1A-Adrenoceptors Activate Glucose Uptake in L6 Mus
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内分泌学杂志 2005年第2期
Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, SE 10691 Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Tore Bengtsson, Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratory F3, Stockholm University, SE 10691 Stockholm, Sweden. E-mail: tore.bengtsson@zoofys.su.se.
Abstract
The role of 1-adrenoceptor activation on glucose uptake in L6 cells was investigated. The 1-adrenoceptor agonist phenylephrine [pEC50 (–log10 EC50), 5.27 ± 0.30] or cirazoline (pEC50, 5.00 ± 0.23) increased glucose uptake in a concentration-dependent manner, as did insulin (pEC50, 7.16 ± 0.21). The 2-adrenoceptor agonist clonidine was without any stimulatory effect on glucose uptake. The stimulatory effect of cirazoline was inhibited by the 1-adrenoceptor antagonist prazosin, but not by the ?-adrenoceptor antagonist propranolol. RT-PCR showed that the 1A-adrenoceptor was the sole 1-adrenoceptor subtype expressed in L6 cells. Cirazoline- or insulin-mediated glucose uptake was inhibited by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting a possible interaction between the 1-adrenoceptor and insulin pathways. Cirazoline or insulin stimulated phosphatidylinositol-3 kinase activity, but 1-adrenoceptor activation did not phosphorylate Akt. Both cirazoline- and insulin-mediated glucose uptake were inhibited by protein kinase C (PKC), phospholipase C, and p38 kinase inhibitors, but not by Erk1/2 inhibitors (despite both treatments being able to phosphorylate Erk1/2). Insulin and cirazoline were able to activate and phosphorylate p38 kinase. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the calcium ionophore A23187 produced significant increases in glucose uptake, indicating roles for PKC and calcium in glucose uptake. Down-regulation of conventional PKC isoforms inhibited glucose uptake mediated by 12-O-tetradecanoylphorbol-13-acetate, but not by insulin or cirazoline. This study demonstrates that 1-adrenoceptors mediate increases in glucose uptake in L6 muscle cells. This effect appears to be related to activation of phospholipase C, phosphatidylinositol-3 kinase, p38 kinase, and PKC.
Introduction
A RELATIVELY LITTLE-studied response to adrenoceptor activation is facilitation of glucose uptake. Adrenoceptors are classified into three main subtypes: 1-, 2-, and ?-adrenoceptors. ?-Adrenoceptor stimulation increases glucose uptake in rodent skeletal muscle (1, 2, 3, 4) and brown adipose tissue (5, 6). This effect is mediated primarily by the ?3-adrenoceptor in brown adipose tissue (5, 6), but is mediated by ?2-adrenoceptors in skeletal muscle cells (4). There is relatively little known about the presence/effect of 1-adrenoceptors in skeletal muscle or about -adrenoceptor-mediated regulation of glucose uptake.
1-Adrenoceptors are located in a large number of tissues from a large number of species. In the central nervous system, they perform excitatory functions and perform numerous functions in the periphery (contraction of vascular and nonvascular smooth muscle, relaxation of gastrointestinal smooth muscle, positive inotropic effects in the heart, and hepatic glycogenolysis). Previous studies showed the presence of 1-adrenoceptors in rat skeletal muscle through quantitative autoradiography (7), ribonuclease protection assays (8), and radioligand binding (9) studies. Recent reports indicate that 1-adrenoceptors may modulate glucose uptake in the mouse skeletal muscle C2C12 cell line (10) and rat white adipose tissue (11, 12). However, the C2C12 cell line is probably not the best model to study mechanisms of insulin-stimulated glucose uptake in muscle, because these cells have been reported to be insulin unresponsive (or have a very small effect on glucose uptake) due to a postulated defective postreceptor signaling pathway (13), suggested to be a lack of an insulin-responsive vesicular glucose transporter 4 (GLUT4) compartment (14). L6 cells represent a better model for glucose uptake because they have been used extensively to elucidate the mechanisms of glucose uptake in muscle, have an intact insulin signaling pathway, and express the insulin-sensitive GLUT4.
The intracellular mechanisms involved in adrenergically mediated glucose uptake in skeletal muscle are still unclear. ?-Adrenoceptors are Gs-coupled receptors, and their activation results in the production of cAMP, although cAMP may not be needed for ?-adrenoceptor stimulation of glucose uptake (4). An earlier report (3) indicated that in L6 muscle cells, insulin and ?2-adrenoceptors increase glucose uptake by two distinct mechanisms, but a recent report (4) indicates that insulin and ?2-adrenoceptors use two pathways for increases in glucose uptake, but these pathways overlap, probably at the level of phosphatidylinositol 3-kinase (PI3K). 1-Adrenoceptors are Gq-coupled receptors. Their activation leads to phospholipase C activation, resulting in the hydrolysis of phosphatidylinositol(4, 5)-bisphosphate to produce inositol-1,4,5-phosphate, which releases calcium from intracellular stores, and diacylglycerol, which can activate protein kinase C (PKC) (15). The phospholipase C-PKC pathway has been linked to glucose uptake (16, 17, 18), but there is little evidence for 1-adrenoceptor mediation of glucose uptake through this pathway. Lipids in the phosphatidylinositol(4, 5)-bisphosphate pathway can be used as substrates for PI3K (a kinase important for insulin’s effects on glucose uptake), and 1-adrenoceptor-mediated glucose uptake in white adipose tissue and heart is mediated through PI3K (12, 19).
The present study investigated the -adrenergic control of glucose uptake in L6 muscle cells. We show that 1-adrenoceptor, but not 2-adrenoceptor, stimulation results in increases in glucose uptake in L6 cells through a mechanism dependent upon phospholipase C, PI3K, p38 kinase, and PKC. The actions of insulin and 1-adrenoceptors on glucose uptake use two pathways, but converge at the level of PKC.
Materials and Methods
Cell culture
Rat L6 cells (obtained from American Type Culture Collection, Manassas, VA) were grown as a monolayer in DMEM (1 g/liter glucose) containing GlutaMAX (Invitrogen Life Technologies, Inc., Carlsbad, CA), 10% (vol/vol) fetal bovine serum, 10 mM HEPES buffer, fungizone (2.5 μg/ml), and gentamicin sulfate (80 μg/ml) under 8% CO2 at 37 C. To differentiate, cells were allowed to reach confluence and the media changed to that containing 2% (vol/vol) fetal bovine serum for 7 d, with medium changes every second day. Experiments were restricted to cells from passages 2–15, and undifferentiated cells were not allowed to grow more than 60–70% confluence.
RNA isolation and determination of 1-adrenoceptor mRNA levels
RNA was extracted from L6 cells after 7 d differentiation, and from atria, ventricle, liver, brain, soleus muscle or prostate from one male Sprague Dawley rat (280 g; anesthetized with 20% O2/80% CO2 and decapitated). This was performed with ethical permission from the North Stockholm animal ethics committee. Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer’s protocol. The yield and quality of RNA was assessed by measuring absorbance at 260 and 280 nm and electrophoresis on 1.3% agarose gels. There was no degradation of any RNA samples.
cDNAs were synthesized by RT of 1 μg of each total RNA using oligo(deoxythymidine)15 (Invitrogen Life Technologies, Inc.) as a primer (20). PCR amplification was carried out on cDNA equivalent to 100 ng starting RNA using primers specific for ?-actin or 1A-, 1B-, or 1D-adrenoceptor, which amplify 559-, 404-, 452-, and 513-bp fragments, respectively (Invitrogen Life Technologies, Inc.; see Table 1). All primers were intron-spanning to eliminate possible contamination by genomic DNA. Reactions were carried out in a Primus 96 Plus thermocycler (MWG Biotech AG, Ebersberg, Germany). For 1A-adrenoceptor or ?-actin PCR, PCR mixes contained cDNA, 1 U Taq DNA polymerase (Invitrogen Life Technologies, Inc.), 1x PCR buffer, 200 μM deoxy-NTPs (Amersham Biosciences), 2 mM Mg-acetate, forward primer (2.8 pmol 1A-adrenoceptor or 1.5 pmol ?-actin) and reverse primer (2.8 pmol 1A-adrenoceptor or 1.5 pmol ?-actin). For 1D-adrenoceptor PCR, PCR mixes contained cDNA, 0.5 U Platinum Taq DNA polymerase (Invitrogen Life Technologies, Inc.), 1x AMP buffer, and 1x Enhancer solution (supplied by Invitrogen Life Technologies, Inc.), deoxy-NTPs (130 μM), MgSO4 (1.5 mM), 5.8 pmol forward primer, and 5.8 pmol reverse primer. For 1B-adrenoceptor PCR, PCR mixes were the same as for 1D-adrenoceptor PCR, except no Enhancer solution was added. The annealing temperature for all PCRs was 64 C, and 30 cycles were performed (16 for ?-actin). After amplification, PCR products were electrophoresed on 1.3% agarose gels and visualized.
TABLE 1. Oligonucleotides used as PCR primers
Immunoblotting
Cells were serum-starved overnight before each experiment on d 7 and were exposed to drugs for the times and concentrations indicated with the data. Extraction of cells and subsequent Western blotting were performed as previously described (21), except samples were electrotransferred to Hybond-C Extra nitrocellulose membranes (pore size, 0.45 μm; Amersham Biosciences, Arlington Heights, IL) for Erk1/2 studies or Hybond-P polyvinylidene difluoride membranes (pore size, 0.45 μm; Amersham Biosciences) for Akt or p38 kinase studies. The primary antibodies used were p44/42 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), Akt, phospho-Akt (Ser473), or phospho-Akt (Thr308) antibody diluted 1:1000, which were detected using a secondary antibody (horseradish peroxidase-linked antirabbit IgG) diluted 1:2000 and enhanced chemiluminescence (ECL, Amersham Biosciences).
2-Deoxy-[3H]D-glucose uptake assay
Glucose uptake was measured using the 2-deoxy-[3H]D-glucose method (3) with minor modifications. Briefly, cells were serum-stared overnight before each experiment and glucose uptake was measured on d 7. Medium was replaced in the morning (serum-free medium), and cells were exposed to drugs for 2–2.5 h. Cells were washed twice in warm PBS before media and drugs were placed in DMEM devoid of glucose for 20 min. 2-Deoxy-[3H]D-glucose (50 nM) was added for 15 min at 37 C, and the reactions were terminated by washing twice in ice-cold PBS. Cells were digested in 0.2 M NaOH for 1 h at 60 C, and samples were transferred to scintillation vials with scintillant and allowed to sit at room temperature for 1 h before being counted. When inhibitors were used, the time indicated with the results represents the time cells were preequilibrated with the inhibitors before agonists were added.
Immunoprecipitation of PI3K and ELISA for detection of PI3K activity
L6 cells (d 7) were serum-starved overnight before cells were treated with drugs for 10 min. The cells were washed three times with ice-cold buffer A [137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM CaCl2, 1 mM MgCl2, and 0.1 mM sodium orthovanadate] before solubilization for 20 min at 4 C in lysis buffer [buffer A containing 1% (vol/vol) Nonidet P-40, and 1 mM phenylmethylsulfonylfluoride]. After centrifugation at 10,000 x g for 10 min at 4 C, the supernatant (cell lysates) were incubated for 1 h at 4 C with anti-PI3K p85 antibody (5 μl antibody/sample), followed by addition of 60 μl of a 50% slurry of protein A-agarose beads in PBS for 1 h at 4 C with mixing. The beads were washed three times in wash buffer 1 [buffer A containing 1% (vol/vol) Nonidet P-40], three times with wash buffer 2 [0.1 M Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium orthovanadate], and twice in wash buffer 3 [10 mM Tris-HCl (pH 7.4), 150 mM LiCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate].
PI3K activity was measured in vitro using a competitive ELISA format (Echelon Biosciences, Inc., Salt Lake City, UT) according to the manufacturer’s instructions. Briefly, the bead-bound immunoprecipitated enzyme was incubated with phosphatidylinositol (4, 5)-biphosphate [PI(4, 5)P2] substrate (100 pmol) in kinase reaction buffer [4 mM MgCl2, 20 mM Tris (pH 7.4), 10 mM NaCl, and 25 μM ATP] for 2 h at room temperature with shaking. The supernatant was then incubated with a PI(3, 4, 5)P3 detector protein for 1 h at room temperature, and the reaction mixes were transferred to PI(3, 4, 5)P3-coated detection plates for 1 h at room temperature. After washing in wash buffer [150 mM NaCl, 10 mM Tris (pH 7.5), and 0.05% (vol/vol) Tween 20], secondary detection reagent (supplied with the kit) was added, plates were washed again, developing solution (supplied with the kit) was added, and PI(3, 4, 5)P3 detector protein binding to the plate was determined by measuring the absorbance at 450 nm.
Measurement of p38 kinase activity
L6 cells (d 7) were serum-starved for 4 h before cells were treated with drugs as indicated with the data. Preparation of cell lysates and immunoprecipitation of phospho-p38 MAPK (Thr180/Tyr184) were performed, and subsequent p38 kinase activity was measured using a nonradioactive p38 MAPK assay kit (Cell Signaling Technology, Beverly, MA) according to the manufacturer’s detailed instructions. The assay is based on the ability of an immobilized phospho-p38 MAPK (Thr180/Tyr182) monoclonal antibody to immunoprecipitate p38 MAPK, followed by an in vitro kinase assay using activating transcription factor-2 (ATF-2) as a substrate. ATF-2 phosphorylation is assessed by immunoblotting using a phospho-ATF-2 (Thr71) antibody.
Statistical analysis
Immunoblotting results are expressed in a graph format as the ratio between phosphorylated and total protein, with the ratio normalized in each experiment to that of control samples. All experiments were performed in singlicate or duplicate; n refers to the number of independent experiments performed.
For glucose uptake experiments, all experiments were performed in duplicate and expressed as the mean ± SEM of n independent experiments. Data were analyzed using nonlinear curve fitting (PRISM version 3.03, GraphPad, Inc., San Diego, CA) to obtain pEC50 (–log10 EC50) values, where appropriate, and statistical significance was determined by t test where appropriate. P 0.05 was considered significant.
For PI3K assay results, the activity of PI3K was expressed as a percentage of the activity measured in control treated cells as suggested by the manufacturer (Echelon Biosciences, Inc.). For p38 kinase assay results, p38 kinase activity was expressed as a percentage of the activity measured in control treated cells.
Drugs and reagents
Drugs and reagents were purchased as follows: 2-deoxy-[3H]D-glucose (12 Ci/mmol; Amersham Biosciences, Little Chalfont, UK); G?6973, Ro-318220, SB202190, U73122, and U73343 (Calbiochem, La Jolla, CA); PD98059 (Cell Signaling Technology, Beverly, MA); insulin (Actrapid, Novo Nordisk, Bagsvaerd, Denmark); A23187, cirazoline, (±)-isoprenaline, LY294002, (–)-phenylephrine, phenylmethylsulfonylfluoride, prazosin, (–)-propranolol, 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich Co., St. Louis, MO).
All cell culture medium and supplements were obtained from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). All antibodies, except anti-PI3K p85 (Upstate Biotechnology, Inc., Lake Placid, NY) were obtained from Cell Signaling Technology. All other drugs and reagents were of analytical grade.
Results
Measurement of 1-adrenoceptor subtype mRNA expression
RT-PCR experiments were conducted to examine which subtype(s) of the 1-adrenoceptor are expressed in differentiated L6 cells. 1A-Adrenoceptor mRNA was expressed at moderate levels compared with prostate or brain (Fig. 1). 1A-Adrenoceptor mRNA from rat soleus muscle was also detectable, consistent with the findings of another study (8). No 1B- or 1D-adrenoceptor mRNA was detected in differentiated L6 cells, although 1B-adrenoceptor mRNA was readily detectable in liver and ventricle, and 1D-adrenoceptor mRNA was readily detectable in brain and atria.
FIG. 1. 1A- (404 bp), 1B- (452 bp), and 1D-adrenoceptor (513 bp) PCR products from rat prostate, brain, liver, ventricle, soleus muscle and atria, and 1A-adrenoceptor PCR products from L6 myotubes. Levels of expression were measured using RT-PCR analysis. Also shown are ?-actin (559 bp) PCR products.
Effects of insulin and adrenergic agonists on glucose uptake in L6 cells
Concentration-response curves to adrenergic agonists were constructed to determine whether -adrenergic agonists were capable of increasing glucose uptake in differentiated L6 cells (Fig. 2). Insulin, the nonselective ?-adrenoceptor agonist (±)-isoprenaline, the nonselective -adrenoceptor agonist phenylephrine, and the 1-adrenoceptor agonist cirazoline stimulated glucose uptake (Fig. 2 and Table 2). Maximal glucose uptake responses to either cirazoline or phenylephrine were significantly lower than glucose uptake mediated by either (±)-isoprenaline or insulin. The 2-adrenoceptor agonist clonidine was without effect (n = 5). Insulin and (±)-isoprenaline produced the same degree of maximum response of glucose uptake in these cells, consistent with our previous study (4).
FIG. 2. Concentration-response curve for insulin or the ?-adrenoceptor agonist (±)-isoprenaline (A) and for the -adrenoceptor agonist phenylephrine, the 1-adrenoceptor agonist cirazoline, or the 2-adrenoceptor agonist clonidine (B) on 2-deoxy-[3H]D-glucose uptake in differentiated L6 cells. Points show means, and vertical lines indicate the SEM of five to 10 experiments performed in duplicate.
TABLE 2. Effects of adrenergic agonists on glucose uptake in L6 cells
Inhibition of adrenergically mediated glucose uptake by propranolol or prazosin
To determine whether the effects of isoprenaline or cirazoline were mediated by -adrenoceptor or ?-adrenoceptor stimulation, cells were preincubated with either the 1-adrenoceptor antagonist prazosin or the ?-adrenoceptor antagonist (–)-propranolol (Fig. 3). Isoprenaline-mediated glucose uptake was inhibited by propranolol, but not by prazosin, confirming that its effects on glucose uptake are mediated by ?-adrenoceptor stimulation. Conversely, cirazoline-mediated glucose uptake was inhibited by prazosin, but not by propranolol, confirming 1-adrenoceptor stimulation. Phenylephrine (10 μM), a conventional -adrenoceptor agonist, stimulated glucose uptake (basal glucose uptake, 100%; phenylephrine, 141 ± 5%; n = 5), but its effects, although totally inhibited by prazosin [prazosin (1 μM), 108 ± 4%; plus phenylephrine, 114 ± 6%; n = 5], were also partly inhibited by propranolol [propranolol (1 μM), 103 ± 5%; plus phenylephrine, 122 ± 6%; n = 5], indicating that its actions are mediated by both - and ?-adrenoceptor stimulation; hence, investigation of 1-adrenoceptor-mediated glucose uptake was performed using cirazoline as the agonist. Preincubation of cells with either propranolol or prazosin did not significantly affect basal glucose uptake.
FIG. 3. Glucose uptake in differentiated L6 cells in response to isoprenaline (10 μM; A) or cirazoline (10 μM; B) in the absence or presence of either propranolol (1 μM, 1 h) or prazosin (1 μM, 1 h). The histograms are the mean ± SEM of six experiments performed in duplicate.
Effect of combined insulin and adrenergic agonists on glucose uptake
To examine the potential interaction between insulin and either 1-adrenceptor or ?-adrenoceptor agonists on glucose uptake, cells were incubated with cirazoline (10 μM) or (±)-isoprenaline (10 μM) in the absence or presence of insulin (1 μM; Table 3). There was no additive increase in insulin-mediated glucose uptake when cells were coincubated with either cirazoline or isoprenaline.
TABLE 3. Effects of insulin on adrenergically stimulated glucose uptake in L6 cells
PI3K activity is increased by insulin, TPA, or cirazoline in L6 cells
PI3K activity is increased after insulin receptor stimulation and is thought to be a downstream mediator of glucose uptake. We measured PI3K activity after 10 min of stimulation by insulin (1 μM), cirazoline (10 μM), or TPA (100 nM). Insulin stimulated PI3K activity approximately 2.6-fold over basal levels. TPA stimulated PI3K activity 2-fold, and cirazoline 1.7-fold over basal levels (Fig. 4).
FIG. 4. PI3K activity is increased after insulin (1 μM, 10 min), TPA (100 nM, 10 min), or cirazoline (10 μM, 10 min) stimulation of L6 cells (n = 4).
Insulin, but not cirazoline or TPA, phosphorylates Akt at both Ser473 and Thr308 in L6 cells
Akt is activated after insulin receptor stimulation (22) and is implicated in insulin-mediated glucose uptake in adipocytes and muscle (23, 24, 25). Hence, we examined whether cirazoline is capable of activating Akt in L6 cells. Insulin phosphorylated Akt on residues Ser473 and Thr308 (Fig. 5), which are important for Akt activation. Cirazoline or TPA was unable to phosphorylate Akt on either residue at any time point examined (Fig. 5, A and B). Cirazoline also had no effect on the ability of insulin to phosphorylate Akt (Fig. 5C)
FIG. 5. Akt is phosphorylated on residues Thr308 and Ser473 after insulin (1 μM, 10 min), but not cirazoline (A; 10 μM, 5–120 min as indicated) or TPA (B; 100 nM, 5–120 min as indicated) stimulation. C, Cirazoline (10 μM, 10 min) has no effect on the ability of insulin (1 μM, 10 min) to phosphorylate Akt. Western blots are representative of four individual experiments.
Insulin and cirazoline phosphorylate Erk1/2 and p38 kinase in a transient manner in L6 cells
Several studies have proposed a potential role for Erk1/2 (26, 27, 28, 29) or p38 kinase (30, 31, 32, 33) in glucose uptake. We therefore examined whether cirazoline or insulin was able to phosphorylate either of these kinases in L6 cells. Cirazoline and insulin both led to a very marked increase in Erk1/2 phosphorylation (5- and 12-fold stimulation over basal levels, respectively; Fig. 6). The response to insulin was sustained over the time period examined, whereas the response to cirazoline was transient in nature. The Erk1/2 inhibitor PD98059 inhibited insulin- or cirazoline-mediated Erk1/2 phosphorylation (data not shown). Insulin was able to phosphorylate p38 kinase (2-fold) in a transient manner, whereas cirazoline phosphorylated p38 kinase after 15- and 30-min stimulation. The p38 kinase inhibitor SB202190 inhibited insulin- or cirazoline-mediated p38 kinase phosphorylation (data not shown). In all experiments examining phosphorylation of p38 kinase, glucose oxidase (50 mU, 30 min) was used as a positive control (Fig. 7).
FIG. 6. Time-response curve of Erk1/2 phosphorylation by either insulin (1 μM) or cirazoline (10 μM) in differentiated L6 cells. A, Graph of the ratio of phosphorylated Erk1/2 to total Erk1/2. Values represent the mean ± SEM obtained from five experiments preformed in singlicate. Data are presented as a percentage of the response to control cells at 0 min. B, Representative Western blot of Erk1/2 phosphorylation by insulin or cirazoline.
FIG. 7. p38 kinase is phosphorylated by either insulin (1 μM) or cirazoline (10 μM) in differentiated L6 cells (A and B) and increases p38 kinase activity after insulin (1 μM, 5 min) or cirazoline (10 μM, 15 min) treatment. A, Graph of the ratio of phosphorylated p38 to total p38 kinase. Values represent the mean ± SEM obtained from five experiments performed in singlicate. Data are presented as a percentage of the response to control cells at 0 min. B, Representative Western blot of p38 kinase phosphorylation by insulin or cirazoline [glucose oxidase (50 mU/ml, 30 min) was used as a positive control]. C, Representative Western blot of phosphorylated ATF-2 as a measure of p38 kinase activity as described in Materials and Methods. D, Graph of p38 kinase activity. Values represent the mean ± SEM obtained from three experiments performed in singlicate. Data are presented as a percentage of the response to control cells.
p38 kinase activity is increased after insulin or cirazoline stimulation in L6 cells
We measured p38 kinase activity after stimulation by insulin (5 min, 1 μM), or cirazoline (5 or 15 min, 10 μM). Insulin stimulated p38 kinase activity approximately 2.3-fold over basal levels. Cirazoline (15 min, but not 5 min) stimulated p38 kinase activity 1.6 times over basal (Fig. 7, C and D).
Elucidation of signaling molecules involved in cirazoline- or insulin-mediated glucose uptake
Phospholipase C.
1-Adrenoceptor activation increases phospholipase C activity (15), which has been linked to glucose uptake (16, 17, 18). Hence, we investigated whether phospholipase C is involved in 1-adrenergic or insulin-mediated increases in glucose uptake by use of the specific phospholipase C inhibitor U73122 (we have also used its inactive analog U73343). U73122, but not U73343, inhibited both cirazoline- and insulin-mediated glucose uptake (Fig. 8). This inhibition was complete in the presence of cirazoline, but was only partial in the presence of insulin. To mimic 1-adrenoceptor activation, we used the PKC activator TPA and the calcium ionophore A23187, which increases intracellular calcium levels. The phorbol ester TPA and the calcium ionophore A23187 significantly increased glucose uptake, and these effects were not inhibited by phospholipase C inhibition (Fig. 8), suggesting that phospholipase C, although downstream of the 1-adrenoceptor, is upstream of PKC and intracellular calcium with regard to glucose uptake.
FIG. 8. Effects of phospholipase C inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the phospholipase C inhibitor U73122 (1 μM, 5 min) or its inactive analog U73343 (1 μM, 5 min) before agonist stimulation. The histograms are the mean ± SEM of three to 12 experiments performed in duplicate. Asterisks represent values that are significantly different (***, P < 0.001) from samples of the agonists.
PI3K.
PI3K is a key kinase involved in insulin-mediated glucose uptake in both adipose tissue and skeletal muscle. To determine whether 1-adrenoceptor signaling increases glucose uptake through this kinase, we used a specific PI3K inhibitor, LY294002. Glucose uptake mediated by either cirazoline or insulin was inhibited by LY294002 (Fig. 9), although this inhibition was only partial with respect to insulin. Furthermore, LY294002 partially inhibited TPA- or A23187-mediated glucose uptake (Fig. 9).
FIG. 9. Effects of PI3K inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the PI3K inhibitor LY294002 (1 μM, 1 h) before agonist stimulation. The histograms are the mean ± SEM of six to nine experiments performed in duplicate. Asterisks represent values that are significantly different (**, P < 0.01; ***, P < 0.001) from samples of the agonists.
PKC.
PKC has been linked to glucose uptake (16, 17, 18) and is a key downstream mediator of insulin-mediated glucose uptake in both adipose tissue and skeletal muscle (34). We investigated whether PKCs are involved in 1-adrenoceptor-activated glucose uptake (1-adrenoceptors are capable of activating PKC) (15). We employed G?6983, which inhibits conventional (, ?, and ), novel (), and atypical () PKC isoforms. G?6983 markedly inhibited glucose uptake mediated by cirazoline and partially inhibited insulin-mediated glucose uptake (Fig. 9). The phorbol ester TPA, which activates conventional and novel PKC isoforms, increased glucose uptake, and this effect was totally abolished by G?6983 (glucose uptake mediated by A23187 was also inhibited by G?6983; Fig. 10). This indicates that PKCs are very important in 1-adrenoceptor- and insulin-mediated glucose uptake.
FIG. 10. Effects of PKC inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the PKC inhibitor G?6983 (1 μM, 5 min) before agonist stimulation. The histograms are the mean ± SEM of six to eight experiments performed in duplicate. Asterisks represent values that are significantly different (***, P < 0.001) from samples of the agonists.
MAPKs Erk1/2 and p38 kinase.
The MAPKs Erk1/2 and p38 kinase have been implicated in glucose uptake. In this study we investigated their potential roles. As shown in Fig. 6, both cirazoline and insulin were able to phosphorylate Erk1/2 in L6 cells. To assess whether Erk1/2 was involved in glucose uptake, we used the MAPK kinase 1/2 inhibitor PD98059. Glucose uptake in response to cirazoline, insulin, TPA, or A23187 was not affected by PD98059 (Fig. 11), suggesting that Erk1/2, although markedly phosphorylated by these treatments, is not implicated in glucose uptake. However, inhibition of p38 kinase by SB202190 partially inhibited glucose uptake in response to insulin, TPA, or A23187 and fully inhibited cirazoline-mediated glucose uptake (Fig. 11). This indicates a role for p38 kinase in glucose uptake by insulin or cirazoline (because both treatments are able to phosphorylate and activate p38 kinase in these cells).
FIG. 11. Effects of Erk1/2 or p38 kinase inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the Erk1/2 inhibitor PD98059 (10 μM, 1 h) or the p38 kinase inhibitor SB202190 (10 μM, 1 h) before agonist stimulation. The histograms are the mean ± SEM of five to seven experiments performed in duplicate. Asterisks represent values that are significantly different (**, P < 0.01; ***, P < 0.001) from samples of the agonists.
PKC and calcium mediate increases in glucose uptake in L6 cells
PKC has been implicated as an important mediator of insulin-stimulated glucose uptake (for review, see Ref.35), whereas the role for calcium is less established, with studies both for (36, 37) and against (38, 39) its involvement. In this study we have shown that the PKC activator TPA or the calcium ionophore A23187 significantly increased glucose uptake (Figs. 8–11). The magnitude of response to TPA was similar to that obtained with insulin, whereas the maximal response to A23187 was slightly more than that obtained with 1-adrenoceptor stimulation. This indicates that PKC and calcium can significantly increase glucose uptake in L6 cells. We were able to inhibit glucose uptake mediated by insulin, cirazoline, TPA, or A23187 by preincubating cells with the cell-permeable calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Table 4; but not with BAPTA; data not shown), suggesting that intracellular, not extracellular, calcium is vital to increases in glucose uptake. Basal glucose uptake was inhibited slightly by BAPTA-AM (32%), but its ability to inhibit glucose uptake mediated by insulin, cirazoline, TPA, or A23187 was very clear when the results were expressed as absolute differences. BAPTA-AM treatment was not detrimental to the cells.
TABLE 4. Effects of BAPTA-AM on glucose uptake in L6 cells
Diacylglycerol (DAG)-sensitive protein kinase Cs are not involved in insulin or cirazoline-mediated glucose uptake: role for atypical protein kinase C isoforms
TPA (100 nM, overnight) treatment down-regulates DAG-sensitive PKCs, and this method has been used to distinguish responses mediated though DAG-sensitive or -insensitive PKC isoforms (40). This treatment by itself had no significant effect on basal glucose uptake levels. TPA does not activate atypical PKC isoforms and therefore was used as a positive control to determine whether the treatment was effective. TPA-mediated glucose uptake was inhibited by TPA treatment overnight, whereas insulin- or cirazoline-mediated glucose uptake was not affected by TPA, indicating a role for DAG-insensitive PKCs (Fig. 12).
FIG. 12. 2-Deoxy-[3H]D-glucose uptake in differentiated L6 cells in response to cirazoline (10 μM), insulin (1 μM), or TPA (100 nM) after down-regulation of conventional PKCs (100 nM TPA, 16 h). The histograms are the mean ± SEM of four to eight experiments performed in duplicate.
Discussion
Insulin-stimulated glucose uptake is severely impaired in type II diabetics, and there is considerable interest in identifying insulin-independent mechanisms of glucose uptake. Numerous studies have focused on the potential of ?-adrenoceptor agonists (1, 2, 3, 4) as a potential treatment for obesity and type II diabetes. 1-Adrenoceptor effects on glucose uptake have been primarily limited to studies in white adipocytes (11, 12) and cardiac preparations (19, 41, 42, 43, 44). In skeletal muscle, 1-adrenoceptors have been identified (7, 8, 9), but only recently have their effects on glucose homeostasis been reported.
Role of -adrenoceptors in glucose uptake in skeletal muscle
We have previously shown that insulin or ?2-adrenoceptor stimulation mediates increases in glucose uptake in L6 cells (4). Interestingly, 1-adrenoceptor stimulation by the nonspecific -adrenoceptor agonist phenylephrine or the specific 1-adrenoceptor agonist cirazoline also increased glucose uptake in L6 cells (the 2-adrenoceptor agonist clonidine was without any stimulatory effect on glucose uptake). The maximal activation of glucose uptake by 1-adrenoceptor stimulation is substantial, but not as great as that obtained with either insulin or ?-adrenoceptor activation. The concentrations needed for 1- or ?2-adrenoceptor activation of glucose uptake are significantly different, with higher concentrations needed for the stimulatory effect by 1-adrenoceptors with the agonists used in this study. These high concentrations have been reported for phenylephrine in other studies performed in muscle cells (12, 42, 45). In addition, phenylephrine-mediated glucose uptake in this study was mediated by 1- and partly by ?-adrenoceptor activation. High concentrations of phenylephrine have previously been reported to have actions on ?-adrenoceptors (46, 47, 48). Cirazoline-mediated glucose uptake was not inhibited by ?-adrenoceptor blockade, but was inhibited with a 1-adrenoceptor antagonist, confirming a role for 1-adrenoceptors in glucose uptake in L6 cells (this is presumably through the 1A-adrenoceptor, which is the sole 1-adrenoceptor subtype expressed in L6 cells, as determined by RT-PCR). Because the effect of phenylephrine can be mediated partly by ?-adrenoceptor activation, all further studies used cirazoline. Isoprenaline-mediated glucose uptake was due solely to ?-adrenoceptor activation, as reported previously (4).
Elucidation of the signaling pathways mediating 1-adrenoceptor glucose uptake in L6 cells
Phospholipase C is important for 1-adrenoceptor-mediated glucose uptake.
Stimulation of 1-adrenoceptors results in the activation of both phospholipase C and PKC (15), and these proteins have been extensively linked to glucose uptake (16, 17, 18). In this study we investigated whether 1-adrenoceptor-mediated glucose uptake in L6 cells was mediated by phospholipase C using the inhibitor U73122 (49). 1-Adrenoceptor-mediated glucose uptake was fully inhibited by U73122 (but not by its inactive analog, U73343), indicating an important role for phospholipase C in cirazoline-mediated glucose uptake. Similarly, insulin-mediated glucose uptake was inhibited by phospholipase C inhibition, although the inhibition with U73122 was only partial. A role for insulin activation of phospholipase C in glucose uptake is less well studied. Insulin has been shown to activate phospholipase C in a PI3K-dependent manner (50), and inhibition of phospholipase C by U73122 inhibits insulin-mediated glucose uptake in other studies (18, 50, 51). Hence, our results show that insulin-mediated glucose uptake could be dependent, at least partially, upon phospholipase C. Because TPA- or A23187-mediated glucose uptake was not affected by phospholipase C inhibition, this suggests that PKC isoforms sensitive to TPA and calcium are downstream of phospholipase C.
PKC and calcium mediate glucose uptake by insulin or 1-adrenoceptor activation.
PKC has been studied extensively as a key mediator of insulin-stimulated glucose uptake (52), and a role for PKC in insulin-mediated glucose uptake has been established (53, 54, 55 ; for review, see Ref.35). However, there is still much debate regarding which isoforms of PKC are involved. Both insulin and TPA stimulate conventional (, ?1, ?2, and ) and novel ( and ) PKC isoforms, but atypical () PKC isoforms are stimulated by insulin and not by TPA (56, 57). Both insulin and TPA are potent activators of glucose uptake in L6 cells as is, to a lesser extent, the calcium ionophore A23187, implying that PKC and calcium are involved in glucose uptake. The role for intracellular calcium was also supported by studies using the membrane-permeable calcium chelator BAPTA-AM, which totally abolished glucose uptake mediated by insulin, cirazoline, TPA, or A23187. BAPTA, which is not cell permeable, had no effect on glucose uptake mediated by any of these agents (data not shown), suggesting that it is intracellular, not extracellular, calcium that is involved in glucose uptake.
We investigated whether PKCs are involved in 1-adrenoceptor-mediated glucose uptake in L6 cells using G?6983 (58), which is an inhibitor of all PKC isoforms. Glucose uptake mediated by TPA or A23187 was totally abolished by G?6983, whereas insulin-mediated glucose uptake was partially inhibited. The partial inhibition of insulin-stimulated glucose uptake by G?6983 indicates that PKC is involved in glucose uptake, but that other pathways must exist for insulin’s effect. 1-Adrenoceptor-mediated glucose uptake was fully inhibited by G?6983, indicating that PKC is very significant in 1-adrenoceptor-mediated glucose uptake.
A method to discriminate between conventional and novel PKCs from atypical PKCs is down-regulation of calcium/DAG-dependent PKCs (achieved with prolonged treatment of phorbol esters). TPA-mediated glucose uptake was inhibited approximately 70% by down-regulation of DAG-sensitive PKCs, indicating that this protocol was effective in down-regulating calcium/DAG isoforms of PKC (40). Down-regulation of these PKC isoforms did not inhibit cirazoline- or insulin-mediated glucose uptake, suggesting that calcium/DAG-dependent PKCs are not involved. This finding was consistent with other studies showing a noninvolvement of DAG-sensitive PKC in insulin-stimulated glucose uptake (39, 55, 59). Additional studies are needed to elucidate which atypical PKCs are involved.
PI3K is important for 1-adrenoceptor- and insulin-mediated glucose uptake.
PI3K is an important mediator of insulin-stimulated glucose uptake, and its activity is increased after insulin stimulation of L6 cells. Akt is rapidly and persistently activated by insulin in skeletal muscle (including L6 cells) and adipose tissue, and there is strong evidence to suggest that Akt is activated by a PI3K-dependent mechanism (reviewed in Ref.60). There is conflicting evidence for adrenergic activation of PI3K or Akt. ?1/?2-Adrenoceptor activation leads to Akt activation in a PI3K-dependent manner in cardiac myocytes (61, 62), whereas in Rat-1 fibroblasts and cardiac myocytes, 1-adrenoceptor activation has no effect on Akt activation (63, 64, 65, 66) and can inhibit insulin activation of Akt (65). 1-Adrenoceptor activation can also stimulate PI3K activity in NIH-3T3 cells (67). In this study cirazoline was able to activate PI3K, but was unable to phosphorylate Akt (or inhibit insulin activation of Akt), indicating a potential difference in the mechanisms of 1-adrenoceptor- and insulin-mediated glucose uptake as well as PI3K activation. It is presently unclear how cirazoline activates PI3K, which PI3K isoforms are responsible, and the physiological relevance of activating PI3K, but not Akt. In addition, we found that TPA increased PI3K activity similar to other reports for muscle and adipose tissue (40, 68, 69), but was unable to phosphorylate Akt.
Akt is a well recognized downstream target of PI3K, but although insulin was able to phosphorylate Akt in L6 cells, cirazoline was without effect, even though cirazoline-mediated glucose uptake was inhibited by LY294002. Other studies have shown, with the use of PI3K inhibitors such as LY294002 or wortmannin (70, 71, 72), a role for PI3K in 1-adrenoceptor glucose uptake (10, 19, 44). Our results suggest that PI3K activity is involved in glucose uptake mediated by cirazoline, because LY294002 totally inhibits this uptake, and cirazoline activates PI3K activity. This discrepancy in 1-adrenoceptors’ inability to activate Akt, but its ability to produce responses that are PI3K dependent is not new [such as 1-adrenoceptor activation of p70 S6 kinase 2 (63, 64, 66) or eukaryotic initiation factor 4E-binding protein 1 (64)] and should be further characterized.
To elucidate whether insulin and 1-adrenoceptor agonists use the same signaling pathways to stimulate glucose uptake, cells were exposed to cirazoline or isoprenaline in the presence of insulin. These responses were not additive, indicating that insulin, 1-adrenoceptors, and ?2-adrenoceptors use distinct pathways that probably overlap at the level of PI3K. One mechanism by which 1-adrenceptors may activate PI3K is through the release of G?-subunits after receptor stimulation, because class IB PI3Ks can be stimulated by G?-subunits (73). Gq proteins have been shown in 3T3-L1 adipocytes to associate with the insulin receptor, and this interaction was necessary for insulin-mediated GLUT4 translocation and glucose uptake (74). This interaction was upstream of PI3K (74) and may provide a basis for 1-adrenoceptor-mediated glucose uptake.
LY294002 was able to partially inhibit glucose uptake mediated by either TPA or the calcium ionophore A23187. This result was unexpected, because it is generally believed that PKC and calcium are downstream effectors of PI3K. Other reports show that glucose uptake mediated by phorbol esters can be inhibited by PI3K inhibitors (40, 57, 69). This discrepancy may be explained by findings in fat and muscle cells (and our results here) that phorbol esters are capable of activating PI3K (40, 57, 68, 69), although glucose uptake mediated by TPA is still mediated primarily through PKC as opposed to PI3K (57).
p38 kinase, but not Erk1/2, is involved in glucose uptake mediated by either insulin or 1-adrenoceptor activation.
We have shown that both insulin and cirazoline are able to phosphorylate Erk1/2. Classically Erk1/2 activation has been considered to be limited to activation by peptide growth factors acting via receptor tyrosine kinases (75, 76), but more recently it has been appreciated that G protein-coupled receptors, such as adrenoceptors, are capable of activating Erk1/2 in tissues important for glucose homeostasis, such as adipocytes (21, 77, 78, 79) and skeletal muscle (80). Because insulin or 1-adrenoceptor stimulation caused a rapid phosphorylation of Erk1/2, we investigated whether Erk1/2 may be involved in glucose uptake by use of the specific upstream kinase (MAPK kinase 1/2) inhibitor PD98059 (81). Glucose uptake mediated by insulin or cirazoline was not inhibited by PD98059, suggesting that Erk1/2 activity is not required for increases in glucose uptake, consistent with other studies (82, 83). It cannot be ruled out that Erk1/2 may be involved in other consequential actions of insulin and cirazoline, such as changes in mRNA and protein levels, because Erk1/2 is known to affect gene transcription.
Recently, there has been enormous focus on the potential role of p38 kinase in insulin-stimulated glucose uptake. There are reported discrepancies in the literature as to whether insulin is able to phosphorylate or activate p38 kinase itself, with several studies showing a positive action of insulin (31, 33) and others showing no effect (84, 85). Inhibition of p38 kinase by selective inhibitors such as SB202190 (86) inhibits insulin-mediated glucose uptake in skeletal muscle and adipose tissue (30, 31, 32, 33), although the mechanism of this is highly debated. Some studies postulate that p38 kinase activation may enhance GLUT4 intrinsic activity (30, 32), p38 kinase inhibitors (such as SB203580) are nonspecific and may act on Akt or GLUT4 itself (87), or that p38 kinase dephosphorylates intracellular pools of GLUT4 by a p38 kinase-dependent phosphatase to result in GLUT4 translocation (88). Our results indicate that insulin and cirazoline are able to phosphorylate and activate p38 kinase, and glucose uptake mediated by either agent is sensitive to p38 kinase inhibition. This supports a role for p38 kinase in glucose uptake in L6 cells.
Conclusions
We have demonstrated that 1-adrenoceptor activation increases glucose uptake in L6 muscle cells. We hypothesize that 1-adrenoceptor activation of glucose uptake is through activation of phospholipase C, which produces both DAG and inositol trisphosphate. This causes the release of intracellular calcium, which by some mechanism not yet clarified causes the activation of PI3K. Although DAG can then theoretically activate DAG-sensitive isoforms of PKC and increase glucose uptake, this is not involved in glucose uptake mediated by 1-adrenoceptor activation. 1-Adrenoceptor stimulation leads to activation of PI3K, but not phosphorylation of Akt. Even though activation of 1-adrenoceptors leads to some of the same signal transduction pathways via PI3K and atypical PKCs that are stimulated by insulin, it is likely that the differences between the 1-adrenoceptor and insulin pathways upstream could be exploited in the search for insulin-independent mechanisms of glucose uptake.
Acknowledgments
We gratefully acknowledge Dr. Bronwyn A. Evans (Monash University, Melbourne, Australia) for assistance in designing primers and PCR design, and Profs. Roger J. Summers (Monash University), Barbara Cannon, and Jan Nedergaard (Stockholm University, Stockholm, Sweden) for helpful discussions.
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Address all correspondence and requests for reprints to: Dr. Tore Bengtsson, Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratory F3, Stockholm University, SE 10691 Stockholm, Sweden. E-mail: tore.bengtsson@zoofys.su.se.
Abstract
The role of 1-adrenoceptor activation on glucose uptake in L6 cells was investigated. The 1-adrenoceptor agonist phenylephrine [pEC50 (–log10 EC50), 5.27 ± 0.30] or cirazoline (pEC50, 5.00 ± 0.23) increased glucose uptake in a concentration-dependent manner, as did insulin (pEC50, 7.16 ± 0.21). The 2-adrenoceptor agonist clonidine was without any stimulatory effect on glucose uptake. The stimulatory effect of cirazoline was inhibited by the 1-adrenoceptor antagonist prazosin, but not by the ?-adrenoceptor antagonist propranolol. RT-PCR showed that the 1A-adrenoceptor was the sole 1-adrenoceptor subtype expressed in L6 cells. Cirazoline- or insulin-mediated glucose uptake was inhibited by the phosphatidylinositol-3 kinase inhibitor LY294002, suggesting a possible interaction between the 1-adrenoceptor and insulin pathways. Cirazoline or insulin stimulated phosphatidylinositol-3 kinase activity, but 1-adrenoceptor activation did not phosphorylate Akt. Both cirazoline- and insulin-mediated glucose uptake were inhibited by protein kinase C (PKC), phospholipase C, and p38 kinase inhibitors, but not by Erk1/2 inhibitors (despite both treatments being able to phosphorylate Erk1/2). Insulin and cirazoline were able to activate and phosphorylate p38 kinase. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the calcium ionophore A23187 produced significant increases in glucose uptake, indicating roles for PKC and calcium in glucose uptake. Down-regulation of conventional PKC isoforms inhibited glucose uptake mediated by 12-O-tetradecanoylphorbol-13-acetate, but not by insulin or cirazoline. This study demonstrates that 1-adrenoceptors mediate increases in glucose uptake in L6 muscle cells. This effect appears to be related to activation of phospholipase C, phosphatidylinositol-3 kinase, p38 kinase, and PKC.
Introduction
A RELATIVELY LITTLE-studied response to adrenoceptor activation is facilitation of glucose uptake. Adrenoceptors are classified into three main subtypes: 1-, 2-, and ?-adrenoceptors. ?-Adrenoceptor stimulation increases glucose uptake in rodent skeletal muscle (1, 2, 3, 4) and brown adipose tissue (5, 6). This effect is mediated primarily by the ?3-adrenoceptor in brown adipose tissue (5, 6), but is mediated by ?2-adrenoceptors in skeletal muscle cells (4). There is relatively little known about the presence/effect of 1-adrenoceptors in skeletal muscle or about -adrenoceptor-mediated regulation of glucose uptake.
1-Adrenoceptors are located in a large number of tissues from a large number of species. In the central nervous system, they perform excitatory functions and perform numerous functions in the periphery (contraction of vascular and nonvascular smooth muscle, relaxation of gastrointestinal smooth muscle, positive inotropic effects in the heart, and hepatic glycogenolysis). Previous studies showed the presence of 1-adrenoceptors in rat skeletal muscle through quantitative autoradiography (7), ribonuclease protection assays (8), and radioligand binding (9) studies. Recent reports indicate that 1-adrenoceptors may modulate glucose uptake in the mouse skeletal muscle C2C12 cell line (10) and rat white adipose tissue (11, 12). However, the C2C12 cell line is probably not the best model to study mechanisms of insulin-stimulated glucose uptake in muscle, because these cells have been reported to be insulin unresponsive (or have a very small effect on glucose uptake) due to a postulated defective postreceptor signaling pathway (13), suggested to be a lack of an insulin-responsive vesicular glucose transporter 4 (GLUT4) compartment (14). L6 cells represent a better model for glucose uptake because they have been used extensively to elucidate the mechanisms of glucose uptake in muscle, have an intact insulin signaling pathway, and express the insulin-sensitive GLUT4.
The intracellular mechanisms involved in adrenergically mediated glucose uptake in skeletal muscle are still unclear. ?-Adrenoceptors are Gs-coupled receptors, and their activation results in the production of cAMP, although cAMP may not be needed for ?-adrenoceptor stimulation of glucose uptake (4). An earlier report (3) indicated that in L6 muscle cells, insulin and ?2-adrenoceptors increase glucose uptake by two distinct mechanisms, but a recent report (4) indicates that insulin and ?2-adrenoceptors use two pathways for increases in glucose uptake, but these pathways overlap, probably at the level of phosphatidylinositol 3-kinase (PI3K). 1-Adrenoceptors are Gq-coupled receptors. Their activation leads to phospholipase C activation, resulting in the hydrolysis of phosphatidylinositol(4, 5)-bisphosphate to produce inositol-1,4,5-phosphate, which releases calcium from intracellular stores, and diacylglycerol, which can activate protein kinase C (PKC) (15). The phospholipase C-PKC pathway has been linked to glucose uptake (16, 17, 18), but there is little evidence for 1-adrenoceptor mediation of glucose uptake through this pathway. Lipids in the phosphatidylinositol(4, 5)-bisphosphate pathway can be used as substrates for PI3K (a kinase important for insulin’s effects on glucose uptake), and 1-adrenoceptor-mediated glucose uptake in white adipose tissue and heart is mediated through PI3K (12, 19).
The present study investigated the -adrenergic control of glucose uptake in L6 muscle cells. We show that 1-adrenoceptor, but not 2-adrenoceptor, stimulation results in increases in glucose uptake in L6 cells through a mechanism dependent upon phospholipase C, PI3K, p38 kinase, and PKC. The actions of insulin and 1-adrenoceptors on glucose uptake use two pathways, but converge at the level of PKC.
Materials and Methods
Cell culture
Rat L6 cells (obtained from American Type Culture Collection, Manassas, VA) were grown as a monolayer in DMEM (1 g/liter glucose) containing GlutaMAX (Invitrogen Life Technologies, Inc., Carlsbad, CA), 10% (vol/vol) fetal bovine serum, 10 mM HEPES buffer, fungizone (2.5 μg/ml), and gentamicin sulfate (80 μg/ml) under 8% CO2 at 37 C. To differentiate, cells were allowed to reach confluence and the media changed to that containing 2% (vol/vol) fetal bovine serum for 7 d, with medium changes every second day. Experiments were restricted to cells from passages 2–15, and undifferentiated cells were not allowed to grow more than 60–70% confluence.
RNA isolation and determination of 1-adrenoceptor mRNA levels
RNA was extracted from L6 cells after 7 d differentiation, and from atria, ventricle, liver, brain, soleus muscle or prostate from one male Sprague Dawley rat (280 g; anesthetized with 20% O2/80% CO2 and decapitated). This was performed with ethical permission from the North Stockholm animal ethics committee. Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer’s protocol. The yield and quality of RNA was assessed by measuring absorbance at 260 and 280 nm and electrophoresis on 1.3% agarose gels. There was no degradation of any RNA samples.
cDNAs were synthesized by RT of 1 μg of each total RNA using oligo(deoxythymidine)15 (Invitrogen Life Technologies, Inc.) as a primer (20). PCR amplification was carried out on cDNA equivalent to 100 ng starting RNA using primers specific for ?-actin or 1A-, 1B-, or 1D-adrenoceptor, which amplify 559-, 404-, 452-, and 513-bp fragments, respectively (Invitrogen Life Technologies, Inc.; see Table 1). All primers were intron-spanning to eliminate possible contamination by genomic DNA. Reactions were carried out in a Primus 96 Plus thermocycler (MWG Biotech AG, Ebersberg, Germany). For 1A-adrenoceptor or ?-actin PCR, PCR mixes contained cDNA, 1 U Taq DNA polymerase (Invitrogen Life Technologies, Inc.), 1x PCR buffer, 200 μM deoxy-NTPs (Amersham Biosciences), 2 mM Mg-acetate, forward primer (2.8 pmol 1A-adrenoceptor or 1.5 pmol ?-actin) and reverse primer (2.8 pmol 1A-adrenoceptor or 1.5 pmol ?-actin). For 1D-adrenoceptor PCR, PCR mixes contained cDNA, 0.5 U Platinum Taq DNA polymerase (Invitrogen Life Technologies, Inc.), 1x AMP buffer, and 1x Enhancer solution (supplied by Invitrogen Life Technologies, Inc.), deoxy-NTPs (130 μM), MgSO4 (1.5 mM), 5.8 pmol forward primer, and 5.8 pmol reverse primer. For 1B-adrenoceptor PCR, PCR mixes were the same as for 1D-adrenoceptor PCR, except no Enhancer solution was added. The annealing temperature for all PCRs was 64 C, and 30 cycles were performed (16 for ?-actin). After amplification, PCR products were electrophoresed on 1.3% agarose gels and visualized.
TABLE 1. Oligonucleotides used as PCR primers
Immunoblotting
Cells were serum-starved overnight before each experiment on d 7 and were exposed to drugs for the times and concentrations indicated with the data. Extraction of cells and subsequent Western blotting were performed as previously described (21), except samples were electrotransferred to Hybond-C Extra nitrocellulose membranes (pore size, 0.45 μm; Amersham Biosciences, Arlington Heights, IL) for Erk1/2 studies or Hybond-P polyvinylidene difluoride membranes (pore size, 0.45 μm; Amersham Biosciences) for Akt or p38 kinase studies. The primary antibodies used were p44/42 MAPK, phospho-p44/42 MAPK (Thr202/Tyr204), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), Akt, phospho-Akt (Ser473), or phospho-Akt (Thr308) antibody diluted 1:1000, which were detected using a secondary antibody (horseradish peroxidase-linked antirabbit IgG) diluted 1:2000 and enhanced chemiluminescence (ECL, Amersham Biosciences).
2-Deoxy-[3H]D-glucose uptake assay
Glucose uptake was measured using the 2-deoxy-[3H]D-glucose method (3) with minor modifications. Briefly, cells were serum-stared overnight before each experiment and glucose uptake was measured on d 7. Medium was replaced in the morning (serum-free medium), and cells were exposed to drugs for 2–2.5 h. Cells were washed twice in warm PBS before media and drugs were placed in DMEM devoid of glucose for 20 min. 2-Deoxy-[3H]D-glucose (50 nM) was added for 15 min at 37 C, and the reactions were terminated by washing twice in ice-cold PBS. Cells were digested in 0.2 M NaOH for 1 h at 60 C, and samples were transferred to scintillation vials with scintillant and allowed to sit at room temperature for 1 h before being counted. When inhibitors were used, the time indicated with the results represents the time cells were preequilibrated with the inhibitors before agonists were added.
Immunoprecipitation of PI3K and ELISA for detection of PI3K activity
L6 cells (d 7) were serum-starved overnight before cells were treated with drugs for 10 min. The cells were washed three times with ice-cold buffer A [137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM CaCl2, 1 mM MgCl2, and 0.1 mM sodium orthovanadate] before solubilization for 20 min at 4 C in lysis buffer [buffer A containing 1% (vol/vol) Nonidet P-40, and 1 mM phenylmethylsulfonylfluoride]. After centrifugation at 10,000 x g for 10 min at 4 C, the supernatant (cell lysates) were incubated for 1 h at 4 C with anti-PI3K p85 antibody (5 μl antibody/sample), followed by addition of 60 μl of a 50% slurry of protein A-agarose beads in PBS for 1 h at 4 C with mixing. The beads were washed three times in wash buffer 1 [buffer A containing 1% (vol/vol) Nonidet P-40], three times with wash buffer 2 [0.1 M Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium orthovanadate], and twice in wash buffer 3 [10 mM Tris-HCl (pH 7.4), 150 mM LiCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate].
PI3K activity was measured in vitro using a competitive ELISA format (Echelon Biosciences, Inc., Salt Lake City, UT) according to the manufacturer’s instructions. Briefly, the bead-bound immunoprecipitated enzyme was incubated with phosphatidylinositol (4, 5)-biphosphate [PI(4, 5)P2] substrate (100 pmol) in kinase reaction buffer [4 mM MgCl2, 20 mM Tris (pH 7.4), 10 mM NaCl, and 25 μM ATP] for 2 h at room temperature with shaking. The supernatant was then incubated with a PI(3, 4, 5)P3 detector protein for 1 h at room temperature, and the reaction mixes were transferred to PI(3, 4, 5)P3-coated detection plates for 1 h at room temperature. After washing in wash buffer [150 mM NaCl, 10 mM Tris (pH 7.5), and 0.05% (vol/vol) Tween 20], secondary detection reagent (supplied with the kit) was added, plates were washed again, developing solution (supplied with the kit) was added, and PI(3, 4, 5)P3 detector protein binding to the plate was determined by measuring the absorbance at 450 nm.
Measurement of p38 kinase activity
L6 cells (d 7) were serum-starved for 4 h before cells were treated with drugs as indicated with the data. Preparation of cell lysates and immunoprecipitation of phospho-p38 MAPK (Thr180/Tyr184) were performed, and subsequent p38 kinase activity was measured using a nonradioactive p38 MAPK assay kit (Cell Signaling Technology, Beverly, MA) according to the manufacturer’s detailed instructions. The assay is based on the ability of an immobilized phospho-p38 MAPK (Thr180/Tyr182) monoclonal antibody to immunoprecipitate p38 MAPK, followed by an in vitro kinase assay using activating transcription factor-2 (ATF-2) as a substrate. ATF-2 phosphorylation is assessed by immunoblotting using a phospho-ATF-2 (Thr71) antibody.
Statistical analysis
Immunoblotting results are expressed in a graph format as the ratio between phosphorylated and total protein, with the ratio normalized in each experiment to that of control samples. All experiments were performed in singlicate or duplicate; n refers to the number of independent experiments performed.
For glucose uptake experiments, all experiments were performed in duplicate and expressed as the mean ± SEM of n independent experiments. Data were analyzed using nonlinear curve fitting (PRISM version 3.03, GraphPad, Inc., San Diego, CA) to obtain pEC50 (–log10 EC50) values, where appropriate, and statistical significance was determined by t test where appropriate. P 0.05 was considered significant.
For PI3K assay results, the activity of PI3K was expressed as a percentage of the activity measured in control treated cells as suggested by the manufacturer (Echelon Biosciences, Inc.). For p38 kinase assay results, p38 kinase activity was expressed as a percentage of the activity measured in control treated cells.
Drugs and reagents
Drugs and reagents were purchased as follows: 2-deoxy-[3H]D-glucose (12 Ci/mmol; Amersham Biosciences, Little Chalfont, UK); G?6973, Ro-318220, SB202190, U73122, and U73343 (Calbiochem, La Jolla, CA); PD98059 (Cell Signaling Technology, Beverly, MA); insulin (Actrapid, Novo Nordisk, Bagsvaerd, Denmark); A23187, cirazoline, (±)-isoprenaline, LY294002, (–)-phenylephrine, phenylmethylsulfonylfluoride, prazosin, (–)-propranolol, 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich Co., St. Louis, MO).
All cell culture medium and supplements were obtained from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). All antibodies, except anti-PI3K p85 (Upstate Biotechnology, Inc., Lake Placid, NY) were obtained from Cell Signaling Technology. All other drugs and reagents were of analytical grade.
Results
Measurement of 1-adrenoceptor subtype mRNA expression
RT-PCR experiments were conducted to examine which subtype(s) of the 1-adrenoceptor are expressed in differentiated L6 cells. 1A-Adrenoceptor mRNA was expressed at moderate levels compared with prostate or brain (Fig. 1). 1A-Adrenoceptor mRNA from rat soleus muscle was also detectable, consistent with the findings of another study (8). No 1B- or 1D-adrenoceptor mRNA was detected in differentiated L6 cells, although 1B-adrenoceptor mRNA was readily detectable in liver and ventricle, and 1D-adrenoceptor mRNA was readily detectable in brain and atria.
FIG. 1. 1A- (404 bp), 1B- (452 bp), and 1D-adrenoceptor (513 bp) PCR products from rat prostate, brain, liver, ventricle, soleus muscle and atria, and 1A-adrenoceptor PCR products from L6 myotubes. Levels of expression were measured using RT-PCR analysis. Also shown are ?-actin (559 bp) PCR products.
Effects of insulin and adrenergic agonists on glucose uptake in L6 cells
Concentration-response curves to adrenergic agonists were constructed to determine whether -adrenergic agonists were capable of increasing glucose uptake in differentiated L6 cells (Fig. 2). Insulin, the nonselective ?-adrenoceptor agonist (±)-isoprenaline, the nonselective -adrenoceptor agonist phenylephrine, and the 1-adrenoceptor agonist cirazoline stimulated glucose uptake (Fig. 2 and Table 2). Maximal glucose uptake responses to either cirazoline or phenylephrine were significantly lower than glucose uptake mediated by either (±)-isoprenaline or insulin. The 2-adrenoceptor agonist clonidine was without effect (n = 5). Insulin and (±)-isoprenaline produced the same degree of maximum response of glucose uptake in these cells, consistent with our previous study (4).
FIG. 2. Concentration-response curve for insulin or the ?-adrenoceptor agonist (±)-isoprenaline (A) and for the -adrenoceptor agonist phenylephrine, the 1-adrenoceptor agonist cirazoline, or the 2-adrenoceptor agonist clonidine (B) on 2-deoxy-[3H]D-glucose uptake in differentiated L6 cells. Points show means, and vertical lines indicate the SEM of five to 10 experiments performed in duplicate.
TABLE 2. Effects of adrenergic agonists on glucose uptake in L6 cells
Inhibition of adrenergically mediated glucose uptake by propranolol or prazosin
To determine whether the effects of isoprenaline or cirazoline were mediated by -adrenoceptor or ?-adrenoceptor stimulation, cells were preincubated with either the 1-adrenoceptor antagonist prazosin or the ?-adrenoceptor antagonist (–)-propranolol (Fig. 3). Isoprenaline-mediated glucose uptake was inhibited by propranolol, but not by prazosin, confirming that its effects on glucose uptake are mediated by ?-adrenoceptor stimulation. Conversely, cirazoline-mediated glucose uptake was inhibited by prazosin, but not by propranolol, confirming 1-adrenoceptor stimulation. Phenylephrine (10 μM), a conventional -adrenoceptor agonist, stimulated glucose uptake (basal glucose uptake, 100%; phenylephrine, 141 ± 5%; n = 5), but its effects, although totally inhibited by prazosin [prazosin (1 μM), 108 ± 4%; plus phenylephrine, 114 ± 6%; n = 5], were also partly inhibited by propranolol [propranolol (1 μM), 103 ± 5%; plus phenylephrine, 122 ± 6%; n = 5], indicating that its actions are mediated by both - and ?-adrenoceptor stimulation; hence, investigation of 1-adrenoceptor-mediated glucose uptake was performed using cirazoline as the agonist. Preincubation of cells with either propranolol or prazosin did not significantly affect basal glucose uptake.
FIG. 3. Glucose uptake in differentiated L6 cells in response to isoprenaline (10 μM; A) or cirazoline (10 μM; B) in the absence or presence of either propranolol (1 μM, 1 h) or prazosin (1 μM, 1 h). The histograms are the mean ± SEM of six experiments performed in duplicate.
Effect of combined insulin and adrenergic agonists on glucose uptake
To examine the potential interaction between insulin and either 1-adrenceptor or ?-adrenoceptor agonists on glucose uptake, cells were incubated with cirazoline (10 μM) or (±)-isoprenaline (10 μM) in the absence or presence of insulin (1 μM; Table 3). There was no additive increase in insulin-mediated glucose uptake when cells were coincubated with either cirazoline or isoprenaline.
TABLE 3. Effects of insulin on adrenergically stimulated glucose uptake in L6 cells
PI3K activity is increased by insulin, TPA, or cirazoline in L6 cells
PI3K activity is increased after insulin receptor stimulation and is thought to be a downstream mediator of glucose uptake. We measured PI3K activity after 10 min of stimulation by insulin (1 μM), cirazoline (10 μM), or TPA (100 nM). Insulin stimulated PI3K activity approximately 2.6-fold over basal levels. TPA stimulated PI3K activity 2-fold, and cirazoline 1.7-fold over basal levels (Fig. 4).
FIG. 4. PI3K activity is increased after insulin (1 μM, 10 min), TPA (100 nM, 10 min), or cirazoline (10 μM, 10 min) stimulation of L6 cells (n = 4).
Insulin, but not cirazoline or TPA, phosphorylates Akt at both Ser473 and Thr308 in L6 cells
Akt is activated after insulin receptor stimulation (22) and is implicated in insulin-mediated glucose uptake in adipocytes and muscle (23, 24, 25). Hence, we examined whether cirazoline is capable of activating Akt in L6 cells. Insulin phosphorylated Akt on residues Ser473 and Thr308 (Fig. 5), which are important for Akt activation. Cirazoline or TPA was unable to phosphorylate Akt on either residue at any time point examined (Fig. 5, A and B). Cirazoline also had no effect on the ability of insulin to phosphorylate Akt (Fig. 5C)
FIG. 5. Akt is phosphorylated on residues Thr308 and Ser473 after insulin (1 μM, 10 min), but not cirazoline (A; 10 μM, 5–120 min as indicated) or TPA (B; 100 nM, 5–120 min as indicated) stimulation. C, Cirazoline (10 μM, 10 min) has no effect on the ability of insulin (1 μM, 10 min) to phosphorylate Akt. Western blots are representative of four individual experiments.
Insulin and cirazoline phosphorylate Erk1/2 and p38 kinase in a transient manner in L6 cells
Several studies have proposed a potential role for Erk1/2 (26, 27, 28, 29) or p38 kinase (30, 31, 32, 33) in glucose uptake. We therefore examined whether cirazoline or insulin was able to phosphorylate either of these kinases in L6 cells. Cirazoline and insulin both led to a very marked increase in Erk1/2 phosphorylation (5- and 12-fold stimulation over basal levels, respectively; Fig. 6). The response to insulin was sustained over the time period examined, whereas the response to cirazoline was transient in nature. The Erk1/2 inhibitor PD98059 inhibited insulin- or cirazoline-mediated Erk1/2 phosphorylation (data not shown). Insulin was able to phosphorylate p38 kinase (2-fold) in a transient manner, whereas cirazoline phosphorylated p38 kinase after 15- and 30-min stimulation. The p38 kinase inhibitor SB202190 inhibited insulin- or cirazoline-mediated p38 kinase phosphorylation (data not shown). In all experiments examining phosphorylation of p38 kinase, glucose oxidase (50 mU, 30 min) was used as a positive control (Fig. 7).
FIG. 6. Time-response curve of Erk1/2 phosphorylation by either insulin (1 μM) or cirazoline (10 μM) in differentiated L6 cells. A, Graph of the ratio of phosphorylated Erk1/2 to total Erk1/2. Values represent the mean ± SEM obtained from five experiments preformed in singlicate. Data are presented as a percentage of the response to control cells at 0 min. B, Representative Western blot of Erk1/2 phosphorylation by insulin or cirazoline.
FIG. 7. p38 kinase is phosphorylated by either insulin (1 μM) or cirazoline (10 μM) in differentiated L6 cells (A and B) and increases p38 kinase activity after insulin (1 μM, 5 min) or cirazoline (10 μM, 15 min) treatment. A, Graph of the ratio of phosphorylated p38 to total p38 kinase. Values represent the mean ± SEM obtained from five experiments performed in singlicate. Data are presented as a percentage of the response to control cells at 0 min. B, Representative Western blot of p38 kinase phosphorylation by insulin or cirazoline [glucose oxidase (50 mU/ml, 30 min) was used as a positive control]. C, Representative Western blot of phosphorylated ATF-2 as a measure of p38 kinase activity as described in Materials and Methods. D, Graph of p38 kinase activity. Values represent the mean ± SEM obtained from three experiments performed in singlicate. Data are presented as a percentage of the response to control cells.
p38 kinase activity is increased after insulin or cirazoline stimulation in L6 cells
We measured p38 kinase activity after stimulation by insulin (5 min, 1 μM), or cirazoline (5 or 15 min, 10 μM). Insulin stimulated p38 kinase activity approximately 2.3-fold over basal levels. Cirazoline (15 min, but not 5 min) stimulated p38 kinase activity 1.6 times over basal (Fig. 7, C and D).
Elucidation of signaling molecules involved in cirazoline- or insulin-mediated glucose uptake
Phospholipase C.
1-Adrenoceptor activation increases phospholipase C activity (15), which has been linked to glucose uptake (16, 17, 18). Hence, we investigated whether phospholipase C is involved in 1-adrenergic or insulin-mediated increases in glucose uptake by use of the specific phospholipase C inhibitor U73122 (we have also used its inactive analog U73343). U73122, but not U73343, inhibited both cirazoline- and insulin-mediated glucose uptake (Fig. 8). This inhibition was complete in the presence of cirazoline, but was only partial in the presence of insulin. To mimic 1-adrenoceptor activation, we used the PKC activator TPA and the calcium ionophore A23187, which increases intracellular calcium levels. The phorbol ester TPA and the calcium ionophore A23187 significantly increased glucose uptake, and these effects were not inhibited by phospholipase C inhibition (Fig. 8), suggesting that phospholipase C, although downstream of the 1-adrenoceptor, is upstream of PKC and intracellular calcium with regard to glucose uptake.
FIG. 8. Effects of phospholipase C inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the phospholipase C inhibitor U73122 (1 μM, 5 min) or its inactive analog U73343 (1 μM, 5 min) before agonist stimulation. The histograms are the mean ± SEM of three to 12 experiments performed in duplicate. Asterisks represent values that are significantly different (***, P < 0.001) from samples of the agonists.
PI3K.
PI3K is a key kinase involved in insulin-mediated glucose uptake in both adipose tissue and skeletal muscle. To determine whether 1-adrenoceptor signaling increases glucose uptake through this kinase, we used a specific PI3K inhibitor, LY294002. Glucose uptake mediated by either cirazoline or insulin was inhibited by LY294002 (Fig. 9), although this inhibition was only partial with respect to insulin. Furthermore, LY294002 partially inhibited TPA- or A23187-mediated glucose uptake (Fig. 9).
FIG. 9. Effects of PI3K inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the PI3K inhibitor LY294002 (1 μM, 1 h) before agonist stimulation. The histograms are the mean ± SEM of six to nine experiments performed in duplicate. Asterisks represent values that are significantly different (**, P < 0.01; ***, P < 0.001) from samples of the agonists.
PKC.
PKC has been linked to glucose uptake (16, 17, 18) and is a key downstream mediator of insulin-mediated glucose uptake in both adipose tissue and skeletal muscle (34). We investigated whether PKCs are involved in 1-adrenoceptor-activated glucose uptake (1-adrenoceptors are capable of activating PKC) (15). We employed G?6983, which inhibits conventional (, ?, and ), novel (), and atypical () PKC isoforms. G?6983 markedly inhibited glucose uptake mediated by cirazoline and partially inhibited insulin-mediated glucose uptake (Fig. 9). The phorbol ester TPA, which activates conventional and novel PKC isoforms, increased glucose uptake, and this effect was totally abolished by G?6983 (glucose uptake mediated by A23187 was also inhibited by G?6983; Fig. 10). This indicates that PKCs are very important in 1-adrenoceptor- and insulin-mediated glucose uptake.
FIG. 10. Effects of PKC inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the PKC inhibitor G?6983 (1 μM, 5 min) before agonist stimulation. The histograms are the mean ± SEM of six to eight experiments performed in duplicate. Asterisks represent values that are significantly different (***, P < 0.001) from samples of the agonists.
MAPKs Erk1/2 and p38 kinase.
The MAPKs Erk1/2 and p38 kinase have been implicated in glucose uptake. In this study we investigated their potential roles. As shown in Fig. 6, both cirazoline and insulin were able to phosphorylate Erk1/2 in L6 cells. To assess whether Erk1/2 was involved in glucose uptake, we used the MAPK kinase 1/2 inhibitor PD98059. Glucose uptake in response to cirazoline, insulin, TPA, or A23187 was not affected by PD98059 (Fig. 11), suggesting that Erk1/2, although markedly phosphorylated by these treatments, is not implicated in glucose uptake. However, inhibition of p38 kinase by SB202190 partially inhibited glucose uptake in response to insulin, TPA, or A23187 and fully inhibited cirazoline-mediated glucose uptake (Fig. 11). This indicates a role for p38 kinase in glucose uptake by insulin or cirazoline (because both treatments are able to phosphorylate and activate p38 kinase in these cells).
FIG. 11. Effects of Erk1/2 or p38 kinase inhibition on insulin (A; 1 μM), cirazoline (B; 10 μM), TPA (C; 100 nM), or A23187 (D; 100 nM)-induced glucose uptake. Cells were pretreated with the Erk1/2 inhibitor PD98059 (10 μM, 1 h) or the p38 kinase inhibitor SB202190 (10 μM, 1 h) before agonist stimulation. The histograms are the mean ± SEM of five to seven experiments performed in duplicate. Asterisks represent values that are significantly different (**, P < 0.01; ***, P < 0.001) from samples of the agonists.
PKC and calcium mediate increases in glucose uptake in L6 cells
PKC has been implicated as an important mediator of insulin-stimulated glucose uptake (for review, see Ref.35), whereas the role for calcium is less established, with studies both for (36, 37) and against (38, 39) its involvement. In this study we have shown that the PKC activator TPA or the calcium ionophore A23187 significantly increased glucose uptake (Figs. 8–11). The magnitude of response to TPA was similar to that obtained with insulin, whereas the maximal response to A23187 was slightly more than that obtained with 1-adrenoceptor stimulation. This indicates that PKC and calcium can significantly increase glucose uptake in L6 cells. We were able to inhibit glucose uptake mediated by insulin, cirazoline, TPA, or A23187 by preincubating cells with the cell-permeable calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM; Table 4; but not with BAPTA; data not shown), suggesting that intracellular, not extracellular, calcium is vital to increases in glucose uptake. Basal glucose uptake was inhibited slightly by BAPTA-AM (32%), but its ability to inhibit glucose uptake mediated by insulin, cirazoline, TPA, or A23187 was very clear when the results were expressed as absolute differences. BAPTA-AM treatment was not detrimental to the cells.
TABLE 4. Effects of BAPTA-AM on glucose uptake in L6 cells
Diacylglycerol (DAG)-sensitive protein kinase Cs are not involved in insulin or cirazoline-mediated glucose uptake: role for atypical protein kinase C isoforms
TPA (100 nM, overnight) treatment down-regulates DAG-sensitive PKCs, and this method has been used to distinguish responses mediated though DAG-sensitive or -insensitive PKC isoforms (40). This treatment by itself had no significant effect on basal glucose uptake levels. TPA does not activate atypical PKC isoforms and therefore was used as a positive control to determine whether the treatment was effective. TPA-mediated glucose uptake was inhibited by TPA treatment overnight, whereas insulin- or cirazoline-mediated glucose uptake was not affected by TPA, indicating a role for DAG-insensitive PKCs (Fig. 12).
FIG. 12. 2-Deoxy-[3H]D-glucose uptake in differentiated L6 cells in response to cirazoline (10 μM), insulin (1 μM), or TPA (100 nM) after down-regulation of conventional PKCs (100 nM TPA, 16 h). The histograms are the mean ± SEM of four to eight experiments performed in duplicate.
Discussion
Insulin-stimulated glucose uptake is severely impaired in type II diabetics, and there is considerable interest in identifying insulin-independent mechanisms of glucose uptake. Numerous studies have focused on the potential of ?-adrenoceptor agonists (1, 2, 3, 4) as a potential treatment for obesity and type II diabetes. 1-Adrenoceptor effects on glucose uptake have been primarily limited to studies in white adipocytes (11, 12) and cardiac preparations (19, 41, 42, 43, 44). In skeletal muscle, 1-adrenoceptors have been identified (7, 8, 9), but only recently have their effects on glucose homeostasis been reported.
Role of -adrenoceptors in glucose uptake in skeletal muscle
We have previously shown that insulin or ?2-adrenoceptor stimulation mediates increases in glucose uptake in L6 cells (4). Interestingly, 1-adrenoceptor stimulation by the nonspecific -adrenoceptor agonist phenylephrine or the specific 1-adrenoceptor agonist cirazoline also increased glucose uptake in L6 cells (the 2-adrenoceptor agonist clonidine was without any stimulatory effect on glucose uptake). The maximal activation of glucose uptake by 1-adrenoceptor stimulation is substantial, but not as great as that obtained with either insulin or ?-adrenoceptor activation. The concentrations needed for 1- or ?2-adrenoceptor activation of glucose uptake are significantly different, with higher concentrations needed for the stimulatory effect by 1-adrenoceptors with the agonists used in this study. These high concentrations have been reported for phenylephrine in other studies performed in muscle cells (12, 42, 45). In addition, phenylephrine-mediated glucose uptake in this study was mediated by 1- and partly by ?-adrenoceptor activation. High concentrations of phenylephrine have previously been reported to have actions on ?-adrenoceptors (46, 47, 48). Cirazoline-mediated glucose uptake was not inhibited by ?-adrenoceptor blockade, but was inhibited with a 1-adrenoceptor antagonist, confirming a role for 1-adrenoceptors in glucose uptake in L6 cells (this is presumably through the 1A-adrenoceptor, which is the sole 1-adrenoceptor subtype expressed in L6 cells, as determined by RT-PCR). Because the effect of phenylephrine can be mediated partly by ?-adrenoceptor activation, all further studies used cirazoline. Isoprenaline-mediated glucose uptake was due solely to ?-adrenoceptor activation, as reported previously (4).
Elucidation of the signaling pathways mediating 1-adrenoceptor glucose uptake in L6 cells
Phospholipase C is important for 1-adrenoceptor-mediated glucose uptake.
Stimulation of 1-adrenoceptors results in the activation of both phospholipase C and PKC (15), and these proteins have been extensively linked to glucose uptake (16, 17, 18). In this study we investigated whether 1-adrenoceptor-mediated glucose uptake in L6 cells was mediated by phospholipase C using the inhibitor U73122 (49). 1-Adrenoceptor-mediated glucose uptake was fully inhibited by U73122 (but not by its inactive analog, U73343), indicating an important role for phospholipase C in cirazoline-mediated glucose uptake. Similarly, insulin-mediated glucose uptake was inhibited by phospholipase C inhibition, although the inhibition with U73122 was only partial. A role for insulin activation of phospholipase C in glucose uptake is less well studied. Insulin has been shown to activate phospholipase C in a PI3K-dependent manner (50), and inhibition of phospholipase C by U73122 inhibits insulin-mediated glucose uptake in other studies (18, 50, 51). Hence, our results show that insulin-mediated glucose uptake could be dependent, at least partially, upon phospholipase C. Because TPA- or A23187-mediated glucose uptake was not affected by phospholipase C inhibition, this suggests that PKC isoforms sensitive to TPA and calcium are downstream of phospholipase C.
PKC and calcium mediate glucose uptake by insulin or 1-adrenoceptor activation.
PKC has been studied extensively as a key mediator of insulin-stimulated glucose uptake (52), and a role for PKC in insulin-mediated glucose uptake has been established (53, 54, 55 ; for review, see Ref.35). However, there is still much debate regarding which isoforms of PKC are involved. Both insulin and TPA stimulate conventional (, ?1, ?2, and ) and novel ( and ) PKC isoforms, but atypical () PKC isoforms are stimulated by insulin and not by TPA (56, 57). Both insulin and TPA are potent activators of glucose uptake in L6 cells as is, to a lesser extent, the calcium ionophore A23187, implying that PKC and calcium are involved in glucose uptake. The role for intracellular calcium was also supported by studies using the membrane-permeable calcium chelator BAPTA-AM, which totally abolished glucose uptake mediated by insulin, cirazoline, TPA, or A23187. BAPTA, which is not cell permeable, had no effect on glucose uptake mediated by any of these agents (data not shown), suggesting that it is intracellular, not extracellular, calcium that is involved in glucose uptake.
We investigated whether PKCs are involved in 1-adrenoceptor-mediated glucose uptake in L6 cells using G?6983 (58), which is an inhibitor of all PKC isoforms. Glucose uptake mediated by TPA or A23187 was totally abolished by G?6983, whereas insulin-mediated glucose uptake was partially inhibited. The partial inhibition of insulin-stimulated glucose uptake by G?6983 indicates that PKC is involved in glucose uptake, but that other pathways must exist for insulin’s effect. 1-Adrenoceptor-mediated glucose uptake was fully inhibited by G?6983, indicating that PKC is very significant in 1-adrenoceptor-mediated glucose uptake.
A method to discriminate between conventional and novel PKCs from atypical PKCs is down-regulation of calcium/DAG-dependent PKCs (achieved with prolonged treatment of phorbol esters). TPA-mediated glucose uptake was inhibited approximately 70% by down-regulation of DAG-sensitive PKCs, indicating that this protocol was effective in down-regulating calcium/DAG isoforms of PKC (40). Down-regulation of these PKC isoforms did not inhibit cirazoline- or insulin-mediated glucose uptake, suggesting that calcium/DAG-dependent PKCs are not involved. This finding was consistent with other studies showing a noninvolvement of DAG-sensitive PKC in insulin-stimulated glucose uptake (39, 55, 59). Additional studies are needed to elucidate which atypical PKCs are involved.
PI3K is important for 1-adrenoceptor- and insulin-mediated glucose uptake.
PI3K is an important mediator of insulin-stimulated glucose uptake, and its activity is increased after insulin stimulation of L6 cells. Akt is rapidly and persistently activated by insulin in skeletal muscle (including L6 cells) and adipose tissue, and there is strong evidence to suggest that Akt is activated by a PI3K-dependent mechanism (reviewed in Ref.60). There is conflicting evidence for adrenergic activation of PI3K or Akt. ?1/?2-Adrenoceptor activation leads to Akt activation in a PI3K-dependent manner in cardiac myocytes (61, 62), whereas in Rat-1 fibroblasts and cardiac myocytes, 1-adrenoceptor activation has no effect on Akt activation (63, 64, 65, 66) and can inhibit insulin activation of Akt (65). 1-Adrenoceptor activation can also stimulate PI3K activity in NIH-3T3 cells (67). In this study cirazoline was able to activate PI3K, but was unable to phosphorylate Akt (or inhibit insulin activation of Akt), indicating a potential difference in the mechanisms of 1-adrenoceptor- and insulin-mediated glucose uptake as well as PI3K activation. It is presently unclear how cirazoline activates PI3K, which PI3K isoforms are responsible, and the physiological relevance of activating PI3K, but not Akt. In addition, we found that TPA increased PI3K activity similar to other reports for muscle and adipose tissue (40, 68, 69), but was unable to phosphorylate Akt.
Akt is a well recognized downstream target of PI3K, but although insulin was able to phosphorylate Akt in L6 cells, cirazoline was without effect, even though cirazoline-mediated glucose uptake was inhibited by LY294002. Other studies have shown, with the use of PI3K inhibitors such as LY294002 or wortmannin (70, 71, 72), a role for PI3K in 1-adrenoceptor glucose uptake (10, 19, 44). Our results suggest that PI3K activity is involved in glucose uptake mediated by cirazoline, because LY294002 totally inhibits this uptake, and cirazoline activates PI3K activity. This discrepancy in 1-adrenoceptors’ inability to activate Akt, but its ability to produce responses that are PI3K dependent is not new [such as 1-adrenoceptor activation of p70 S6 kinase 2 (63, 64, 66) or eukaryotic initiation factor 4E-binding protein 1 (64)] and should be further characterized.
To elucidate whether insulin and 1-adrenoceptor agonists use the same signaling pathways to stimulate glucose uptake, cells were exposed to cirazoline or isoprenaline in the presence of insulin. These responses were not additive, indicating that insulin, 1-adrenoceptors, and ?2-adrenoceptors use distinct pathways that probably overlap at the level of PI3K. One mechanism by which 1-adrenceptors may activate PI3K is through the release of G?-subunits after receptor stimulation, because class IB PI3Ks can be stimulated by G?-subunits (73). Gq proteins have been shown in 3T3-L1 adipocytes to associate with the insulin receptor, and this interaction was necessary for insulin-mediated GLUT4 translocation and glucose uptake (74). This interaction was upstream of PI3K (74) and may provide a basis for 1-adrenoceptor-mediated glucose uptake.
LY294002 was able to partially inhibit glucose uptake mediated by either TPA or the calcium ionophore A23187. This result was unexpected, because it is generally believed that PKC and calcium are downstream effectors of PI3K. Other reports show that glucose uptake mediated by phorbol esters can be inhibited by PI3K inhibitors (40, 57, 69). This discrepancy may be explained by findings in fat and muscle cells (and our results here) that phorbol esters are capable of activating PI3K (40, 57, 68, 69), although glucose uptake mediated by TPA is still mediated primarily through PKC as opposed to PI3K (57).
p38 kinase, but not Erk1/2, is involved in glucose uptake mediated by either insulin or 1-adrenoceptor activation.
We have shown that both insulin and cirazoline are able to phosphorylate Erk1/2. Classically Erk1/2 activation has been considered to be limited to activation by peptide growth factors acting via receptor tyrosine kinases (75, 76), but more recently it has been appreciated that G protein-coupled receptors, such as adrenoceptors, are capable of activating Erk1/2 in tissues important for glucose homeostasis, such as adipocytes (21, 77, 78, 79) and skeletal muscle (80). Because insulin or 1-adrenoceptor stimulation caused a rapid phosphorylation of Erk1/2, we investigated whether Erk1/2 may be involved in glucose uptake by use of the specific upstream kinase (MAPK kinase 1/2) inhibitor PD98059 (81). Glucose uptake mediated by insulin or cirazoline was not inhibited by PD98059, suggesting that Erk1/2 activity is not required for increases in glucose uptake, consistent with other studies (82, 83). It cannot be ruled out that Erk1/2 may be involved in other consequential actions of insulin and cirazoline, such as changes in mRNA and protein levels, because Erk1/2 is known to affect gene transcription.
Recently, there has been enormous focus on the potential role of p38 kinase in insulin-stimulated glucose uptake. There are reported discrepancies in the literature as to whether insulin is able to phosphorylate or activate p38 kinase itself, with several studies showing a positive action of insulin (31, 33) and others showing no effect (84, 85). Inhibition of p38 kinase by selective inhibitors such as SB202190 (86) inhibits insulin-mediated glucose uptake in skeletal muscle and adipose tissue (30, 31, 32, 33), although the mechanism of this is highly debated. Some studies postulate that p38 kinase activation may enhance GLUT4 intrinsic activity (30, 32), p38 kinase inhibitors (such as SB203580) are nonspecific and may act on Akt or GLUT4 itself (87), or that p38 kinase dephosphorylates intracellular pools of GLUT4 by a p38 kinase-dependent phosphatase to result in GLUT4 translocation (88). Our results indicate that insulin and cirazoline are able to phosphorylate and activate p38 kinase, and glucose uptake mediated by either agent is sensitive to p38 kinase inhibition. This supports a role for p38 kinase in glucose uptake in L6 cells.
Conclusions
We have demonstrated that 1-adrenoceptor activation increases glucose uptake in L6 muscle cells. We hypothesize that 1-adrenoceptor activation of glucose uptake is through activation of phospholipase C, which produces both DAG and inositol trisphosphate. This causes the release of intracellular calcium, which by some mechanism not yet clarified causes the activation of PI3K. Although DAG can then theoretically activate DAG-sensitive isoforms of PKC and increase glucose uptake, this is not involved in glucose uptake mediated by 1-adrenoceptor activation. 1-Adrenoceptor stimulation leads to activation of PI3K, but not phosphorylation of Akt. Even though activation of 1-adrenoceptors leads to some of the same signal transduction pathways via PI3K and atypical PKCs that are stimulated by insulin, it is likely that the differences between the 1-adrenoceptor and insulin pathways upstream could be exploited in the search for insulin-independent mechanisms of glucose uptake.
Acknowledgments
We gratefully acknowledge Dr. Bronwyn A. Evans (Monash University, Melbourne, Australia) for assistance in designing primers and PCR design, and Profs. Roger J. Summers (Monash University), Barbara Cannon, and Jan Nedergaard (Stockholm University, Stockholm, Sweden) for helpful discussions.
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