett.
298,
285-290[CrossRef][Medline]
[Order article via Infotrieve]
-
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254[CrossRef][Medline]
[Order article via Infotrieve]
-
Felsenfeld, D. P.,
and Sweadner, K. J.
(1988)
J. Biol. Chem.
263,
10932-10942[Abstract/Free Full Text]
-
van der Kaay, J.,
Batty, I. H.,
Cross, D. A. E.,
Watt, P. W.,
and Downes, C. P.
(1997)
J. Biol. Chem.
272,
5477-5481[Abstract/Free Full Text]
-
Cross, D. A. E.,
Alessi, D. R.,
Cohen, P.,
Andjelkovic, M.,
and Hemmings, B. A.
(1995)
Nature
378,
785-789[CrossRef][Medline]
[Order article via Infotrieve]
-
Martin, F. J.,
Miguez, J. M.,
Aldegunde, M.,
and Atienza, G.
(1995)
Life Sci.
56,
51-59[CrossRef][Medline]
[Order article via Infotrieve]
-
Monopoli, A.,
Conti, A.,
Forlani, A.,
Ongini, E.,
Antona, C.,
and Biglioli, P.
(1990)
Cardiovasc. Drugs Ther.
4,
59-61
-
Tsakiridis, T.,
McDowell, H. E.,
Walker, T.,
Downes, C. P.,
Hundal, H. S.,
Vranic, M.,
and Klip, A.
(1995)
Endocrinology
136,
4315-4322[Abstract]
-
Yang, J.,
Clarke, J. F.,
Ester, C. J.,
Young, P. W.,
Kasuga, M.,
and Holman, G. D.
(1996)
Biochem. J.
313,
125-131[Medline]
[Order article via Infotrieve]
-
Clarke, J. F.,
Young, P. W.,
Yonezawa, K.,
Kasuga, M.,
and Holman, G. D.
(1994)
Biochem. J.
300,
631-635[Medline]
[Order article via Infotrieve]
-
Stephens, L.,
Smrcka, A.,
Cooke, F. T.,
Jackson, T. R.,
Sternweis, P. C.,
and Hawkins, P. T.
(1994)
Cell
77,
83-93[Medline]
[Order article via Infotrieve]
-
Tilton, B.,
Andjelkovic, M.,
Didichenko, S. A.,
Hemmings, B. A.,
and Thelen, M.
(1997)
J. Biol. Chem.
272,
28096-28101[Abstract/Free Full Text]
-
Murga, C.,
Laguinge, L.,
Wetzker, R.,
Cuadrado, A.,
and Gutkind, J. S.
(1998)
J. Biol. Chem.
273,
19080-19085[Abstract/Free Full Text]
-
Kohn, A. D.,
Summers, S. A.,
Birnbaum, M. J.,
and Roth, R. A.
(1996)
J. Biol. Chem.
271,
31372-31378[Abstract/Free Full Text]
-
Tanti, J. F.,
Grillo, S.,
Gremeaux, T.,
Coffer, P. J.,
Vanobberghen, E.,
and Lemarchandbrustel, Y.
(1997)
Endocrinology
138,
2005-2010[Abstract/Free Full Text]
-
Van Epps-Fung, M.,
Gupta, K.,
Hardy, R. W.,
and Wells, A.
(1997)
Endocrinology
138,
5170-5175[Abstract/Free Full Text]
-
Newsholme, E. A.,
and Blomstrand, E.
(1995)
Adv. Exp. Med. Biol.
384,
315-320[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.