GLUT-4 expression in skeletal muscle Chronic

Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle

S. Park, T. L. Scheffler, A. M. Gunawan, H. Shi, C. Zeng, K. M. Hannon, A. L. Grant and D. E. Gerrard Am J Physiol Cell Physiol 296:106-115, 2009. First published Oct 29, 2008; doi:10.1152/ajpcell.00114.2008 You might find this additional information useful... This article cites 64 articles, 37 of which you can access free at: http://ajpcell.physiology.org/cgi/content/full/296/1/C106#BIBL Updated information and services including high-resolution figures, can be found at: http://ajpcell.physiology.org/cgi/content/full/296/1/C106 Additional material and information about AJP - Cell Physiology can be found at: http://www.the-aps.org/publications/ajpcell

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Am J Physiol Cell Physiol 296: C106–C115, 2009. First published November 5, 2008; doi:10.1152/ajpcell.00114.2008.

Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle S. Park,1 T. L. Scheffler,1 A. M. Gunawan,1 H. Shi,1 C. Zeng,1 K. M. Hannon,2 A. L. Grant,1 and D. E. Gerrard1 Departments of 1Animal Sciences and 2Basic Medical Sciences, Purdue University, West Lafayette, Indiana Submitted 22 February 2008; accepted in final form 30 September 2008

SKELETAL MUSCLE IS A MAJOR site of glucose uptake and is critical to whole body glucose metabolism. The skeletal muscle-specific glucose transporter, GLUT-4 (14, 23, 57), mediates both insulin and contraction-stimulated glucose transport (33) by translocating to the cell membrane and facilitating glucose uptake into cells (6). Glucose transport is highly correlated with GLUT-4 mRNA and protein expression (27). The changes in gene and protein expression that contribute to muscle plasticity are the result of interplay between signaling pathways. For instance, exercise is recommended for controlling Type 2 diabetes because of its positive effect on GLUT-4 gene expression, glucose uptake, and glucose metabolism independent of the stimulation of the insulin receptor (9, 59). Therefore, signaling mechanisms related to muscle contraction may offer further insight into pathways regulating GLUT-4 expression.

Ihleman et al. (22) proposed that muscle contraction induces glucose uptake through two distinct mechanisms. First, calcium acts through a feed-forward mechanism. During contraction, membrane depolarization stimulates the release of calcium from the sarcoplasmic reticulum (SR), which leads to muscle contraction. The increased cytosolic calcium, in turn, serves as a second messenger to trigger pathways that will stimulate glucose transport before muscle senses changes in energy level (19). Although calcium can feed-forward to stimulate glucose uptake, more work is needed to determine how prolonged exposure to calcium affects gene transcription and expression. Several studies have used caffeine, an agent that causes calcium release from the SR, to mimic the effects of muscle contraction on glucose transport. Holloszy et al. (19) showed that 4 mM caffeine treatment for 120 min activates glucose transport in frog sartorius muscle. Furthermore, caffeine treatment in combination with agents that prevent calcium release from the SR, such as EGTA or dantrolene, inhibits calciuminduced GLUT-4 expression (62). In contrast, other studies (13, 31) have shown that sustained increases in cytosolic calcium caused by caffeine or calcium ionophores either inhibited or had no effect on basal glucose transport in skeletal muscle. These conflicting results are likely attributed to differences in the amplitude and duration of cytosolic calcium levels. To better establish the relationship between calcium concentration and duration of exposure on glucose transport, it may be advantageous to use an animal model with a genetic mutation that specifically affects calcium efflux from the SR. Malignant hyperthermia-susceptible pigs possess a point mutation in the halothane gene that leads to a mutated skeletal muscle ryanodine receptor (2). The calcium release channels of pigs mutant for the halothane gene (Hal) are more sensitive to agents that stimulate channel opening, thus allowing longer open time probability and resulting in enhanced calcium release (37, 38). Ihleman et al. (22) also suggested that glucose transport is induced by a feedback mechanism, whereby altered metabolic rate causes changes in energy charge, ion balance, and substrate levels. Contraction is an energetically demanding process that requires ATP to maintain energy homeostasis. Low AMP-to-ATP ratio enhances AMP-activated protein kinase (AMPK) activity, which stimulates energy producing pathways and downregulates energy-consuming pathways to preserve cellular ATP (26). AMPK is a heterotrimeric serine-threonine protein kinase composed of a catalytic ␣-subunit and regulatory ␤- and ␥-subunits. Threonine 172 in the ␣-subunit has

Address for reprint requests and other correspondence: D. E. Gerrard, 915 W. State St., Dept. of Animal Sciences, Purdue Univ., West Lafayette, IN 47907 (e-mail: [email protected]).

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adenosine 5⬘-monophosphate-activated protein kinase; glucose transporter; C2C12 cells; caffeine; ryanodine receptor

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Park S, Scheffler TL, Gunawan AM, Shi H, Zeng C, Hannon KM, Grant AL, Gerrard DE. Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle. Am J Physiol Cell Physiol 296: C106 –C115, 2009. First published November 5, 2008; doi:10.1152/ajpcell.00114.2008.—Muscle contraction stimulates glucose transport independent of insulin. Glucose uptake into muscle cells is positively related to skeletal muscle-specific glucose transporter (GLUT-4) expression. Therefore, our objective was to determine the effects of the contraction-mediated signals, calcium and AMP-activated protein kinase (AMPK), on glucose uptake and GLUT-4 expression under acute and chronic conditions. To accomplish this, we used pharmacological agents, cell culture, and pigs possessing genetic mutations for increased cytosolic calcium and constitutively active AMPK. In C2C12 myotubes, caffeine, a sarcoplasmic reticulum calcium-releasing agent, had a biphasic effect on GLUT-4 expression and glucose uptake. Low-concentration (1.25 to 2 mM) or short-term (4 h) caffeine treatment together with the AMPK activator, 5-aminoimidazole4-carboxamide-1-␤-D-ribonucleoside (AICAR), had an additive effect on GLUT-4 expression. However, high-concentration (2.5 to 5 mM) or long-term (4 to 30 h) caffeine treatment decreased AMPK-induced GLUT-4 expression without affecting cell viability. The negative effect of caffeine on AICAR-induced GLUT-4 expression was reduced by dantrolene, which desensitizes the ryanodine receptor. Consistent with cell culture data, increases in GLUT-4 mRNA and protein expression induced by AMPK were blunted in pigs possessing genetic mutations for both increased cytosolic calcium and constitutively active AMPK. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPKinduced GLUT-4 expression in skeletal muscle.

CALCIUM BLOCKS GLUCOSE UPTAKE

MATERIALS AND METHODS

Cell culture. C2C12 mouse muscle cells were plated at 4 ⫻ 104 cells per 35-mm well in 10% fetal bovine serum in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with antibiotics [1% antibiotic antimycotic solution (Sigma) and 0.1% gentamycin (Gibco)] at 37°C and 5% CO2 in air. At 80% confluence, cells were switched to a 5% heat-inactivated horse serum in DMEM with antibiotics for differentiation of myoblasts into myotubes. During this time, the medium was changed approximately every 24 h, and chemical treatments began at 5 days, when myoblasts were fully differentiated. Dose- and time-dependent treatments. After cells were fully differentiated, they were treated with 2 mM AICAR, 3 mM caffeine, 1 ␮M A-23187, or 1 mM dantrolene. The dose-dependent effects of caffeine and A-23187 on AICAR-induced GLUT-4 gene expression were analyzed at final concentrations up to 5 mM and 1 ␮M, respectively. For the time course experiment, myotubes were incubated with 3 mM caffeine for 1, 2, 4, 8, 12, 24, 48, and 72 h before the RNA extraction with or without AICAR. Trypan blue staining. To check cell viability, we performed Trypan blue staining following the procedure of Freshney (12). Myotubes AJP-Cell Physiol • VOL

were washed with PBS, submerged in 0.2 ml of 0.4% Trypan blue stain, and then incubated at room temperature for 5 min. Nonviable myotubes stained blue, whereas viable cells did not stain. Visualization of glucose uptake. A fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2NBDG; Invitrogen), was used to measure glucose uptake in C2C12 myotubes. C2C12 cells were exposed to 2 mM AICAR, 3 mM caffeine, or 1 mM dantrolene for 1 h (acute) or 30 h (chronic) at 37°C. 2-NBDG (200 ␮M) was then added to the culture medium and incubated at 37°C for 10 min. After cells were washed, fluorescence intensity of 2-NBDG at 520- to 560-nm wavelength (480-nm excitation wavelength) was measured and collected by target camera mounted on a Leica DMI6000B microscope. Images of 15–50 cells per treatment were randomly selected and digitized in 8bi. After subtracting for background, fluorescence intensity was calculated as the difference in the average fluorescence of cells before and after application of 2-NBDG using NIH ImageJ software. All cell culture results represent at least two independent experiments. Animals. Experimental procedures with the animals used in this study were approved by the Purdue University Animal Care and Use Committee. For the Hal and RN pig study, only pigs homozygous for both mutations (NN/ rn⫹rn⫹, NN/RN⫺RN⫺, nn/ rn⫹rn⫹, nn/ RN⫺RN⫺; n ⫽ 10 per treatment) were used for this experiment. Genotypes were determined using polymerase chain reaction (PCR) restriction fragment length polymorphism technique. Hal and RN PCR products were digested with appropriate restriction enzymes and separated on agarose gel following the procedures from O’Brien et al. (44) and Meadus et al. (34), respectively. At ⬃120 kg body wt, pigs were euthanized and exsanguinated following standard industry procedures. Muscle samples from longissimus dorsi (LD) were removed and frozen in liquid nitrogen. RNA isolation, cDNA synthesis, and real-time PCR. RNA from cell cultures was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with isopropanol and ethanol. Total RNA from LD muscle was extracted using the Single Step RNA isolation method (5) with modifications. cDNA was made using the random primed cDNA synthesis method. A mixture of 5 ␮g RNA, 100 ng/␮l random hexamers, and 100 ␮M dNTPs was denatured at 65°C, after which a cDNA mixture containing 10 mM DTT, 5 units Moloney murine leukemia virus reverse transcriptase (Invitrogen), 0.5 units SUPERase-In (Ambion, Indianapolis, IN), and 5⫻ First Strand Buffer (Invitrogen) was added. The cDNA mixture was incubated in a thermal cycler at 25°C for 10 min, 37°C for 50 min, and 70°C for 10 min, and was diluted to 50 ␮g/5 ␮l. Afterward, PCR master mix containing 10 pmol/␮l gene-specific primers and iQ SYBR Green Supermix (Bio-Rad, Richmond, CA) was added. GLUT-4 primer sequences for pig (forward, ACC CTT GTC CTC GCC GTC TTC TC; reverse, ACC TTC TCC GGG GCA TTC ATG A) and for C2C12 cells (forward, CAA CGT GGC TGG GTA GGC AAG GT; reverse, CGG AGA GAG CCC AGA GCG TAG TA) produced amplicons of 86 bp and 81 bp, respectively. Sequences for fatty acid translocase (FAT)/CD36 primers are ATC GTG CCT ATC CTC TGG (forward) and CCA GGC CAA GGA GGT TAA (reverse), and for peroxisome proliferator-activated receptor-␥ coactivator (PGC)-1␣ are GAG ATT CCG TAT CAC CAC C (forward) and CTT TCA GAC TCC CGC TTC (reverse). ␤-Actin and 18S were used as internal controls for mice and pigs, respectively, and primer sequences are AGG AAG TCC CTC ACC CTC CCA AAA (forward) and CAG AAG CAA TGC TGT CAC CTT CCC (reverse), and CCA TCC AAT CGG TAG TAG CG (forward) and GTA ACC CGT TGA ACC CCA TT (reverse), respectively. Real-time quantification of GLUT-4 mRNA was performed using iCycler PCR machine (Bio-Rad). Briefly, samples were amplified in separate tubes, and the increase in fluorescence was measured in real time. The relative amount of mRNA was calculated using comparative cycle threshold method. Water was used as negative control, and no PCR product was detected.

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been identified as the phosphorylation site for AMPK activity. The ␤-subunit functions as a scaffold and contains a glycogen binding domain. The ␥-subunit possesses a binding site for the allosteric activator, AMP, thus making AMPK a better substrate for upstream kinases. The compound 5-aminoimidazole4-carboxamide-1-␤-D-ribonucleoside (AICAR) is metabolized to an AMP analog, and therefore is used experimentally to increase AMPK activity (16, 35). Chronic activation of the metabolic sensor AMPK is associated with endurance exercise and AICAR-induced increases in GLUT-4 expression and translocation in skeletal muscle (20, 24, 29, 41, 45, 46). Additionally, various genetic mutations have been used in animal models to investigate the effects of AMPK. Overexpressing a kinase-dead ␣2-AMPK plasmid or knocking out the catalytic ␣2-isoform completely abolishes AICAR-stimulated glucose uptake (25, 41). A point mutation in the Rendement Napole (RN) gene in pigs causes an amino acid change in the muscle-specific isoform of the ␥-regulatory subunit of AMPK (39). The skeletal muscle of RN pigs has higher glycogen content (11) and greater oxidative capacity than skeletal muscle of wild-type pigs (10, 30). Transgenic mice with the mutant form (R225Q) of AMPK ␥3-subunit show enhanced work performance and glycogen resynthesis rate after exercise (42). Additionally, mutation in ␥-subunit eliminates allosteric regulation by AMP/ATP, resulting in increased basal AMPK activity (1, 3, 15). The role of AMPK in sensing energy status as well as the profound effects of the ␥3-mutations on muscle phenotype suggest that AMPK is a key regulator of GLUT-4 expression. The combined effects of calcium and AMPK activity on GLUT-4 expression are not well established. It seems plausible that both duration of exposure and calcium concentration, in combination with AMPK activation, differentially affect GLUT-4 expression. Our data show that chronic caffeine exposure blunts increased GLUT-4 expression caused by AICAR-induced AMPK activation in C2C12 myotubes. These results were confirmed using Hal and RN mutant pigs as an animal model. Altogether, these data support that chronic exposure to elevated cytosolic calcium concentration blunts the effects of AMPK activation on GLUT-4 expression.

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RESULTS

Quantitative PCR for GLUT-4 mRNA expression. ␤-Actin and 18S gene expression, the controls to test for experimental variation, did not differ between treatments throughout this study (data not shown). GLUT-4 mRNA expression levels were assessed in C2C12 myotubes treated with 2 mM AICAR, 3 mM caffeine, or 1 mM dantrolene for 30 h (Fig. 1A). AICAR, but not caffeine, increased GLUT-4 mRNA expression (P ⬍ 0.05) in myotubes. Interestingly, AICAR-induced GLUT-4 mRNA expression was decreased to control level in the presence of caffeine. The inhibitory effect of caffeine on AICARinduced GLUT-4 mRNA expression was numerically reduced, but it was not significantly different with inclusion of dantrolene, a chelating agent that blocks calcium release from the SR (Fig. 1A). Correspondingly, incubation of caffeine (30 h) AJP-Cell Physiol • VOL

Fig. 1. Glucose transporter 4 (GLUT-4) mRNA and protein expression in C2C12 myotubes. A: effects of 30-h treatment of 2 mM 5-aminoimidazole-4carboxamide-1-␤-D-ribonucleoside (AICAR) and 3 mM caffeine in the absence or presence of 1 mM dantrolene on GLUT-4 mRNA expression of C2C12 myotubes. Means with different lowercase letters are statistically different (P ⬍ 0.05). B: GLUT-4 protein level from myotubes treated with 2 mM AICAR, 3 mM caffeine, and both (AIC⫹caf) for 30 h. *P ⬍ 0.05, significantly different from all other groups. Data are means ⫾ SE of 6 samples per group.

alone did not significantly affect GLUT-4 protein expression in C2C12 myotubes (Fig. 1B). In contrast, GLUT-4 protein expression was increased by 30 h incubation with AICAR (P ⬍ 0.05). However, GLUT-4 protein expression in C2C12 myotubes treated with caffeine in combination with AICAR was similar to control (Fig. 1B). Time- and dose-dependent response. GLUT-4 mRNA expression in myotubes was increased (P ⬍ 0.05) within 4 h of AICAR or caffeine treatment compared with control (Fig. 2). With AICAR treatment, GLUT-4 mRNA expression remained elevated after 4 h (P ⬍ 0.05). However, at 8 h, GLUT-4 mRNA expression in caffeine- and AICAR ⫹ caffeine-treated cells was decreased to control levels and was lower compared with AICAR-treated cells (P ⬍ 0.05; Fig. 2). When C2C12 cells were incubated for 30 h in medium containing 4 mM AICAR with different levels of caffeine, lower concentrations of caffeine (1.25 and 1.875 mM) further increased AICAR-induced GLUT-4 gene expression (P ⬍ 0.05; Fig. 3A). Higher concentrations of caffeine (2.5 to 5 mM)

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Western blot analysis. Muscle tissues powdered in liquid nitrogen and myotubes cultured in six-well plates were homogenized in icecold RIPA buffer containing 50 mM Tris 䡠 HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mM Na3VO4, 1 mM NaF, and 1 ␮g/ml aprotinin, leupeptin, and pepstatin with phosphatase inhibitor cocktail 1 and 2 (Sigma, St. Louis, MO). Samples were sonicated on ice for 5 s and centrifuged for 10 min at 10,000 g at 4°C. Protein concentration was determined using BCA Protein Assay kit (Pierce, Rockford, MI). Then, 30 ␮g protein per sample was resolved by SDS-PAGE. Separated proteins were transferred to polyvinylidene difluoride membranes, blocked, and immunoblotted with primary antibodies specific for acetyl CoA-carboxylase (ACC), phosphorylated ACC (pACC), AMPK, phosphorylated AMPK (pAMPK), Ca2⫹/calmodulin-dependent kinase II (CaMKII), phosphorylated CaMKII, ␣-tubulin, and GLUT-4. Secondary antibodies conjugated with horseradish peroxidase (1:1,000) were applied for 1 h. Bands were visualized using enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) and quantified using NIH ImageJ software. AMPK activity. AMPK activity was measured using SAMS peptide (54, 60). Briefly, powdered LD muscles were homogenized in a Polytron homogenizer with lysis buffer. After centrifugation at 13,000 g for 5 min at 4°C, 10 ␮l of supernatant was incubated for 10 min at 37°C in 40 mM HEPES, 0.2 mM SAMS peptide (HMRSAMSGLHLVKRR; GenScript, Piscataway, NJ), 80 mM NaCl, 8% wt/vol glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, and 0.2 mM ATP ⫹ 2 ␮Ci [32P]ATP (PerkinElmer, Waltham, MA), pH 7.0. An aliquot was spotted on a Whatman P81 paper. Filter papers were washed three times with 1% phosphoric acid for 10 min, and radioactivity was quantified. The activity was expressed as the phosphorylation of mM peptide 䡠 min⫺1 䡠 g muscle⫺1. Chemicals. AICAR was purchased from Toronto Research Chemicals (Toronto, ON, Canada). Antibodies for ACC, pACC, AMPK, pAMPK, CaMKII, and pCaMKII were purchased from Cell Signaling (Beverly, MA). Anti-␣-tubulin was purchased from AbCam (Cambridge, MA). Anti-GLUT-4 antibody was purchased from Biogenesis (Brentwood, NH). The horseradish peroxidase-conjugated donkey anti-rabbit IgG was purchased from Jackson ImmunoResearch (West Grove, PA). ECL reagents were obtained from Amersham. 2-NBDG was purchased from Invitrogen. Calcium-releasing agents (caffeine and A-23187), insulin, dantrolene, and all other chemicals were obtained from Sigma. Statistical analysis. Data are represented as means ⫾ SE. Differences between individual means were assessed with the PROC GLM procedure of SAS. Least square means were generated using the LSMEANS and Tukey’s adjustment for multiple comparisons. The PROC MIXED procedure was used to evaluate the effect of caffeine on GLUT-4 mRNA expression at different times. Statistical significance was established at P ⬍ 0.05.


with AICAR decreased GLUT-4 gene expression compared with AICAR treatment alone (P ⬍ 0.05). AICAR-induced GLUT-4 gene expression levels were similar (0.1, 0.2, and 0.8 ␮M) or higher (0.4 ␮M) with inclusion of A-23187. Similar to caffeine, higher concentrations of A-23187 (1 ␮M) decreased AICAR-induced GLUT-4 expression to control level (Fig. 3B). Cell viability and number. C2C12 myotubes were stained with Trypan blue (Fig. 4). After cells were treated with methanol, the majority of cells were not viable as indicated by absorption of Trypan blue stain (Fig. 4A). Control cells and those treated with 2 mM AICAR, 4 mM caffeine, or combination did not take up Trypan blue stain, and no obvious differences in cell morphology were observed (Fig. 4, B–E). However, when C2C12 myotubes were treated with both 4 mM AICAR and 6.25 mM caffeine, some staining was observed, indicating that greater than 4 mM caffeine may harm cell viability (Fig. 4F). Phosphorylation of AMPK and ACC and AMPK activity in C2C12 myotubes. Phosphorylation of AMPK ␣-subunit at Thr172 site, which is an indication of AMPK activity, was measured from control, AICAR-, caffeine-, and AICAR ⫹ caffeine-treated C2C12 myotubes (Fig. 5A). Thirty-hour incubation of C2C12 myotubes with 2 mM AICAR increased AMPK phosphorylation (P ⬍ 0.05; Fig. 5A). Administration of 3 mM caffeine alone for 30 h did not significantly affect AMPK phosphorylation. The combination of AICAR and caffeine resulted in similar AMPK activation compared with control (Fig. 5A), suggesting that caffeine inhibits AICARinduced AMPK phosphorylation. Phosphorylation of ACC on its regulatory Ser79, which is substrate of AMPK, was similar to that of AMPK (Fig. 5B). AMPK activity using SAMS peptide corresponded to phosphorylation of AMPK and ACC (Fig. 5C). Real-time fluorescence 2-NBDG uptake. Acute (1 h) treatment of AICAR, caffeine, or both increased 2-NBDG uptake (Fig. 6A) in C2C12 myotubes by 1.8-fold compared with control (P ⬍ 0.01). The addition of dantrolene to caffeine inhibited caffeine-stimulated 2-NBDG uptake (P ⬍ 0.01 vs. caffeine), whereas it did not inhibit AICAR-induced 2-NBDG AJP-Cell Physiol • VOL

uptake. 2-NBDG uptake in the dantrolene ⫹ AICAR ⫹ caffeine group remained higher (1.6-fold compared with control level; P ⬍ 0.01). After 30-h incubation (Fig. 6B), however, 2-NBDG uptake in caffeine- and AICAR ⫹ caffeine-treated cells was decreased to control levels. Dantrolene reduced the inhibitory effect of caffeine on AICAR-induced 2-NBDG uptake (Fig. 6B). The patterns in glucose transport observed after 30 h parallel the changes observed in GLUT-4 mRNA expression. Expression of GLUT-4, phosphorylation of AMPK and ACC from mutant pigs. Interestingly, RN mutant pigs had increased GLUT-4 gene expression compared with control (P ⬍ 0.08), Hal (P ⬍ 0.05), and Hal-RN (P ⬍ 0.05; Fig. 7A). The membrane fraction of GLUT-4 protein was consistent with the GLUT-4 mRNA expression (Fig. 7B). Activated AMPK from Hal and Hal-RN mutant pigs was not different from that of wild-type pigs (Fig. 8A). However, muscle from RN mutant pigs showed increased AMPK phosphorylation (P ⬍ 0.05; Fig. 8A). Phosphorylation of ACC exhibited a similar pattern as AMPK phosphorylation (Fig. 8B). AMPK activity measured in assay media without AMP was significantly higher in RN pigs compared with wild-type pigs (Fig. 8C). AMPK activity from Hal pigs was not different from that of wild-type pigs (Fig. 8C). In Hal-RN double-mutant pigs, RN-induced AMPK ac-

Fig. 3. A: GLUT-4 expression in myotubes incubated for 30 h in the absence (black bar) or presence (open bar) of 4 mM AICAR and with AICAR and different concentrations of caffeine (hatched bars). B: expression of GLUT-4 in myotubes incubated for 30 h in the absence (black bars) or presence (white bar and hatched bars) of 2 mM AICAR and different concentrations of calcium ionophore (A-23187). Data are means ⫾ SE of 4 samples per group. Means with different lowercase letters are statistically different (P ⬍ 0.05).

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Fig. 2. Time-dependent effects of 2 mM AICAR and 3 mM caffeine on relative GLUT-4 gene expression in C2C12 myotubes. Data are means ⫾ SE of 4 samples per group (F, control; E, AICAR; Œ, caffeine; ‚, AICAR and caffeine-treated myotubes). *P ⬍ 0.05 vs. all other groups within each time point. Means with different lowercase letters are statistically different (P ⬍ 0.05) within each time point.

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tivity was decreased to the level observed in wild-type pigs (Fig. 8C). Quantitative PCR for PGC-1␣ and CD36 mRNA expression. Gene expression of PGC-1␣, which functions in mitochondrial biogenesis, was significantly increased in muscles from mutant pigs (RN, Hal, and Hal-RN) compared with muscles from normal pigs (Fig. 9A; P ⬍ 0.05). RN and Hal mutant pigs showed significantly higher fatty acid translocase (FAT/CD36) mRNA expression than muscles from normal pigs (P ⬍ 0.05), and the effect of these mutations on FAT/CD36 was additive (Fig. 9B; P ⬍ 0.01). Phosphorylation of CaMKII. CaMKII is one of the calcium signals modulating skeletal muscle function and adaptation. CaMKII was detected at 56 kDa and 75 kDa (Fig. 10); these bands correspond to ␥/␦-isoforms and muscle-specific ␤-isoform, respectively (48). Hal-bearing muscles had higher CaMKII phosphorylation compared with muscles from normal pigs (P ⬍ 0.05). DISCUSSION

Our results suggest the biphasic effect of calcium on AMPK induced GLUT-4 expression is due to the amplitude and duration of the calcium transient. With short- or long-term exposure to AICAR, C2C12 myotubes showed an increase in AMPK and ACC phosphorylation, thereby increasing GLUT-4 expression and 2-NBDG uptake. At 30 h, lower concentrations (⬃2 mM) of caffeine had an additive effect on AICAR-induced GLUT-4 expression, consistent with results from Wright et al. (62). Short-term treatment of caffeine (3 mM) also had a positive effect on 2-NBDG uptake but was not additive to AICAR. However, the increase in GLUT-4 expression induced by AICAR was blocked by continuous exposure (30 h) of C2C12 myotubes to 1 ␮M of the calcium ionophore A-23187 or 3 mM caffeine, with no effect on cell viability. Dantrolene, which blocks the increases in cytosolic calcium by insensitizing the ryanodine receptor, attenuated the decrease in GLUT-4 expression induced by caffeine in myotubes treated with AJP-Cell Physiol • VOL

AICAR and caffeine. Dantrolene also weakened the decremental effect of caffeine on AICAR-induced 2-NBDG uptake. Thus, dantrolene supports the function of cytosolic calcium on AMPK-induced GLUT-4 expression. It has been reported that the GLUT-4 protein is detected at very low levels, which are insufficient to establish insulininduced glucose transport in C2C12 myotubes (28, 56). However, others (52, 53) have found C2C12 myotubes contain GLUT-4 and have showed insulin responsiveness of glucose uptake in these cells. Furthermore, insulin-induced glucose transport has been shown to be involved with GLUT-4 (43, 52). This result was confirmed by showing that indinavir, a human immunodeficiency virus protease inhibitor that also blocks GLUT-4, inhibits GLUT-4 translocation and insulinstimulated glucose transport in muscle tissue and C2C12 cell culture. In the present study, C2C12 myotubes showed detectable GLUT-4 protein, and AICAR increased GLUT-4 protein in accordance with the increase in GLUT-4 mRNA level and 2-NBDG uptake. Although it was not tested in our study, AICAR-induced GLUT-4 expression could be related to increase in PGC-1␣ content, which interacts with several nuclear transcription factors and promotes GLUT-4 expression (36, 49, 55). Thus, further studies are necessary to resolve the discrepancies in GLUT-4-induced glucose transport in the C2C12 cell line. The calcium-releasing agents used in our study, calcium ionophore (A-23187) and caffeine, possess different mechanisms for increasing cytosolic calcium. A-23187 transports calcium across biological membranes and is a known metabolic uncoupler, whereas caffeine specifically sensitizes the ryanodine receptor (51). To address the possibility that our results were influenced by a side effect of caffeine, inhibition of phosphodiesterase (PDE), we confirmed that a PDE inhibitor, pentoxifylline, did not alter GLUT-4 expression (data not shown). The stimulation of calcium efflux from the SR into the cytosol by caffeine in cell culture is more similar mechanistically to the Hal mutation because both affect sensitivity of the

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Fig. 4. Phase-contrast microscopy of differentiated C2C12 cells after Trypan blue staining. Media were treated for 30 h with methanol (positive control; A), control (B), 2 mM AICAR (C), 4 mM caffeine (D), 2 mM AICAR and 4 mM caffeine (E), or 2 mM AICAR and 6.25 mM caffeine (F).


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Fig. 5. Effect of 30-h incubation with AICAR (2 mM), caffeine (3 mM), and both on phosphorylation of AMPK (pAMPK; A) and acetyl CoA-carboxylase (pACC; B) and AMPK activity using SAMS peptide (C) in C2C12 myotubes. Data are means ⫾ SE of 4 samples per group. *P ⬍ 0.05 vs. all other groups.

ryanodine receptor. Together, these studies demonstrate that chronic calcium treatment inhibits the ability of AMPK to stimulate GLUT-4 expression. Previous studies (13, 31) have reported that sustained increases in cytosolic calcium by calcium ionophores or caffeine inhibit or have no effect on basal GLUT-4 expression in skeletal muscle. However, the abrogation of AMPK-induced GLUT-4 expression by chronic calAJP-Cell Physiol • VOL

Fig. 6. 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose (2NBDG) uptake in C2C12 myotubes. A: effects of 1-h treatment of 2 mM AICAR and 3 mM caffeine in the absence or presence of 1 mM dantrolene on 2-NBDG uptake of C2C12 myotubes. B: effects of 30-h treatment on 2-NBDG uptake of C2C12 myotubes. *P ⬍ 0.05 vs. all other groups.

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cium is novel, and our findings demonstrate a possible interaction between calcium-related signaling pathways and AMPK activation. Pigs with the RN mutation possess a missense mutation (Arg225 3 Gln) in the domain of ␥3 responsible for AMP binding. Adams et al. (1) and Barnes et al. (3) suggested that the RN mutation leads to a loss of AMP dependency for phosphorylation of AMPK, resulting in higher basal AMPK activity. In our study, muscle from RN mutant pigs had increased AMPK phosphorylation, which corresponded with enhanced GLUT-4 expression, and is consistent with the high glycogen content in muscles from RN pigs (7). Also, the pattern of AMPK activity measured with SAMS peptide was similar to data for phosphorylation of AMPK (Fig. 8C). Because AMP would be expected to induce activation in wildtype AMPK, we measured AMPK activity in assay buffer without AMP. AMPK activity was significantly increased in RN pigs. Hal tended to blunt the RN effect, evidenced by the numerically reduced AMPK activity in Hal-RN double mutant pigs. Increased AMPK activity in Hal pigs may be the result of the stress susceptibility of these mutant pigs (50). The Hal mutation may contribute to higher basal calcium that blunts the increase in AMPK activity due to the RN mutation, but the stress of the slaughtering process may have been sufficient to increase the AMP-to-ATP ratio and partly activate AMPK. There are several conflicting reports regarding the effect of the RN mutation on AMPK activity. Milan et al. (39) reported

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a portion of the exercise-induced increase in glucose transport. It seems, therefore, that calcium is also involved with mediating contraction-induced glucose transport. Taking this into account, it is particularly interesting that chronic calcium treatment alone does not affect basal AMPK activity or GLUT-4 expression in either caffeine-treated myotubes or skeletal muscle from Hal mutant pigs. In normal cells, basal calcium level is tightly maintained close to 0.1 ␮M, which is approximately 10,000-fold lower than that in SR (4). In Hal mutant muscle, the ryanodine receptor is “leaky,” leading to chronically elevated cytosolic calcium (63). The


Fig. 7. GLUT-4 gene expression (A) and protein abundance of the membrane portion (B) of the longissimus dorsi muscle from normal, Rendement Napole (RN), halothane (Hal), and Hal-RN pigs. Data are means ⫾ SE of observations from 10 animals (A) and 3 animals (B) per group. Means with different lowercase letters are statistically different (P ⬍ 0.05). *P ⬍ 0.05 vs. all other groups.

decreased AMPK activity in RN mutant pigs. Limited information is provided regarding muscle sample collection in Milan et al.’s (39) study. Given that metabolic processes in muscle are still occurring after slaughter, phosphorylation status and activity of AMPK may change during the postmortem period. Therefore, sampling time may contribute to these discrepancies. Yu et al. (65) also showed decreased ␣2-specific isoform activity in ␥3 R225Q mutant mice. However, Barnes et al. (3) demonstrated that the R225Q mutation is associated with higher basal AMPK activity with diminished AMPK dependence. Moreover, Costford et al. (8) recently identified ␥3 R225W mutation in humans. Differentiated satellite cells from ␥3 R225W carriers possess nearly a twofold increase in basal and AMP-activated AMPK activity, and muscle from these subjects contains a 90% increase in glycogen. Regardless, the inconsistency between phosphorylation status and AMPK activity in our data is surprising. This may be due to the dynamic nature of energy metabolism in muscle. Further investigation of the mechanisms of temporal and spatial regulation of AMPK activity is needed. Similarly, administration of AICAR promotes AMPK phosphorylation and increases GLUT-4 mRNA expression and protein. Activation of glucose uptake by AICAR is completely abolished in transgenic mice expressing a dominant-negative form of AMPK (40). However, contraction by electrical stimulation partially restores glucose uptake in these kinase-dead mice, indicating that activation of AMPK may account for only AJP-Cell Physiol • VOL

Fig. 8. Phosphorylation of AMPK (A) and ACC (B) and AMPK activity (C) in the longissimus dorsi muscle from normal, RN, Hal, and Hal-RN mutant pigs. Data are means ⫾ SE of 3 muscles per group. Means with different lowercase letters are statistically different (P ⬍ 0.05). *P ⬍ 0.05 vs. all other groups.

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calcium level in Hal mutant pigs is estimated at 0.33– 0.48 ␮M, which is ⬃3.5 times higher than normal, yet calcium threshold for contraction is 0.75–1.00 ␮M (32). Rose and Hargreaves (47) reported that exercise increases activity of CaMKII, one of the Ca2⫹/calmodulin-regulated protein kinase, in skeletal muscle. We showed increase in phosphorylation of CaMKII in Hal-bearing muscles (Fig. 10), implying that increase in calcium level by Hal mutation can cause CaMKII activation. This suggests that perhaps the transients in calcium amplitude are necessary to induce changes in glucose transport. Additionally, studies (62, 64) have shown that treatment of myotubes with caffeine (3–3.5 mM for 15–30 min) can induce calcium release and glucose transport without triggering contraction. In our study, continuous exposure of caffeine over 12 h did not change GLUT-4 expression, providing evidence that cytosolic calcium has different effects on glucose transport depending on its duration and amplitude. Therefore, it seems plausible that the stress response or exercise in Hal mutant pigs would provide adequate calcium concentration to stimulate contraction-mediated glucose transport pathways. Once Hal mutant pigs are stimulated, calcium floods the cytoplasm, and resequestering this excess calcium and maintaining muscle relaxation consumes large amounts of ATP. Consequently, cellular energy charge is lowered and aerobic and anaerobic metabolism is hastened. This contraction-induced, heightened calcium level would likely increase glucose uptake. AJP-Cell Physiol • VOL

Interestingly, RN induced increases in AMPK phosphorylation and GLUT-4 expression are blunted in Hal-RN mutant pigs. Although elevated calcium caused by the Hal mutation presumably may not be enough to trigger calcium-related contraction pathways, this does not exclude the possibility that calcium acts as a second messenger and affects AMPK activity via other signaling pathways. Because AMPK activity is regulated by phosphorylation and dephosphorylation events, the relationships between calcium and upstream kinases and phosphatases are particularly intriguing. Ca2⫹/calmodulin-dependent kinase kinase (CaMKK) and LKB1 are proposed upstream kinases of AMPK (17, 18, 21, 61). Under chronic conditions, cytosolic calcium may help block AMPK phosphorylation through these upstream kinases. Alternatively, calcium may enhance AMPK dephosphorylation by increasing protein phosphatase activity. In addition, the possibility exists that there is a “set” minimum AMPK activity that cannot be diminished. If the control level and minimum level of phosphorylated AMPK are similar, this would conceal any negative effects induced by chronic, elevated calcium on AMPK phosphorylation and glucose transport. Moreover, calcium may indirectly affect AMPK or phosphatase activity by affecting metabolic pathways. AMPK and calcium may facilitate adaptation by contributing to transitions in energy metabolism and muscle phenotype. Curiously, Hal-RN pigs have higher glycogen level than normal pigs (7). We expected that lower GLUT-4 expression level in Hal-RN pigs would correspond with lower glycogen levels since glucose transport is considered the rate-limiting step for glycogen synthesis. We hypothesized that elevated glycogen in Hal-RN pigs is due to a greater reliance on fatty acid metabolism, thus increasing energy efficiency and sparing glycogen. Expression of fatty acid transporter, FAT/CD36, is related to fatty acid uptake in skeletal muscle (58). Our data show increased FAT/ CD36 in Hal-RN double-mutant pigs (Fig. 9B). Increased mRNA expression of PGC-1␣ (Fig. 9A), which functions in mitochondrial biogenesis, supports increased fatty acid oxidation in Hal-RN pigs. However, the increased stress suscepti-

Fig. 10. Phosphorylation of Ca2⫹/calmodulin-dependent kinase II (CaMKII) in longissimus dorsi muscles from normal, RN, Hal, and Hal-RN mutant pigs. Data are means ⫾ SE of 3 muscles per group. *P ⬍ 0.05 vs. all other groups.

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Fig. 9. Peroxisome proliferator-activated receptor-␥ coactivator (PGC-1␣; A) and fatty acid translocase (FAT)/CD36 (B) mRNA expression in longissimus dorsi muscles from wild-type, RN, Hal, and Hal-RN pigs by real-time PCR. Means with different lowercase letters are statistically different (P ⬍ 0.05). *P ⬍ 0.05 vs. normal (n ⫽ 4 in each group).

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bility of Hal pigs suggests that glycolysis would be preferable. Certainly, more work will be necessary to elucidate the complex relationship between AMPK, calcium and muscle energy metabolism. In conclusion, this study provides valuable insights into how cytosolic calcium levels affect AMPK phosphorylation and GLUT-4 gene expression. Using C2C12 myotubes, we have shown that calcium affects GLUT-4 expression in a time- and dose-dependent manner. Prolonged exposure to elevated caffeine concentrations inhibits AICAR-induced AMPK phosphorylation and GLUT-4 expression. Consistent with the cell culture study, abnormal calcium homeostasis blunts AMPK phosphorylation and GLUT-4 expression in Hal-RN mutant pigs. Altogether, these data suggest that chronic exposure to elevated cytosolic calcium concentration blocks AMPK-induced increases in GLUT-4 mRNA and protein expression in skeletal muscle. Further investigation into the mechanisms by which calcium modulates AMPK phosphorylation will provide a better understanding of how muscle contraction mediates improvements in GLUT-4 expression and glucose metabolism.


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