AMP-Activated Protein Kinase Activation by Adrenoceptors in L6 Skeletal Muscle Cells
http://www.100md.com
糖尿病学杂志 2006年第3期
the Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratory F3, Stockholm University, Stockholm, Sweden
ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide 1--D-ribonucleoside; AMPK, AMP-activated protein kinase; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate
ABSTRACT
AMP-activated protein kinase (AMPK), which functions as a sensor of cellular energy homeostasis, was phosphorylated after norepinephrine stimulation in L6 skeletal muscle cells. This effect was mediated by 1-adrenoceptors, with no stimulatory effects due to interactions at 2- or -adrenoceptors. 1-Adrenoceptors are Gq-coupled receptors, and calcium but not phorbol esters could mimic the effect of 1-adrenergic stimulation; and we show that protein kinase C is not involved as an upstream signal to AMPK by 1-adrenergic stimulation and that the AMP-to-ATP ratio is unaltered after 1-adrenergic stimulation. We further show that glucose uptake mediated by 1- but not by -adrenoceptors can be inhibited by AMPK inhibition. Acetyl-CoA carboxylase (ACC) is phosphorylated at Ser218 by AMPK, and 1- but not -adrenoceptor stimulation results in phosphorylation of ACC at this residue. These results suggest a novel pathway where 1-adrenoceptor activation, independent of protein kinase C, leads to activation of AMPK in skeletal muscle, which contributes to 1-adrenoceptoreCmediated increases in glucose uptake.
AMP-activated protein kinase (AMPK) has been described as a sensor of cellular and whole-body energy homeostasis and is present at high levels in tissues that regulate energy homeostasis, namely the liver, heart, adipose tissue, pancreas, brain, and skeletal muscle. AMPK is activated by hormonal and nutrient stresses that increase the AMP-to-ATP ratio after depletion of intracellular ATP levels, but some conditions (such as hyperosmotic stress) activate AMPK without ATP reductions (1). Structurally, AMPK is a heterotrimeric protein consisting of an catalytic subunit and - and -regulatory subunits, and activation requires phosphorylation at Thr172 on the catalytic subunit by one or more upstream kinases (for more comprehensive review, refer to 2). Recently two different upstream kinases have been identified, LKB1 (3eC5) and CAMKK (6eC8). Activation of AMPK activates pathways such as glucose transport, glycolysis, and -fatty acid oxidation and inhibits pathways such as fatty acid and cholesterol synthesis through interactions with metabolic enzymes and proteins and effects on gene expression.
Adrenoceptors are classified into three main subtypes: 1-, 2-, and -adrenoceptors, which couple to Gq (increase inositol 1,4,5-trisphosphate and diacylglycerol levels), Gi (inhibit cyclic AMP formation), and Gs (increase cyclic AMP formation) G-proteins respectively. 1- and -adrenoceptors are found in skeletal muscle (9eC15). However, the role of AMPK in adrenergically mediated responses in skeletal muscle has not been investigated to a great extent. With respect to AMPK, the -adrenoceptor agonist phenylephrine increases AMPK activity in isolated mouse soleus muscle (16), and other Gq-coupled receptors activate AMPK in transfected CHO-K1 cells (17). In adipose tissue, -adrenoceptors activate AMPK in white adipocytes (18,19), 3T3-L1 adipocytes (20), and brown adipocytes (21).
Facilitation of glucose uptake in tissues important in glucose homeostasis, such as skeletal muscle, can be accomplished by adrenergic activation. There are many studies showing that epinephrine decreases glucose disposal, primarily by inhibiting insulin secretion via activation of 2-adrenoceptors. Epinephrine inhibits insulin-stimulated glucose uptake in skeletal muscle via -adrenoceptors but in the absence of insulin, can increase glucose uptake in skeletal muscle (22). However, there is much evidence showing that increases of the sympathetic nervous system stimulate glucose uptake in skeletal muscle. One set of key experiments (23,24) shows that electrical stimulation of the ventromedial hypothalamus increases sympathetic activity, resulting in increased glucose uptake in skeletal muscle without alterations in plasma insulin levels. These effects are blocked by guanethidine but not by adrenal medullation, showing that norepinephrine and not epinephrine mediates this response (24). Other studies performed in vivo and in vitro show that - and -adrenoceptor (12,15,25eC29) activation increases glucose uptake in skeletal muscle via an insulin-independent pathway. Hence it is likely that circulating epinephrine has vastly different actions on glucose uptake as opposed to focally released norepinephrine at the synaptic clefts, which can reach high concentrations.
The present study aimed at investigating a possible adrenergic control of AMPK in L6 skeletal muscle cells. We have studied adrenergic phosphorylation of AMPK with the focus on delineating which adrenergic subtypes are involved in the norepinephrine-mediated response and whether AMPK is involved in mediating a biological end point such as glucose uptake response to adrenergic agonists. We show that only 1-adrenergic activation phosphorylates and activates AMPK in these cells, and AMPK activation contributes to 1-adrenergiceCmediated increases in glucose uptake.
RESEARCH DESIGN AND METHODS
Cell culture.
Rat L6 skeletal muscle cells were grown as described previously (12). To differentiate, cells were allowed to reach confluence, and the medium was changed to medium containing 2% FBS for 7 days, with medium changes every 2nd day. Experiments were restricted to cells from passages 2eC15.
CHO-G4myc-1AAR (human 1A-adrenoceptor) and CHO-G4myc cells were grown in Dulbecco’s modified Eagle’s medium-Ham’s F12 (1:1) containing 10% (vol/vol) heat-inactivated FBS, L-glutamine (2 mmol/l), gentamicin sulfate (80 e蘥/ml), and fungizone (2.5 e蘥/ml) under 8% CO2 at 37°C. CHO-G4myc cells were selected with G418 (400 e蘥/ml), whereas CHO-G4myc-1AAR cells were selected with both G418 and blasticidin S (5 e蘥/ml). CHO-K1 and CHO-2AR (human 2-adrenoceptor) cells were grown in Dulbecco’s modified Eagle’s medium-Ham’s F12 (1:1) containing 10% (vol/vol) heat-inactivated FBS, L-glutamine (2 mmol/l), penicillin (100 units/ml), and streptomycin (100 e蘥/ml) under 8% CO2 at 37°C. CHO-2AR medium contained hygromycin B (30 e蘥/ml).
Western blotting.
Cells were serum-starved overnight before each experiment on day 7 and exposed to drugs for times and concentrations indicated with the data. Cells were lysed directly by the addition of 65°C lysis buffer (62.5 mmol/l Tris, pH 6.8, 2% [vol/vol] SDS, 10% [vol/vol] glycerol, 50 mmol/l dithiothreitol, and 1% [vol/vol] bromophenol blue). The lysate was sonicated briefly followed by boiling for 3 min. Aliquots of samples (of same protein amount) were separated on 8 or 12% polyacrylamide gels and electrotransferred to Hybond-P polyvinylidine fluoride membranes (pore size 0.45 e蘭; Amersham Pharmacia Biotech). Primary antibodies used were AMPK antibody (which detects the 1- and 2-isoforms of the catalytic subunit), phospho-AMPK antibody (Thr172), acetyl-CoA carboxylase (ACC) antibody (which detects endogenous levels of all isoforms of ACC), or phospho-ACC (Ser79) antibody (the isoforms of ACC that is expressed in skeletal muscle and L6 cells is ACC2 and hence the phosphorylation site in the rat sequence is at Ser218 and not Ser79, which is the equivalent site in ACC1). Primary antibodies were diluted 1:1,000, which were detected using a secondary antibody (horseradish peroxidaseeClinked anti-rabbit IgG) diluted 1:2,000 and enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). The blots were exposed to Hyperfilm ECL films (Amersham Pharmacia Biotech) and quantified on a Molecular Dynamics densitometer using ImageQuant NT software. Results are expressed as the ratio between the phosphorylated and total protein, with the ratio normalized in each experiment to that of control samples. All experiments were performed singly or in duplicate with n referring to the number of independent experiments performed.
In all experiments performed, 5-aminoimidazole-4-carboxamide 1--D-ribonucleoside (AICAR) was used as a positive control. AICAR is widely used to investigate AMPK actions in vivo and in vitro. It is phosphorylated intracellularly to ZMP, which activates AMPK by mimicking AMP and also by promoting phosphorylation and activation of the upstream kinase, AMPK kinase (30eC32).
2-deoxy-[3H]-D-glucose uptake assay.
Glucose uptake in L6 cells was measured using the 2-deoxy-[3H]-D-glucose method (28) with modifications (12). To measure glucose uptake in CHO-G4myc-1AAR or CHO-2AR cells, cells were seeded at 5 x 105 cells per well and left to adhere overnight. Medium was replaced in the morning (to serum-free medium) for 4 h. For CHO-G4myc-1AAR cells, cells were washed in warm PBS, medium was replaced with glucose-free medium, and drugs were added for 50 min, after which 2-deoxy-[3H]-D-glucose (50 nmol/l) was added for 10 min. For CHO-2AR cells, medium was replaced (to serum-free medium), and drugs were added for 2 h. After this, cells were washed in warm PBS, glucose-free medium was added, and drugs were re-added for 45 min, after which 2-deoxy-[3H]-D-glucose (50 nmol/l) was added for 15 min. Reactions were terminated by washing twice in ice-cold PBS, cells were digested (0.2 mol/l NaOH, 1 h, 60°C), and samples were transferred to scintillation vials with scintillant. When inhibitors were used, the time indicated with the results represents the time cells were pre-equilibrated with the inhibitors before agonists were added. All experiments were performed in duplicate with n referring to the number of independent experiments performed.
AMPK activity.
L6 cells (day 7) were serum-starved overnight, medium was replaced, and cells were treated with drugs for 2 h. Cells were washed twice with ice-cold PBS and lysed in buffer A (20 mmol/l Tris-HCl [pH 7.5], 150 mmol/l NaCl, 1 mmol/l Na2EDTA, 1 mmol/l EGTA, 1% Triton, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l -glycerophosphate, 1 mmol/l Na3VO4, 1 e蘥/ml leupeptin, 1 mmol/l dithiothreitol, and 1 mmol/l phenylmethylsulfonylfluoride) for 5 min on ice. After centrifugation (14,000g, 10 min, 4°C), the supernatant was assayed for protein content, and 200 e蘥 protein (in total volume of 200 e蘬) was incubated overnight at 4°C with AMPK -subunit antibody at 1:80 dilution factor, followed by addition of 20 e蘬 50% slurry of protein-A agarose beads (Upstate Biotechnology, Lake Placid, NY) for 2 h at 4°C. Immunoprecipitates were collected by centrifugation (18,000g, 1 min, 4°C); washed twice with 500 e蘬 buffer A and twice with 500 e蘬 buffer B (240 mmol/l HEPES, pH 7.4, and 480 mmol/l NaCl); and resuspended in 30 e蘬 reaction buffer (40 mmol/l HEPES, pH 7.0, 80 mmol/l NaCl, 0.8 mmol/l dithiothreitol, and 5 mmol/l MgCl2), which contained 100 e蘭ol/l SAMS peptide (HMRSAMSGLHLVKRR). The reaction was started by the addition of 10 e蘬 ATP buffer (75 mmol/l MgCl2, 500 e蘭ol/l free ATP, and 1 e藽i/ml [-32P]ATP [3,000 Ci/mmol]). After 20 min at 30°C, the reaction was stopped by spotting 35-e蘬 samples on P81 Whatman filter papers, which were washed twice in 0.75% (vol/vol) orthophosphoric acid for 5 min and once in acetone for 5 min before drying and scintillation counting. AMPK activity is expressed as the amount of incorporated ATP (picomoles) per immunoprecipitated protein (relative to the amount of protein used for the immunoprecipitation) per minute. The final data are expressed as a percentage of the control values (3.2 ± 1.9 pmol · mineC1 · mg proteineC1).
AMP-to-ATP ratio and ATP level measurement.
L6 cells (day 7) were serum-starved overnight, new medium was added for 2 h, and cells were treated with drugs for 30 min. Cell extracts were isolated and the AMP-to-ATP ratio measured as previously described (21), except that ATP levels were measured in duplicate using a commercial kit (ATP determination kit time stable assay; Biaffin, Kassel, Germany). Results are expressed as the ratio of AMP to ATP and also as nanomoles ATP per milligram protein.
Data analysis.
All results are expressed as means ± SE of n. Data were analyzed using nonlinear curve fitting (GraphPad PRISM version 3.03) to obtain pEC50 values, where appropriate. Statistical significance was determined using paired Student’s t test. P values 0.05 were considered significant.
Drugs and reagents.
The following were gifts: the AMPK inhibitor compound C was a gift obtained from Merck Research Laboratories (Rahway, NJ). Zinterol hydrochloride was obtained from Bristol-Myers Squibb (Noble Park, Victoria, Australia). CHO-G4myc-1AAR, CHO-G4myc, CHO-K1, and CHO-2AR cells were provided by Prof. Roger J Summers (Monash University, Melbourne, Victoria, Australia).
Drugs and reagents were purchased as follows: rosiglitazone (Alexis Biochemicals, Lausen, Switzerland); 2-deoxy-[3H]-D-glucose (12 Ci/mmoleC1; Amersham Biosciences, Buckinghamshire, U.K.); G6976 and G6783 (CalBiochem, La Jolla, CA); 2,4,-dinitrophenol (Merck Schucharat OHG, Hohenbrunn, Germany); insulin (Actrapid) (Novo Nordisk, Bagsvaerd, Denmark); [-32P]ATP (3,000 Ci/mmol) (PerkinElmer Sverige, Upplands Vsby, Sweden); A23187, cirazoline, forskolin, (eC)-isoprenaline, LY294002, (eC)-norepinephrine, phenylephrine, and 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma Chemical, St. Louis, MO); AICAR (Toronto Research Chemicals, North York, Ontario, Canada); and SAMS peptide (Upstate Biotechnology).
All cell culture media and supplements were obtained from Gibco-BRL Life Technologies (Gaithersburg, MD). All antibodies were obtained from Cell Signaling Technology (Beverly, MA). All other drugs and reagents were of analytical grade.
RESULTS
1-Adrenoceptor but not 2- or -adrenoceptor activation increases AMPK phosphorylation in L6 cells and recombinant CHO-K1 cells.
The AMPK activator AICAR phosphorylated AMPK in L6 cells sixfold over basal levels (n = 55eC58), whereas insulin (n = 8) had no effect (Fig. 1A). The endogenous adrenergic ligand norepinephrine phosphorylated AMPK, with an approximate two- to threefold increase in the p-AMPKeCtoeCAMPK ratio (Fig. 1B). This effect was still present after 2 h of norepinephrine stimulation (twofold increase in p-AMPKeCtoeCAMPK ratio; data not shown). To determine which adrenergic receptor subtype(s) mediated the norepinephrine effect, L6 cells were stimulated with different subtype-specific adrenergic agonists. The -adrenoceptor agonist isoprenaline (even after 2 h of incubation; data not shown) and the 2-adrenoceptor agonist zinterol were without effect (Fig. 1C). The adenylate cyclase activator forskolin was also without effect (Fig. 1C). Phenylephrine, an 1-/2-adrenoceptor agonist, phosphorylated AMPK to a similar extent as norepinephrine (Fig. 1D). This effect was mediated by 1-adrenoceptor activation because the 1-adrenoceptor agonist cirazoline phosphorylated AMPK (Fig. 1D, which was sustained for up to 2 h; data not shown), whereas the 2-adrenoceptor agonist clonidine was without effect (Fig. 1D). These phosphorylation studies correlated well with AMPK activity measurements. AICAR and cirazoline increased AMPK activity, which was inhibited by the AMPK inhibitor compound C, whereas isoprenaline and insulin, which did not phosphorylate AMPK, did not increase AMPK activity (Fig. 2). The increases in AMPK activity after 1-adrenergic stimulation are not due to alterations in the AMP-to-ATP ratio or to decreased ATP content because cirazoline failed to significantly affect the AMP-to-ATP ratio or ATP levels compared with the positive controls rosiglitazone and dinitrophenol, which increase the AMP-to-ATP ratio and significantly reduced ATP levels (Tables 1 and 2).
To determine whether the effect of 1- and 2-adrenoceptors on AMPK phosphorylation was confined to muscle cells, we have used CHO-K1 cells transfected with either the human 1a-adrenoceptor (Fig. 3A) or human 2-adrenoceptor (Fig. 3B), which are the predominant subtypes expressed in L6 cells (12,15). The human 1a- rather than the 1b-adrenoceptor (as used by Kishi et al. [17]) was used because the 1b-adrenoceptor is exclusively expressed in liver, whereas the 1a-adrenoceptor is detected in skeletal muscle (10,12). In both cell systems AICAR but not insulin phosphorylated AMPK (threefold increase after 30 min or 2 h of stimulation, n = 4eC8) as observed in the L6 cells above. In CHOh1 cells, norepinephrine and cirazoline phosphorylated AMPK fourfold (Fig. 3A). In CHOh2 cells, norepinephrine, isoprenaline, and forskolin had no effect on basal AMPK phosphorylation levels (Fig. 3B). In CHO-K1 and CHO-G4myc cells, isoprenaline and cirazoline did not phosphorylate AMPK (data not shown).
AMPK is involved in glucose uptake mediated by 1- but not -adrenoceptor activation.
We examined whether cirazoline can increase glucose uptake in L6 cells via AMPK by using an AMPK inhibitor, compound C. AICAR-stimulated but not insulin-stimulated glucose uptake was inhibited by compound C (Fig. 4B), and compound C inhibited the phosphorylation of AMPK by AICAR (Fig. 4A). Norepinephrine-mediated glucose uptake is via both 1- and 2-adrenoceptors in L6 cells (12). Compound C partially inhibited glucose uptake by norepinephrine, but this effect was not statistically different (Fig. 4C; P = 0.16, paired Student’s t test), which could be representative of norepinephrine using both - and -adrenoceptors to increase glucose uptake. Isoprenaline-mediated glucose uptake was not inhibited by compound C (Fig. 4D), but compound C significantly inhibited cirazoline-mediated glucose uptake (Fig. 4D) and cirazoline-mediated AMPK phosphorylation (Fig. 4A).
In CHOh1 cells, cirazoline increased glucose uptake in a concentration-dependent manner (pEC50 8.4 ± 0.2; maximum increase 260 ± 11% over basal; hill slope 0.86; n = 4; data not shown). AICAR, insulin, and cirazoline increased glucose uptake, but compound C only inhibited AICAR and cirazoline-mediated increases in glucose uptake (Fig. 5A). In CHOh2 cells, isoprenaline increased glucose uptake in a concentration-dependent manner (pEC50 7.7 ± 0.4; maximum increase 131 ± 4% over basal; hill slope 0.90; n = 4; data not shown). AICAR, insulin, and isoprenaline increased glucose uptake, but only AICAR-stimulated glucose uptake was inhibited by compound C (Fig. 5B).
Calcium, but not protein kinase C (PKC), is involved in AMPK phosphorylation.
1-Adrenoceptors are Gq-coupled receptors, and their activation results in increased phosphatidylinositol turnover, activation of phospholipase C, and increased intracellular calcium levels. To investigate whether the effect of 1-adrenoceptor stimulation could be mimicked by PKC activation and increases in calcium levels, L6 cells were treated with either TPA (activator of conventional and novel PKCs; 1 e蘭ol/l) or A23187 (calcium ionophore; 1 e蘭ol/l). A23187 but not TPA was able to significantly phosphorylate AMPK (twofold increase over basal) in the time period examined (Fig. 6). Both treatments, however, were able to increase glucose uptake (A23187 1 e蘭ol/l, 207 ± 13% over basal, n = 3; TPA 1 e蘭ol/l, 211 ± 23% over basal, n = 7). TPA also had no effect on basal AMPK phosphorylation levels in CHOh1 cells (data not shown).
Glucose uptake in L6 cells in response to insulin, AICAR, or cirazoline is inhibited by G6983 (inhibits novel, conventional, and atypical PKC isoforms) but not by G6976 (inhibits novel and conventional PKC isoforms), showing an involvement of atypical PKCs (Fig. 7A). To determine whether PKCs are upstream of the signal to AMPK, two different approaches were used. First, downregulation of conventional and novel PKCs can be achieved with long-term stimulation of cells with TPA (33). After downregulation of these PKC isoforms, cirazoline (as well as AICAR) was still able to phosphorylate AMPK to the same level as cells not prestimulated with TPA (Fig. 7B), indicating that conventional and novel PKCs are not involved in the cirazoline and AICAR response. Second, AMPK phosphorylation by AICAR or cirazoline was not inhibited by either G6976 or G6983 (Fig. 7C), suggesting that no isoforms of PKC are involved in an upstream mechanism of AMPK.
1-Adrenoceptor activation phosphorylates ACC at Ser218 in L6 cells.
One downstream target of AMPK is phosphorylation of ACC2 at Ser218. In L6 cells, AICAR and cirazoline but not isoprenaline or insulin (data not shown) phosphorylated ACC2 at Ser218, and this was inhibited largely by compound C (Fig. 8).
DISCUSSION
Insulin-stimulated glucose uptake is severely compromised in type 2 diabetes, and recently, there has been great focus on the potential of AMPK in the treatment of type 2 diabetes. Insulin and AICAR both increase glucose uptake in skeletal muscle but use two distinct signaling pathways to mediate this effect that probably converge at some point. Phosphatidylinositol 3-kinase is necessary for insulin-stimulated but not AICAR-stimulated glucose uptake in skeletal muscle, whereas AMPK is necessary for AICAR-stimulated but not insulin-stimulated glucose uptake (34eC36 and D.S.H., T.B., unpublished data). The effects of AICAR, at least in skeletal muscle, are due to interactions directly resulting from AMPK activation because glucose uptake by AICAR is abolished in skeletal muscle from AMPK inactive mutant mice (37), and overexpression of a dominant-negative AMPK in rat skeletal muscle abolishes AICAR-mediated increases in glucose uptake (38,39). Of interest are the recent discoveries that hormonal and nutritional stresses have the ability to elicit their effects via AMPK, including leptin (16) and adiponectin (36,40,41). Leptin activates AMPK in skeletal muscle via two pathways: directly on skeletal muscle and also by actions in the hypothalamus to increase -adrenergic sympathetic activity to activate AMPK in skeletal muscle (16). To this extent, we have investigated the role of the endogenous ligand norepinephrine on AMPK in L6 skeletal muscle cells.
Norepinephrine stimulated AMPK phosphorylation in L6 skeletal muscle cells via 1-adrenoceptors, but not - or 2-adrenoceptors. 1-Adrenoceptor activation increases AMPK activity in mouse soleus muscle (16) and in CHO cells transfected with the 1a-adrenoceptor (this study) and 1b-adrenoceptor (17). This later study showed that Gq- (1b-adrenoceptor and bradykinin 2 receptor) and not Gi- (2a-adrenoceptor) or Gs- (2-adrenoceptor) coupled receptors cause activation of AMPK. Forskolin, 8-bromo-cAMP, and insulin were also without effect (17), and we also observed similar results in CHO cells. Other Gq-coupled receptors, such as the angiotensin type 2 receptor in rat vascular smooth muscle cells (42) and the histamine (H1) and thrombin (PAR) receptors in human umbilical vein endothelial cells (43), also activate AMPK. The data presented in this study are consistent with the notion that Gq-coupled receptors have the ability to phosphorylate AMPK. However, this is not a general phenomenon that only occurs after stimulation of Gq-coupled receptors because in adipose tissue, Gs-coupled receptors (-adrenoceptors) are capable of phosphorylating and activating AMPK (18eC21). Additionally in brown adipose tissue where 1-adrenoceptors increase glucose uptake in 3-adrenoceptor knockout mice (44), 1-adrenoceptor activation did not phosphorylate AMPK (21). These results in adipose tissue are opposite to what we observe in L6 skeletal muscle cells and CHO cells, which may suggest that in adipose tissue, regulation of AMPK by G-proteineCcoupled receptors differs from other cell types.
AMPK can be activated by two different upstream kinases, LKB1 (3eC5) and CAMKK (6eC8), via different activators, such as decreased ATP and increased AMP-to-ATP ratios for LKB1 and alterations in calcium metabolism for CAMKK. Because 1-adrenoceptor activation leads to phospholipase C activation, which results in the hydrolysis of phosphatidylinositol (4,5)-bisphosphate to produce inositol-1,4,5-phosphate that releases calcium from intracellular stores, and diacylglycerol, which can activate PKC, it is plausible to hypothesize that 1-adrenoceptors may activate AMPK via calcium. The calcium ionophore A23187 activates AMPK in LKB1eC/eC embryo fibroblasts, NIH3T3, and Hela cells (6eC8), and treatment of L6 cells with A23187 phosphorylated AMPK, making it likely that 1-adrenoceptor activation of AMPK is via CAMKK because 1-adrenoceptor activation did not decrease ATP levels or alter the AMP-to-ATP ratio in L6 cells. Further investigation of 1-adrenergic activation of AMPK is currently being investigated.
Activation of AMPK is also not secondary to changes in PKC activity despite PKC being implicated in 1-adrenoceptor mediation of glucose uptake (12,29). The phorbol ester TPA (which activates conventional and novel PKC isoforms) did not phosphorylate AMPK, and downregulation of conventional and novel PKCs with long-term TPA treatment (33) or the use of PKC inhibitors did not affect the ability of cirazoline (or AICAR) to phosphorylate AMPK, suggesting that PKCs are not involved in phosphorylation of AMPK (PKC inhibition was also ineffective in inhibiting AICAR activation of AMPK in rat papillary muscle [45]). However, 1-adrenoceptoreCand AICAR-mediated glucose uptake is very dependent on atypical PKCs, which are presumably downstream of AMPK on glucose uptake effects. These results agree with other studies investigating the interaction between AMPK and PKC. In L6 cells, AICAR treatment activated atypical PKCs, but this effect was downstream of its activation of AMPK, hence raising the possibility that atypical PKCs may be a final activator of glucose uptake for varying agonists, independent of their initial signaling pathways (46). Additionally, dinitrophenol activation of glucose uptake but not AMPK activation was sensitive to calcium chelation and PKC inhibition, suggesting that AMPK activation may be independent of calcium and PKC (47)
Focally released norepinephrine (which can reach very high levels locally in skeletal muscle) and circulating epinephrine (low levels in the blood stream and acts at many different target tissues) may have completely different effects on skeletal muscle glucose uptake and AMPK activation in vivo. Circulating epinephrine inhibits glucose uptake in skeletal muscle by insulin-dependent mechanisms, whereas norepinephrine released from sympathetic nerves (including under conditions of stress such as cold exposure and exercise) increases glucose uptake via insulin-independent mechanisms (rev. in 48). The regulation of AMPK in skeletal muscle is also G-protein dependent, with Gq-coupled receptors activating AMPK (this study; 16). To investigate the role of AMPK of adrenergically mediated glucose uptake, we have used the AMPK inhibitor compound C. This is a potent and small molecule inhibitor of AMPK that acts as a reversible inhibitor of AMPK by binding at the ATP-binding site on AMPK (49). Compound C inhibited AICAR-mediated but not insulin-mediated glucose in all cell types investigated here, and inhibited AICAR-mediated phosphorylation of AMPK, showing that its effects are mediated via AMPK inhibition. 1- but not 2-adrenoceptoreCmediated glucose uptake was inhibited by compound C, which suggests that glucose uptake mediated by 1-adrenoceptors is attributed to AMPK activation. Additionally, TPA-mediated glucose uptake was not inhibited by compound C in L6 cells (data not shown), which correlates with the inability of TPA to phosphorylate AMPK.
One function of AMPK activation is to provide energy to cells by stimulating the use of alternate fuels. AMPK phosphorylates and inactivates ACC2 at Ser218, which is a rate-limiting step in the conversion of acetyl-CoA to malonyl-CoA, resulting in increased free fatty acid oxidation. PKA can also phosphorylate ACC2 but at distinct residues from those phosphorylated by AMPK (rev. in 50). 1-Adrenoceptor and AICAR (but not isoprenaline) stimulation resulted in the phosphorylation of ACC2 at Ser218.
In summary, 1-adrenoceptor activation, but not 2- or -adrenoceptor activation, increases AMPK phosphorylation and activity in L6 skeletal muscle cells through a pathway independent of PKC. Although both 1- and 2-adrenoceptors are able to increase glucose uptake in these cells, AMPK is only involved in 1-adrenoceptoreCmediated glucose uptake.
ACKNOWLEDGMENTS
D.S.H. is a C.J. Martin Fellow from the National Health and Medical Research Council of Australia. This study was supported by the Swedish Natural Science Research Council, the Tore Nilsons Stiftelse for Medicinsk Forskning, and the Jeanssonska funds.
We gratefully acknowledge Prof. Roger J. Summers (Monash University) for providing us with the recombinant CHO-K1 cells and helpful advice. We gratefully acknowledge Prof. Barbara Cannon and Prof. Jan Nedergaard (Stockholm University, Stockholm, Sweden) for helpful discussions, Olof S. Dallner (Stockholm University, Stockholm, Sweden) for advice on AMP-to-ATP measurements, and Dr. Kei Sakamoto (University of Dundee, Dundee, U.K.) for helpful advice on the design of the AMPK activity assay.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Fryer LG, Parbu-Patel A, Carling D: The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct pathways. J Biol Chem 277:25226eC25232, 2002
Hardie DG: The AMP-activated protein kinase pathway: new players upstream and downstream. J Cell Sci 117:5479eC5487, 2004
Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, Hardie DG: Complexes between the LKB1 tumor suppressor, STRAD / and MO25 / are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2:28, 2003
Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley LC: The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 101:3329eC3335, 2004
Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U, Wallimann T, Carlson M, Carling D: LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13:2004eC2008, 2003
Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, Hardie DG: Calmodulin-dependent protein kinase kinase- is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2:9eC19, 2005
Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA: The Ca++/calmoldulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280:29060eC29066, 2005
Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M, Carling D: Ca2+/calmodulin-dependent protein kinase kinase- acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2:21eC33, 2005
Martin WH III, Tolley TK, Saffitz JE: Autoradiographic delineation of skeletal muscle 1-adrenergic receptor distribution. Am J Physiol 259:H1402eCH1408, 1990
Rokosh DG, Bailey BA, Stewart AF, Karns LR, Long CS, Simpson PC: Distribution of 1C-adrenergic receptor mRNA in adult rat tissues by RNase protection assay and comparison with 1B and 1D. Biochem Biophys Res Commun 200:1177eC1184, 1994
Rattigan S, Appleby GJ, Edwards SJ, McKinstry WJ, Colquhoun EQ, Clark MG, Richter EA: -Adrenergic receptors in rat skeletal muscle. Biochem Biophys Res Commun 136:1071eC1077, 1986
Hutchinson DS, Bengtsson T: 1A-Adrenoceptors activate glucose uptake in L6 muscle cells through a phospholipase C-, phosphatidylinositol-3 kinase-, and atypical protein kinase C-dependent pathway. Endocrinology 146:901eC912, 2005
Liggett SB, Shah SD, Cryer PE: Characterization of -adrenergic receptors of human skeletal muscle obtained by needle biopsy. Am J Physiol 254:E795eCE798, 1988
Roberts SJ, Molenaar P, Summers RJ: Characterization of propranolol-resistant (eC)-[125I]-cyanopindolol binding sites in rat soleus muscle. Br J Pharmacol 109:344eC352, 1993
Nevzorova J, Bengtsson T, Evans BA, Summers RJ: Characterization of the -adrenoceptor subtype involved in mediation of glucose transport in L6 cells. Br J Pharmacol 137:9eC18, 2002
Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB: Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339eC343, 2002
Kishi K, Yuasa T, Minami A, Yamada M, Hagi A, Hayashi H, Kemp BE, Witters LA, Ebina Y: AMP-activated protein kinase is activated by the stimulations of G(q)-coupled receptors. Biochem Biophys Res Commun 276:16eC22, 2000
Moule SK, Denton RM: The activation of p38 MAPK by the -adrenergic agonist isoproterenol in rat epididymal fat cells. FEBS Lett 439:287eC290, 1998
Daval M, Diot-Dupuy F, Bazin R, Hainault I, Viollet B, Vaulont S, Hajduch E, Ferre P, Foufelle F: Anti-lipolytic action of AMP-activated protein kinase in rodent adipocytes. J Biol Chem 280:25250eC25257, 2005
Yin W, Mu J, Birnbaum MJ: Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis in 3T3eCL1 adipocytes. J Biol Chem 278:43074eC43080, 2003
Hutchinson DS, Chernogubova E, Dallner OS, Cannon B, Bengtsson T: -adrenoceptors, but not -adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1. Diabetologia 48:2386eC2395, 2005
Laurent D, Petersen KF, Russell RR, Cline GW, Shulman GI: Effect of epinephrine on muscle glycogenolysis and insulin-stimulated muscle glycogen synthesis in humans. Am J Physiol 274:E130eCE138, 1998
Sudo M, Minokoshi Y, Shimazu T: Ventromedial hypothalamic stimulation enhances peripheral glucose uptake in anesthetized rats. Am J Physiol 261:E298eCE303, 1991
Minokoshi Y, Okano Y, Shimazu T: Regulatory mechanism of the ventromedial hypothalamus in enhancing glucose uptake in skeletal muscles. Brain Res 649:343eC347, 1994
Abe H, Minokoshi Y, Shimazu T: Effect of a 3-adrenergic agonist, BRL35135A, on glucose uptake in rat skeletal muscle in vivo and in vitro. J Endocrinol 139:479eC486, 1993
Liu YL, Stock MJ: Acute effects of the 3-adrenoceptor agonist, BRL 35135, on tissue glucose utilisation. Br J Pharmacol 114:888eC894, 1995
Liu YL, Cawthorne MA, Stock MJ: Biphasic effects of the -adrenoceptor agonist, BRL 37344, on glucose utilization in rat isolated skeletal muscle. Br J Pharmacol 117:1355eC1361, 1996
Tanishita T, Shimizu Y, Minokoshi Y, Shimazu T: The 3-adrenergic agonist BRL37344 increases glucose transport into L6 myocytes through a mechanism different from that of insulin. J Biochem (Tokyo) 122:90eC95, 1997
Cheng JT, Liu IM: Stimulatory effect of caffeic acid on 1A-adrenoceptors to increase glucose uptake into cultured C2C12 cells. Naunyn Schmiedebergs Arch Pharmacol 362:122eC127, 2000
Henin N, Vincent MF, Van den Berghe G: Stimulation of rat liver AMP-activated protein kinase by AMP analogues. Biochim Biophys Acta 1290:197eC203, 1996
Corton JM, Gillespie JG, Hawley SA, Hardie DG: 5-aminoimidazole-4-carboxamide ribonucleoside: a specific method for activating AMP-activated protein kinase in intact cells Eur J Biochem 229:558eC565, 1995
Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, Beri RK: Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase. FEBS Lett 353:33eC36, 1994
Bandyopadhyay G, Standaert ML, Galloway L, Moscat J, Farese RV: Evidence for involvement of protein kinase C (PKC)- and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. Endocrinology 138:4721eC4731, 1997
Hayashi T, Hirshman MF, Kurth EJ, Winder WW, Goodyear LJ: Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47:1369eC1373, 1998
Bergeron R, Russell RR III, Young LH, Ren JM, Marcucci M, Lee A, Shulman GI: Effect of AMPK activation on muscle glucose metabolism in conscious rats. Am J Physiol 276:E938eCE944, 1999
Wu X, Motoshima H, Mahadev K, Stalker TJ, Scalia R, Goldstein BJ: Involvement of AMP-activated protein kinase in glucose uptake stimulated by the globular domain of adiponectin in primary rat adipocytes. Diabetes 52:1355eC1363, 2003
Mu J, Brozinick JT Jr, Valladares O, Bucan M, Birnbaum MJ: A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 7:1085eC1094, 2001
Fryer LG, Foufelle F, Barnes K, Baldwin SA, Woods A, Carling D: Characterization of the role of the AMP-activated protein kinase in the stimulation of glucose transport in skeletal muscle cells. Biochem J 363:167eC174, 2002
Sakoda H, Ogihara T, Anai M, Fujishiro M, Ono H, Onishi Y, Katagiri H, Abe M, Fukushima Y, Shojima N, Inukai K, Kikuchi M, Oka Y, Asano T: Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes. Am J Physiol Endocrinol Metab 282:E1239eCE1244, 2002
Tomas E, Tsao TS, Saha AK, Murrey HE, Zhang CC, Itani SI, Lodish HF, Ruderman NB: Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci U S A 99:16309eC16313, 2002
Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T: Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 8:1288eC1295, 2002
Nagata D, Takeda R, Sata M, Satonaka H, Suzuki E, Nagano T, Hirata Y: AMP-activated protein kinase inhibits angiotensin II-stimulated vascular smooth muscle cell proliferation. Circulation 110:444eC451, 2004
Thors B, Halldorsson H, Thorgeirsson G: Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt. FEBS Lett 573:175eC180, 2004
Chernogubova E, Hutchinson DS, Nedergaard J, Bengtsson T: 1- and 1-adrenoceptor signaling fully compensate for 3-adrenoceptor deficiency in brown adipocyte norepinephrine-stimulated glucose uptake. Endocrinology 146:2271eC2284, 2005
Russell RR III, Bergeron R, Shulman GI, Young LH: Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol 277:H643eCH649, 1999
Chen HC, Bandyopadhyay G, Sajan MP, Kanoh Y, Standaert M, Farese RV Jr, Farese RV: Activation of the ERK pathway and atypical protein kinase C isoforms in exercise- and aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR)-stimulated glucose transport. J Biol Chem 277:23554eC23562, 2002
Patel N, Khayat ZA, Ruderman NB, Klip A: Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition. Biochem Biophys Res Commun 285:1066eC1070, 2001
Nonogaki K: New insights into sympathetic regulation of glucose and fat metabolism. Diabetologia 43:533eC549, 2000
Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE: Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167eC1174, 2001
Munday MR: Regulation of mammalian acetyl-CoA carboxylase. Biochem Soc Trans 30:1059eC1064, 2002(Dana S. Hutchinson, and T)
ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4-carboxamide 1--D-ribonucleoside; AMPK, AMP-activated protein kinase; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate
ABSTRACT
AMP-activated protein kinase (AMPK), which functions as a sensor of cellular energy homeostasis, was phosphorylated after norepinephrine stimulation in L6 skeletal muscle cells. This effect was mediated by 1-adrenoceptors, with no stimulatory effects due to interactions at 2- or -adrenoceptors. 1-Adrenoceptors are Gq-coupled receptors, and calcium but not phorbol esters could mimic the effect of 1-adrenergic stimulation; and we show that protein kinase C is not involved as an upstream signal to AMPK by 1-adrenergic stimulation and that the AMP-to-ATP ratio is unaltered after 1-adrenergic stimulation. We further show that glucose uptake mediated by 1- but not by -adrenoceptors can be inhibited by AMPK inhibition. Acetyl-CoA carboxylase (ACC) is phosphorylated at Ser218 by AMPK, and 1- but not -adrenoceptor stimulation results in phosphorylation of ACC at this residue. These results suggest a novel pathway where 1-adrenoceptor activation, independent of protein kinase C, leads to activation of AMPK in skeletal muscle, which contributes to 1-adrenoceptoreCmediated increases in glucose uptake.
AMP-activated protein kinase (AMPK) has been described as a sensor of cellular and whole-body energy homeostasis and is present at high levels in tissues that regulate energy homeostasis, namely the liver, heart, adipose tissue, pancreas, brain, and skeletal muscle. AMPK is activated by hormonal and nutrient stresses that increase the AMP-to-ATP ratio after depletion of intracellular ATP levels, but some conditions (such as hyperosmotic stress) activate AMPK without ATP reductions (1). Structurally, AMPK is a heterotrimeric protein consisting of an catalytic subunit and - and -regulatory subunits, and activation requires phosphorylation at Thr172 on the catalytic subunit by one or more upstream kinases (for more comprehensive review, refer to 2). Recently two different upstream kinases have been identified, LKB1 (3eC5) and CAMKK (6eC8). Activation of AMPK activates pathways such as glucose transport, glycolysis, and -fatty acid oxidation and inhibits pathways such as fatty acid and cholesterol synthesis through interactions with metabolic enzymes and proteins and effects on gene expression.
Adrenoceptors are classified into three main subtypes: 1-, 2-, and -adrenoceptors, which couple to Gq (increase inositol 1,4,5-trisphosphate and diacylglycerol levels), Gi (inhibit cyclic AMP formation), and Gs (increase cyclic AMP formation) G-proteins respectively. 1- and -adrenoceptors are found in skeletal muscle (9eC15). However, the role of AMPK in adrenergically mediated responses in skeletal muscle has not been investigated to a great extent. With respect to AMPK, the -adrenoceptor agonist phenylephrine increases AMPK activity in isolated mouse soleus muscle (16), and other Gq-coupled receptors activate AMPK in transfected CHO-K1 cells (17). In adipose tissue, -adrenoceptors activate AMPK in white adipocytes (18,19), 3T3-L1 adipocytes (20), and brown adipocytes (21).
Facilitation of glucose uptake in tissues important in glucose homeostasis, such as skeletal muscle, can be accomplished by adrenergic activation. There are many studies showing that epinephrine decreases glucose disposal, primarily by inhibiting insulin secretion via activation of 2-adrenoceptors. Epinephrine inhibits insulin-stimulated glucose uptake in skeletal muscle via -adrenoceptors but in the absence of insulin, can increase glucose uptake in skeletal muscle (22). However, there is much evidence showing that increases of the sympathetic nervous system stimulate glucose uptake in skeletal muscle. One set of key experiments (23,24) shows that electrical stimulation of the ventromedial hypothalamus increases sympathetic activity, resulting in increased glucose uptake in skeletal muscle without alterations in plasma insulin levels. These effects are blocked by guanethidine but not by adrenal medullation, showing that norepinephrine and not epinephrine mediates this response (24). Other studies performed in vivo and in vitro show that - and -adrenoceptor (12,15,25eC29) activation increases glucose uptake in skeletal muscle via an insulin-independent pathway. Hence it is likely that circulating epinephrine has vastly different actions on glucose uptake as opposed to focally released norepinephrine at the synaptic clefts, which can reach high concentrations.
The present study aimed at investigating a possible adrenergic control of AMPK in L6 skeletal muscle cells. We have studied adrenergic phosphorylation of AMPK with the focus on delineating which adrenergic subtypes are involved in the norepinephrine-mediated response and whether AMPK is involved in mediating a biological end point such as glucose uptake response to adrenergic agonists. We show that only 1-adrenergic activation phosphorylates and activates AMPK in these cells, and AMPK activation contributes to 1-adrenergiceCmediated increases in glucose uptake.
RESEARCH DESIGN AND METHODS
Cell culture.
Rat L6 skeletal muscle cells were grown as described previously (12). To differentiate, cells were allowed to reach confluence, and the medium was changed to medium containing 2% FBS for 7 days, with medium changes every 2nd day. Experiments were restricted to cells from passages 2eC15.
CHO-G4myc-1AAR (human 1A-adrenoceptor) and CHO-G4myc cells were grown in Dulbecco’s modified Eagle’s medium-Ham’s F12 (1:1) containing 10% (vol/vol) heat-inactivated FBS, L-glutamine (2 mmol/l), gentamicin sulfate (80 e蘥/ml), and fungizone (2.5 e蘥/ml) under 8% CO2 at 37°C. CHO-G4myc cells were selected with G418 (400 e蘥/ml), whereas CHO-G4myc-1AAR cells were selected with both G418 and blasticidin S (5 e蘥/ml). CHO-K1 and CHO-2AR (human 2-adrenoceptor) cells were grown in Dulbecco’s modified Eagle’s medium-Ham’s F12 (1:1) containing 10% (vol/vol) heat-inactivated FBS, L-glutamine (2 mmol/l), penicillin (100 units/ml), and streptomycin (100 e蘥/ml) under 8% CO2 at 37°C. CHO-2AR medium contained hygromycin B (30 e蘥/ml).
Western blotting.
Cells were serum-starved overnight before each experiment on day 7 and exposed to drugs for times and concentrations indicated with the data. Cells were lysed directly by the addition of 65°C lysis buffer (62.5 mmol/l Tris, pH 6.8, 2% [vol/vol] SDS, 10% [vol/vol] glycerol, 50 mmol/l dithiothreitol, and 1% [vol/vol] bromophenol blue). The lysate was sonicated briefly followed by boiling for 3 min. Aliquots of samples (of same protein amount) were separated on 8 or 12% polyacrylamide gels and electrotransferred to Hybond-P polyvinylidine fluoride membranes (pore size 0.45 e蘭; Amersham Pharmacia Biotech). Primary antibodies used were AMPK antibody (which detects the 1- and 2-isoforms of the catalytic subunit), phospho-AMPK antibody (Thr172), acetyl-CoA carboxylase (ACC) antibody (which detects endogenous levels of all isoforms of ACC), or phospho-ACC (Ser79) antibody (the isoforms of ACC that is expressed in skeletal muscle and L6 cells is ACC2 and hence the phosphorylation site in the rat sequence is at Ser218 and not Ser79, which is the equivalent site in ACC1). Primary antibodies were diluted 1:1,000, which were detected using a secondary antibody (horseradish peroxidaseeClinked anti-rabbit IgG) diluted 1:2,000 and enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). The blots were exposed to Hyperfilm ECL films (Amersham Pharmacia Biotech) and quantified on a Molecular Dynamics densitometer using ImageQuant NT software. Results are expressed as the ratio between the phosphorylated and total protein, with the ratio normalized in each experiment to that of control samples. All experiments were performed singly or in duplicate with n referring to the number of independent experiments performed.
In all experiments performed, 5-aminoimidazole-4-carboxamide 1--D-ribonucleoside (AICAR) was used as a positive control. AICAR is widely used to investigate AMPK actions in vivo and in vitro. It is phosphorylated intracellularly to ZMP, which activates AMPK by mimicking AMP and also by promoting phosphorylation and activation of the upstream kinase, AMPK kinase (30eC32).
2-deoxy-[3H]-D-glucose uptake assay.
Glucose uptake in L6 cells was measured using the 2-deoxy-[3H]-D-glucose method (28) with modifications (12). To measure glucose uptake in CHO-G4myc-1AAR or CHO-2AR cells, cells were seeded at 5 x 105 cells per well and left to adhere overnight. Medium was replaced in the morning (to serum-free medium) for 4 h. For CHO-G4myc-1AAR cells, cells were washed in warm PBS, medium was replaced with glucose-free medium, and drugs were added for 50 min, after which 2-deoxy-[3H]-D-glucose (50 nmol/l) was added for 10 min. For CHO-2AR cells, medium was replaced (to serum-free medium), and drugs were added for 2 h. After this, cells were washed in warm PBS, glucose-free medium was added, and drugs were re-added for 45 min, after which 2-deoxy-[3H]-D-glucose (50 nmol/l) was added for 15 min. Reactions were terminated by washing twice in ice-cold PBS, cells were digested (0.2 mol/l NaOH, 1 h, 60°C), and samples were transferred to scintillation vials with scintillant. When inhibitors were used, the time indicated with the results represents the time cells were pre-equilibrated with the inhibitors before agonists were added. All experiments were performed in duplicate with n referring to the number of independent experiments performed.
AMPK activity.
L6 cells (day 7) were serum-starved overnight, medium was replaced, and cells were treated with drugs for 2 h. Cells were washed twice with ice-cold PBS and lysed in buffer A (20 mmol/l Tris-HCl [pH 7.5], 150 mmol/l NaCl, 1 mmol/l Na2EDTA, 1 mmol/l EGTA, 1% Triton, 2.5 mmol/l sodium pyrophosphate, 1 mmol/l -glycerophosphate, 1 mmol/l Na3VO4, 1 e蘥/ml leupeptin, 1 mmol/l dithiothreitol, and 1 mmol/l phenylmethylsulfonylfluoride) for 5 min on ice. After centrifugation (14,000g, 10 min, 4°C), the supernatant was assayed for protein content, and 200 e蘥 protein (in total volume of 200 e蘬) was incubated overnight at 4°C with AMPK -subunit antibody at 1:80 dilution factor, followed by addition of 20 e蘬 50% slurry of protein-A agarose beads (Upstate Biotechnology, Lake Placid, NY) for 2 h at 4°C. Immunoprecipitates were collected by centrifugation (18,000g, 1 min, 4°C); washed twice with 500 e蘬 buffer A and twice with 500 e蘬 buffer B (240 mmol/l HEPES, pH 7.4, and 480 mmol/l NaCl); and resuspended in 30 e蘬 reaction buffer (40 mmol/l HEPES, pH 7.0, 80 mmol/l NaCl, 0.8 mmol/l dithiothreitol, and 5 mmol/l MgCl2), which contained 100 e蘭ol/l SAMS peptide (HMRSAMSGLHLVKRR). The reaction was started by the addition of 10 e蘬 ATP buffer (75 mmol/l MgCl2, 500 e蘭ol/l free ATP, and 1 e藽i/ml [-32P]ATP [3,000 Ci/mmol]). After 20 min at 30°C, the reaction was stopped by spotting 35-e蘬 samples on P81 Whatman filter papers, which were washed twice in 0.75% (vol/vol) orthophosphoric acid for 5 min and once in acetone for 5 min before drying and scintillation counting. AMPK activity is expressed as the amount of incorporated ATP (picomoles) per immunoprecipitated protein (relative to the amount of protein used for the immunoprecipitation) per minute. The final data are expressed as a percentage of the control values (3.2 ± 1.9 pmol · mineC1 · mg proteineC1).
AMP-to-ATP ratio and ATP level measurement.
L6 cells (day 7) were serum-starved overnight, new medium was added for 2 h, and cells were treated with drugs for 30 min. Cell extracts were isolated and the AMP-to-ATP ratio measured as previously described (21), except that ATP levels were measured in duplicate using a commercial kit (ATP determination kit time stable assay; Biaffin, Kassel, Germany). Results are expressed as the ratio of AMP to ATP and also as nanomoles ATP per milligram protein.
Data analysis.
All results are expressed as means ± SE of n. Data were analyzed using nonlinear curve fitting (GraphPad PRISM version 3.03) to obtain pEC50 values, where appropriate. Statistical significance was determined using paired Student’s t test. P values 0.05 were considered significant.
Drugs and reagents.
The following were gifts: the AMPK inhibitor compound C was a gift obtained from Merck Research Laboratories (Rahway, NJ). Zinterol hydrochloride was obtained from Bristol-Myers Squibb (Noble Park, Victoria, Australia). CHO-G4myc-1AAR, CHO-G4myc, CHO-K1, and CHO-2AR cells were provided by Prof. Roger J Summers (Monash University, Melbourne, Victoria, Australia).
Drugs and reagents were purchased as follows: rosiglitazone (Alexis Biochemicals, Lausen, Switzerland); 2-deoxy-[3H]-D-glucose (12 Ci/mmoleC1; Amersham Biosciences, Buckinghamshire, U.K.); G6976 and G6783 (CalBiochem, La Jolla, CA); 2,4,-dinitrophenol (Merck Schucharat OHG, Hohenbrunn, Germany); insulin (Actrapid) (Novo Nordisk, Bagsvaerd, Denmark); [-32P]ATP (3,000 Ci/mmol) (PerkinElmer Sverige, Upplands Vsby, Sweden); A23187, cirazoline, forskolin, (eC)-isoprenaline, LY294002, (eC)-norepinephrine, phenylephrine, and 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma Chemical, St. Louis, MO); AICAR (Toronto Research Chemicals, North York, Ontario, Canada); and SAMS peptide (Upstate Biotechnology).
All cell culture media and supplements were obtained from Gibco-BRL Life Technologies (Gaithersburg, MD). All antibodies were obtained from Cell Signaling Technology (Beverly, MA). All other drugs and reagents were of analytical grade.
RESULTS
1-Adrenoceptor but not 2- or -adrenoceptor activation increases AMPK phosphorylation in L6 cells and recombinant CHO-K1 cells.
The AMPK activator AICAR phosphorylated AMPK in L6 cells sixfold over basal levels (n = 55eC58), whereas insulin (n = 8) had no effect (Fig. 1A). The endogenous adrenergic ligand norepinephrine phosphorylated AMPK, with an approximate two- to threefold increase in the p-AMPKeCtoeCAMPK ratio (Fig. 1B). This effect was still present after 2 h of norepinephrine stimulation (twofold increase in p-AMPKeCtoeCAMPK ratio; data not shown). To determine which adrenergic receptor subtype(s) mediated the norepinephrine effect, L6 cells were stimulated with different subtype-specific adrenergic agonists. The -adrenoceptor agonist isoprenaline (even after 2 h of incubation; data not shown) and the 2-adrenoceptor agonist zinterol were without effect (Fig. 1C). The adenylate cyclase activator forskolin was also without effect (Fig. 1C). Phenylephrine, an 1-/2-adrenoceptor agonist, phosphorylated AMPK to a similar extent as norepinephrine (Fig. 1D). This effect was mediated by 1-adrenoceptor activation because the 1-adrenoceptor agonist cirazoline phosphorylated AMPK (Fig. 1D, which was sustained for up to 2 h; data not shown), whereas the 2-adrenoceptor agonist clonidine was without effect (Fig. 1D). These phosphorylation studies correlated well with AMPK activity measurements. AICAR and cirazoline increased AMPK activity, which was inhibited by the AMPK inhibitor compound C, whereas isoprenaline and insulin, which did not phosphorylate AMPK, did not increase AMPK activity (Fig. 2). The increases in AMPK activity after 1-adrenergic stimulation are not due to alterations in the AMP-to-ATP ratio or to decreased ATP content because cirazoline failed to significantly affect the AMP-to-ATP ratio or ATP levels compared with the positive controls rosiglitazone and dinitrophenol, which increase the AMP-to-ATP ratio and significantly reduced ATP levels (Tables 1 and 2).
To determine whether the effect of 1- and 2-adrenoceptors on AMPK phosphorylation was confined to muscle cells, we have used CHO-K1 cells transfected with either the human 1a-adrenoceptor (Fig. 3A) or human 2-adrenoceptor (Fig. 3B), which are the predominant subtypes expressed in L6 cells (12,15). The human 1a- rather than the 1b-adrenoceptor (as used by Kishi et al. [17]) was used because the 1b-adrenoceptor is exclusively expressed in liver, whereas the 1a-adrenoceptor is detected in skeletal muscle (10,12). In both cell systems AICAR but not insulin phosphorylated AMPK (threefold increase after 30 min or 2 h of stimulation, n = 4eC8) as observed in the L6 cells above. In CHOh1 cells, norepinephrine and cirazoline phosphorylated AMPK fourfold (Fig. 3A). In CHOh2 cells, norepinephrine, isoprenaline, and forskolin had no effect on basal AMPK phosphorylation levels (Fig. 3B). In CHO-K1 and CHO-G4myc cells, isoprenaline and cirazoline did not phosphorylate AMPK (data not shown).
AMPK is involved in glucose uptake mediated by 1- but not -adrenoceptor activation.
We examined whether cirazoline can increase glucose uptake in L6 cells via AMPK by using an AMPK inhibitor, compound C. AICAR-stimulated but not insulin-stimulated glucose uptake was inhibited by compound C (Fig. 4B), and compound C inhibited the phosphorylation of AMPK by AICAR (Fig. 4A). Norepinephrine-mediated glucose uptake is via both 1- and 2-adrenoceptors in L6 cells (12). Compound C partially inhibited glucose uptake by norepinephrine, but this effect was not statistically different (Fig. 4C; P = 0.16, paired Student’s t test), which could be representative of norepinephrine using both - and -adrenoceptors to increase glucose uptake. Isoprenaline-mediated glucose uptake was not inhibited by compound C (Fig. 4D), but compound C significantly inhibited cirazoline-mediated glucose uptake (Fig. 4D) and cirazoline-mediated AMPK phosphorylation (Fig. 4A).
In CHOh1 cells, cirazoline increased glucose uptake in a concentration-dependent manner (pEC50 8.4 ± 0.2; maximum increase 260 ± 11% over basal; hill slope 0.86; n = 4; data not shown). AICAR, insulin, and cirazoline increased glucose uptake, but compound C only inhibited AICAR and cirazoline-mediated increases in glucose uptake (Fig. 5A). In CHOh2 cells, isoprenaline increased glucose uptake in a concentration-dependent manner (pEC50 7.7 ± 0.4; maximum increase 131 ± 4% over basal; hill slope 0.90; n = 4; data not shown). AICAR, insulin, and isoprenaline increased glucose uptake, but only AICAR-stimulated glucose uptake was inhibited by compound C (Fig. 5B).
Calcium, but not protein kinase C (PKC), is involved in AMPK phosphorylation.
1-Adrenoceptors are Gq-coupled receptors, and their activation results in increased phosphatidylinositol turnover, activation of phospholipase C, and increased intracellular calcium levels. To investigate whether the effect of 1-adrenoceptor stimulation could be mimicked by PKC activation and increases in calcium levels, L6 cells were treated with either TPA (activator of conventional and novel PKCs; 1 e蘭ol/l) or A23187 (calcium ionophore; 1 e蘭ol/l). A23187 but not TPA was able to significantly phosphorylate AMPK (twofold increase over basal) in the time period examined (Fig. 6). Both treatments, however, were able to increase glucose uptake (A23187 1 e蘭ol/l, 207 ± 13% over basal, n = 3; TPA 1 e蘭ol/l, 211 ± 23% over basal, n = 7). TPA also had no effect on basal AMPK phosphorylation levels in CHOh1 cells (data not shown).
Glucose uptake in L6 cells in response to insulin, AICAR, or cirazoline is inhibited by G6983 (inhibits novel, conventional, and atypical PKC isoforms) but not by G6976 (inhibits novel and conventional PKC isoforms), showing an involvement of atypical PKCs (Fig. 7A). To determine whether PKCs are upstream of the signal to AMPK, two different approaches were used. First, downregulation of conventional and novel PKCs can be achieved with long-term stimulation of cells with TPA (33). After downregulation of these PKC isoforms, cirazoline (as well as AICAR) was still able to phosphorylate AMPK to the same level as cells not prestimulated with TPA (Fig. 7B), indicating that conventional and novel PKCs are not involved in the cirazoline and AICAR response. Second, AMPK phosphorylation by AICAR or cirazoline was not inhibited by either G6976 or G6983 (Fig. 7C), suggesting that no isoforms of PKC are involved in an upstream mechanism of AMPK.
1-Adrenoceptor activation phosphorylates ACC at Ser218 in L6 cells.
One downstream target of AMPK is phosphorylation of ACC2 at Ser218. In L6 cells, AICAR and cirazoline but not isoprenaline or insulin (data not shown) phosphorylated ACC2 at Ser218, and this was inhibited largely by compound C (Fig. 8).
DISCUSSION
Insulin-stimulated glucose uptake is severely compromised in type 2 diabetes, and recently, there has been great focus on the potential of AMPK in the treatment of type 2 diabetes. Insulin and AICAR both increase glucose uptake in skeletal muscle but use two distinct signaling pathways to mediate this effect that probably converge at some point. Phosphatidylinositol 3-kinase is necessary for insulin-stimulated but not AICAR-stimulated glucose uptake in skeletal muscle, whereas AMPK is necessary for AICAR-stimulated but not insulin-stimulated glucose uptake (34eC36 and D.S.H., T.B., unpublished data). The effects of AICAR, at least in skeletal muscle, are due to interactions directly resulting from AMPK activation because glucose uptake by AICAR is abolished in skeletal muscle from AMPK inactive mutant mice (37), and overexpression of a dominant-negative AMPK in rat skeletal muscle abolishes AICAR-mediated increases in glucose uptake (38,39). Of interest are the recent discoveries that hormonal and nutritional stresses have the ability to elicit their effects via AMPK, including leptin (16) and adiponectin (36,40,41). Leptin activates AMPK in skeletal muscle via two pathways: directly on skeletal muscle and also by actions in the hypothalamus to increase -adrenergic sympathetic activity to activate AMPK in skeletal muscle (16). To this extent, we have investigated the role of the endogenous ligand norepinephrine on AMPK in L6 skeletal muscle cells.
Norepinephrine stimulated AMPK phosphorylation in L6 skeletal muscle cells via 1-adrenoceptors, but not - or 2-adrenoceptors. 1-Adrenoceptor activation increases AMPK activity in mouse soleus muscle (16) and in CHO cells transfected with the 1a-adrenoceptor (this study) and 1b-adrenoceptor (17). This later study showed that Gq- (1b-adrenoceptor and bradykinin 2 receptor) and not Gi- (2a-adrenoceptor) or Gs- (2-adrenoceptor) coupled receptors cause activation of AMPK. Forskolin, 8-bromo-cAMP, and insulin were also without effect (17), and we also observed similar results in CHO cells. Other Gq-coupled receptors, such as the angiotensin type 2 receptor in rat vascular smooth muscle cells (42) and the histamine (H1) and thrombin (PAR) receptors in human umbilical vein endothelial cells (43), also activate AMPK. The data presented in this study are consistent with the notion that Gq-coupled receptors have the ability to phosphorylate AMPK. However, this is not a general phenomenon that only occurs after stimulation of Gq-coupled receptors because in adipose tissue, Gs-coupled receptors (-adrenoceptors) are capable of phosphorylating and activating AMPK (18eC21). Additionally in brown adipose tissue where 1-adrenoceptors increase glucose uptake in 3-adrenoceptor knockout mice (44), 1-adrenoceptor activation did not phosphorylate AMPK (21). These results in adipose tissue are opposite to what we observe in L6 skeletal muscle cells and CHO cells, which may suggest that in adipose tissue, regulation of AMPK by G-proteineCcoupled receptors differs from other cell types.
AMPK can be activated by two different upstream kinases, LKB1 (3eC5) and CAMKK (6eC8), via different activators, such as decreased ATP and increased AMP-to-ATP ratios for LKB1 and alterations in calcium metabolism for CAMKK. Because 1-adrenoceptor activation leads to phospholipase C activation, which results in the hydrolysis of phosphatidylinositol (4,5)-bisphosphate to produce inositol-1,4,5-phosphate that releases calcium from intracellular stores, and diacylglycerol, which can activate PKC, it is plausible to hypothesize that 1-adrenoceptors may activate AMPK via calcium. The calcium ionophore A23187 activates AMPK in LKB1eC/eC embryo fibroblasts, NIH3T3, and Hela cells (6eC8), and treatment of L6 cells with A23187 phosphorylated AMPK, making it likely that 1-adrenoceptor activation of AMPK is via CAMKK because 1-adrenoceptor activation did not decrease ATP levels or alter the AMP-to-ATP ratio in L6 cells. Further investigation of 1-adrenergic activation of AMPK is currently being investigated.
Activation of AMPK is also not secondary to changes in PKC activity despite PKC being implicated in 1-adrenoceptor mediation of glucose uptake (12,29). The phorbol ester TPA (which activates conventional and novel PKC isoforms) did not phosphorylate AMPK, and downregulation of conventional and novel PKCs with long-term TPA treatment (33) or the use of PKC inhibitors did not affect the ability of cirazoline (or AICAR) to phosphorylate AMPK, suggesting that PKCs are not involved in phosphorylation of AMPK (PKC inhibition was also ineffective in inhibiting AICAR activation of AMPK in rat papillary muscle [45]). However, 1-adrenoceptoreCand AICAR-mediated glucose uptake is very dependent on atypical PKCs, which are presumably downstream of AMPK on glucose uptake effects. These results agree with other studies investigating the interaction between AMPK and PKC. In L6 cells, AICAR treatment activated atypical PKCs, but this effect was downstream of its activation of AMPK, hence raising the possibility that atypical PKCs may be a final activator of glucose uptake for varying agonists, independent of their initial signaling pathways (46). Additionally, dinitrophenol activation of glucose uptake but not AMPK activation was sensitive to calcium chelation and PKC inhibition, suggesting that AMPK activation may be independent of calcium and PKC (47)
Focally released norepinephrine (which can reach very high levels locally in skeletal muscle) and circulating epinephrine (low levels in the blood stream and acts at many different target tissues) may have completely different effects on skeletal muscle glucose uptake and AMPK activation in vivo. Circulating epinephrine inhibits glucose uptake in skeletal muscle by insulin-dependent mechanisms, whereas norepinephrine released from sympathetic nerves (including under conditions of stress such as cold exposure and exercise) increases glucose uptake via insulin-independent mechanisms (rev. in 48). The regulation of AMPK in skeletal muscle is also G-protein dependent, with Gq-coupled receptors activating AMPK (this study; 16). To investigate the role of AMPK of adrenergically mediated glucose uptake, we have used the AMPK inhibitor compound C. This is a potent and small molecule inhibitor of AMPK that acts as a reversible inhibitor of AMPK by binding at the ATP-binding site on AMPK (49). Compound C inhibited AICAR-mediated but not insulin-mediated glucose in all cell types investigated here, and inhibited AICAR-mediated phosphorylation of AMPK, showing that its effects are mediated via AMPK inhibition. 1- but not 2-adrenoceptoreCmediated glucose uptake was inhibited by compound C, which suggests that glucose uptake mediated by 1-adrenoceptors is attributed to AMPK activation. Additionally, TPA-mediated glucose uptake was not inhibited by compound C in L6 cells (data not shown), which correlates with the inability of TPA to phosphorylate AMPK.
One function of AMPK activation is to provide energy to cells by stimulating the use of alternate fuels. AMPK phosphorylates and inactivates ACC2 at Ser218, which is a rate-limiting step in the conversion of acetyl-CoA to malonyl-CoA, resulting in increased free fatty acid oxidation. PKA can also phosphorylate ACC2 but at distinct residues from those phosphorylated by AMPK (rev. in 50). 1-Adrenoceptor and AICAR (but not isoprenaline) stimulation resulted in the phosphorylation of ACC2 at Ser218.
In summary, 1-adrenoceptor activation, but not 2- or -adrenoceptor activation, increases AMPK phosphorylation and activity in L6 skeletal muscle cells through a pathway independent of PKC. Although both 1- and 2-adrenoceptors are able to increase glucose uptake in these cells, AMPK is only involved in 1-adrenoceptoreCmediated glucose uptake.
ACKNOWLEDGMENTS
D.S.H. is a C.J. Martin Fellow from the National Health and Medical Research Council of Australia. This study was supported by the Swedish Natural Science Research Council, the Tore Nilsons Stiftelse for Medicinsk Forskning, and the Jeanssonska funds.
We gratefully acknowledge Prof. Roger J. Summers (Monash University) for providing us with the recombinant CHO-K1 cells and helpful advice. We gratefully acknowledge Prof. Barbara Cannon and Prof. Jan Nedergaard (Stockholm University, Stockholm, Sweden) for helpful discussions, Olof S. Dallner (Stockholm University, Stockholm, Sweden) for advice on AMP-to-ATP measurements, and Dr. Kei Sakamoto (University of Dundee, Dundee, U.K.) for helpful advice on the design of the AMPK activity assay.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Fryer LG, Parbu-Patel A, Carling D: The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct pathways. J Biol Chem 277:25226eC25232, 2002
Hardie DG: The AMP-activated protein kinase pathway: new players upstream and downstream. J Cell Sci 117:5479eC5487, 2004
Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, Hardie DG: Complexes between the LKB1 tumor suppressor, STRAD / and MO25 / are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2:28, 2003
Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley LC: The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 101:3329eC3335, 2004
Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D, Schlattner U, Wallimann T, Carlson M, Carling D: LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13:2004eC2008, 2003
Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, Hardie DG: Calmodulin-dependent protein kinase kinase- is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2:9eC19, 2005
Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA: The Ca++/calmoldulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280:29060eC29066, 2005
Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M, Carling D: Ca2+/calmodulin-dependent protein kinase kinase- acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2:21eC33, 2005
Martin WH III, Tolley TK, Saffitz JE: Autoradiographic delineation of skeletal muscle 1-adrenergic receptor distribution. Am J Physiol 259:H1402eCH1408, 1990
Rokosh DG, Bailey BA, Stewart AF, Karns LR, Long CS, Simpson PC: Distribution of 1C-adrenergic receptor mRNA in adult rat tissues by RNase protection assay and comparison with 1B and 1D. Biochem Biophys Res Commun 200:1177eC1184, 1994
Rattigan S, Appleby GJ, Edwards SJ, McKinstry WJ, Colquhoun EQ, Clark MG, Richter EA: -Adrenergic receptors in rat skeletal muscle. Biochem Biophys Res Commun 136:1071eC1077, 1986
Hutchinson DS, Bengtsson T: 1A-Adrenoceptors activate glucose uptake in L6 muscle cells through a phospholipase C-, phosphatidylinositol-3 kinase-, and atypical protein kinase C-dependent pathway. Endocrinology 146:901eC912, 2005
Liggett SB, Shah SD, Cryer PE: Characterization of -adrenergic receptors of human skeletal muscle obtained by needle biopsy. Am J Physiol 254:E795eCE798, 1988
Roberts SJ, Molenaar P, Summers RJ: Characterization of propranolol-resistant (eC)-[125I]-cyanopindolol binding sites in rat soleus muscle. Br J Pharmacol 109:344eC352, 1993
Nevzorova J, Bengtsson T, Evans BA, Summers RJ: Characterization of the -adrenoceptor subtype involved in mediation of glucose transport in L6 cells. Br J Pharmacol 137:9eC18, 2002
Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB: Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339eC343, 2002
Kishi K, Yuasa T, Minami A, Yamada M, Hagi A, Hayashi H, Kemp BE, Witters LA, Ebina Y: AMP-activated protein kinase is activated by the stimulations of G(q)-coupled receptors. Biochem Biophys Res Commun 276:16eC22, 2000
Moule SK, Denton RM: The activation of p38 MAPK by the -adrenergic agonist isoproterenol in rat epididymal fat cells. FEBS Lett 439:287eC290, 1998
Daval M, Diot-Dupuy F, Bazin R, Hainault I, Viollet B, Vaulont S, Hajduch E, Ferre P, Foufelle F: Anti-lipolytic action of AMP-activated protein kinase in rodent adipocytes. J Biol Chem 280:25250eC25257, 2005
Yin W, Mu J, Birnbaum MJ: Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis in 3T3eCL1 adipocytes. J Biol Chem 278:43074eC43080, 2003
Hutchinson DS, Chernogubova E, Dallner OS, Cannon B, Bengtsson T: -adrenoceptors, but not -adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1. Diabetologia 48:2386eC2395, 2005
Laurent D, Petersen KF, Russell RR, Cline GW, Shulman GI: Effect of epinephrine on muscle glycogenolysis and insulin-stimulated muscle glycogen synthesis in humans. Am J Physiol 274:E130eCE138, 1998
Sudo M, Minokoshi Y, Shimazu T: Ventromedial hypothalamic stimulation enhances peripheral glucose uptake in anesthetized rats. Am J Physiol 261:E298eCE303, 1991
Minokoshi Y, Okano Y, Shimazu T: Regulatory mechanism of the ventromedial hypothalamus in enhancing glucose uptake in skeletal muscles. Brain Res 649:343eC347, 1994
Abe H, Minokoshi Y, Shimazu T: Effect of a 3-adrenergic agonist, BRL35135A, on glucose uptake in rat skeletal muscle in vivo and in vitro. J Endocrinol 139:479eC486, 1993
Liu YL, Stock MJ: Acute effects of the 3-adrenoceptor agonist, BRL 35135, on tissue glucose utilisation. Br J Pharmacol 114:888eC894, 1995
Liu YL, Cawthorne MA, Stock MJ: Biphasic effects of the -adrenoceptor agonist, BRL 37344, on glucose utilization in rat isolated skeletal muscle. Br J Pharmacol 117:1355eC1361, 1996
Tanishita T, Shimizu Y, Minokoshi Y, Shimazu T: The 3-adrenergic agonist BRL37344 increases glucose transport into L6 myocytes through a mechanism different from that of insulin. J Biochem (Tokyo) 122:90eC95, 1997
Cheng JT, Liu IM: Stimulatory effect of caffeic acid on 1A-adrenoceptors to increase glucose uptake into cultured C2C12 cells. Naunyn Schmiedebergs Arch Pharmacol 362:122eC127, 2000
Henin N, Vincent MF, Van den Berghe G: Stimulation of rat liver AMP-activated protein kinase by AMP analogues. Biochim Biophys Acta 1290:197eC203, 1996
Corton JM, Gillespie JG, Hawley SA, Hardie DG: 5-aminoimidazole-4-carboxamide ribonucleoside: a specific method for activating AMP-activated protein kinase in intact cells Eur J Biochem 229:558eC565, 1995
Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, Beri RK: Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase. FEBS Lett 353:33eC36, 1994
Bandyopadhyay G, Standaert ML, Galloway L, Moscat J, Farese RV: Evidence for involvement of protein kinase C (PKC)- and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. Endocrinology 138:4721eC4731, 1997
Hayashi T, Hirshman MF, Kurth EJ, Winder WW, Goodyear LJ: Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47:1369eC1373, 1998
Bergeron R, Russell RR III, Young LH, Ren JM, Marcucci M, Lee A, Shulman GI: Effect of AMPK activation on muscle glucose metabolism in conscious rats. Am J Physiol 276:E938eCE944, 1999
Wu X, Motoshima H, Mahadev K, Stalker TJ, Scalia R, Goldstein BJ: Involvement of AMP-activated protein kinase in glucose uptake stimulated by the globular domain of adiponectin in primary rat adipocytes. Diabetes 52:1355eC1363, 2003
Mu J, Brozinick JT Jr, Valladares O, Bucan M, Birnbaum MJ: A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 7:1085eC1094, 2001
Fryer LG, Foufelle F, Barnes K, Baldwin SA, Woods A, Carling D: Characterization of the role of the AMP-activated protein kinase in the stimulation of glucose transport in skeletal muscle cells. Biochem J 363:167eC174, 2002
Sakoda H, Ogihara T, Anai M, Fujishiro M, Ono H, Onishi Y, Katagiri H, Abe M, Fukushima Y, Shojima N, Inukai K, Kikuchi M, Oka Y, Asano T: Activation of AMPK is essential for AICAR-induced glucose uptake by skeletal muscle but not adipocytes. Am J Physiol Endocrinol Metab 282:E1239eCE1244, 2002
Tomas E, Tsao TS, Saha AK, Murrey HE, Zhang CC, Itani SI, Lodish HF, Ruderman NB: Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci U S A 99:16309eC16313, 2002
Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Waki H, Uchida S, Yamashita S, Noda M, Kita S, Ueki K, Eto K, Akanuma Y, Froguel P, Foufelle F, Ferre P, Carling D, Kimura S, Nagai R, Kahn BB, Kadowaki T: Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med 8:1288eC1295, 2002
Nagata D, Takeda R, Sata M, Satonaka H, Suzuki E, Nagano T, Hirata Y: AMP-activated protein kinase inhibits angiotensin II-stimulated vascular smooth muscle cell proliferation. Circulation 110:444eC451, 2004
Thors B, Halldorsson H, Thorgeirsson G: Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt. FEBS Lett 573:175eC180, 2004
Chernogubova E, Hutchinson DS, Nedergaard J, Bengtsson T: 1- and 1-adrenoceptor signaling fully compensate for 3-adrenoceptor deficiency in brown adipocyte norepinephrine-stimulated glucose uptake. Endocrinology 146:2271eC2284, 2005
Russell RR III, Bergeron R, Shulman GI, Young LH: Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol 277:H643eCH649, 1999
Chen HC, Bandyopadhyay G, Sajan MP, Kanoh Y, Standaert M, Farese RV Jr, Farese RV: Activation of the ERK pathway and atypical protein kinase C isoforms in exercise- and aminoimidazole-4-carboxamide-1-beta-D-riboside (AICAR)-stimulated glucose transport. J Biol Chem 277:23554eC23562, 2002
Patel N, Khayat ZA, Ruderman NB, Klip A: Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition. Biochem Biophys Res Commun 285:1066eC1070, 2001
Nonogaki K: New insights into sympathetic regulation of glucose and fat metabolism. Diabetologia 43:533eC549, 2000
Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE: Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167eC1174, 2001
Munday MR: Regulation of mammalian acetyl-CoA carboxylase. Biochem Soc Trans 30:1059eC1064, 2002(Dana S. Hutchinson, and T)