Serotonin (5-Hydroxytryptamine), a Novel Regulator of Glucose Transport in Rat Skeletal Muscle*

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.

stems from the ability of the neurotransmitter to interact with multiple 5-HT receptors (currently classified as 5-HT 1 through to 5-HT 7 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-HT 3 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)(13)(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-HT 2A 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-HT 2A receptor? (ii) Do 5-HT and specific 5-HT 2A 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?
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 g/ml streptomycin, 250 ng/ml amphotericin B) at 37°C in an atmosphere of 5% CO 2 , 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, ϳ5 g of soleus muscle was rapidly frozen in liquid nitrogen and stored at Ϫ80°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 Ϫ80°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-HT 2A 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 (ϳ15 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; CaCl 2 , 2.5 mM; K 2 PO 4 , 1.2 mM, MgSO 4 , 1.2 mM; NaHCO 3 , 25 mM: glucose, 25 mM; bovine serum albumin, 0.1% (w/v), pH 7.4) pregassed with O 2 /CO 2 (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 M 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 M 2-[1,2-3 H]deoxy-D-glucose (1 Ci/ml) and [ 14 C]mannitol (0.2 Ci/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).
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" (GRPRTSS-FAEG) corresponding to the sequence in GSK3 surrounding the Ser residue phosphorylated by MAPKAPK1 and p70 S6 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 Յ 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-HT 2A receptor (18). To test this hypothesis we initially carried out SDS-PAGE and immunoblotting to determine whether the 5-HT 2A 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-HT 2A receptor subtype, a single immunoreactive band of ϳ55 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-HT 2A receptor expression was higher by 3.2 Ϯ 0.6-fold in fully differentiated L6 myotubes (day 8) compared with that in L6 myoblasts (day 3). The apparent difference in 5-HT 2A 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).
Having established that L6 cells and rat skeletal muscle express the 5-HT 2A 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 M), 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 g/ml (equating to ϳ5 M), whereas the platelet-free plasma circulating concentration is ϳ10 nM (29). Thus, the observation that skeletal muscle expresses a 5-HT 2A 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 M) of either 5-HT or the 5-HT 2A agonist methylserotonin (m-HT). Both agents increased 2DG uptake by ϳ50% (Fig. 3A), and identical results were obtained when using 50 M quipazine (another 5-HT 2 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-HT 2A 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-HT 2A antagonist, ketanserin (Fig. 3C). The observed antagonism takes place within the expected K d range reported for the antagonist in the literature (i.e. Ͻ10 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-HT 2 receptor subtype (30).
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 immu-noblotted 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.
To gain some insight into whether components of the insulinsignaling pathway may participate in 5-HT 2A 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 M m-HT following pretreatment with 100 nM wortmannin resulted in a modest, but significant, increase in 2DG uptake by ϳ25% (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-HT 2A mediated signaling. In an attempt to resolve this issue further, we investigated whether 5-HT 2A receptor stimulation modu- H]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 Ϯ S.E. from at least three separate experiments and were all found to be significantly elevated (p Ͻ 0.05) compared with the value obtained with unstimulated cells.
lated the phosphorylation status of IRS1 and the activities of PI3K and protein kinase B␣. Fig. 5B shows an anti-phosphotyrosine blot of IRS1 immunoprecipitates prepared from control and insulin-and m-HTtreated muscle cells. Insulin, but not m-HT, induced tyrosine phosphorylation of IRS1, suggesting that the latter was not a downstream target for the 5-HT 2A 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-HT 2A 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-proteincoupled receptors have been shown to activate PKB in human phagocytes and COS-7 cells ectopically expressing muscarinic acetylcholine receptors that couple to G q and G i (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  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 M 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 g 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." Table I and Fig. 5C does not support such a mechanism. L6 myotubes exposed to 50 M 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 Ser 473 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-HT 2A 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-HT 2A -mediated increase in muscle glucose uptake. An alternative signaling mechanism may involve the phospholipase C/protein kinase C pathway, which the 5-HT 2A 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-HT 2A 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.
In summary, our results show that the 5-HT 2A receptor is expressed in rat and human skeletal muscle. Stimulation of this receptor with 5-HT or a specific 5-HT 2A 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-HT 2A 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-HT 2A 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. 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 M 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 Ϯ 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 Ͻ 0.05). The asterisk indicates a significant difference from the uptake value obtained in the presence of wortmannin alone (p Ͻ 0.05). B, L6 myotubes were stimulated with insulin (Ins) (100 nM) or m-HT (50 M) 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-HTstimulated cells. The left panel shows the same lysates blotted with an antibody that recognizes the phosphorylated Ser 473 residue of PKB.  , 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 M), m-HT (50 M), 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 Ϯ S.E.) and from two separate experiments each conducted in triplicate for PIP3 (mean Ϯ S.D.).