AMPK 2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle

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1 J Appl Physiol 111: , First published May 5, 2011; doi: /japplphysiol AMPK 2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle Marcia J. Abbott, 1 Lindsey D. Bogachus, 1 and Lorraine P. Turcotte 1,2 Departments of 1 Biological Sciences and 2 Kinesiology, University of Southern California, Los Angeles, California Submitted 19 July 2010; accepted in final form 27 April 2011 Abbott MJ, Bogachus LD, Turcotte LP. AMPK 2 deficiency uncovers time dependency in the regulation of contraction-induced palmitate and glucose uptake in mouse muscle. J Appl Physiol 111: , First published May 5, 2011; doi: /japplphysiol AMP-activated protein kinase (AMPK) is a fuel sensor in skeletal muscle with multiple downstream signaling targets that may be triggered by increases in intracellular Ca 2 concentration ([Ca 2 ]). The purpose of this study was to determine whether increases in intracellular [Ca 2 ] induced by caffeine act solely via AMPK 2 and whether AMPK 2 is essential to increase glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle. Hindlimbs from wild-type (WT) or AMPK 2 dominant-negative (DN) transgene mice were perfused during rest (n 11), treatment with 3 mm caffeine (n 10), or muscle contraction (n 11). Time-dependent effects on glucose and FA uptake were uncovered throughout the 20-min muscle contraction perfusion period (P 0.05). Glucose uptake rates did not increase in DN mice during muscle contraction until the last 5 min of the protocol (P 0.05). FA uptake rates were elevated at the onset of muscle contraction and diminished by the end of the protocol in DN mice (P 0.05). FA oxidation rates were abolished in the DN mice during muscle contraction (P 0.05). The DN transgene had no effect on caffeine-induced FA uptake and oxidation (P 0.05). Glucose uptake rates were blunted in caffeine-treated DN mice (P 0.05). The DN transgene resulted in a greater use of intramuscular triglycerides as a fuel source during muscle contraction. The DN transgene did not alter caffeine- or contraction-mediated changes in the phosphorylation of Ca 2 /calmodulin-dependent protein kinase I or ERK1/2 (P 0.05). These data suggest that AMPK 2 is involved in the regulation of substrate uptake in a time-dependent manner in contracting muscle but is not necessary for regulation of FA uptake and oxidation during caffeine treatment. ERK1/2; muscle contraction; caffeine; calcium/calmodulin-dependent protein kinase kinase; acetyl-coa carboxylase Address for reprint requests and other correspondence: L. P. Turcotte, Dept. of Kinesiology, Dept. of Biological Sciences, College of Letters, Arts, and Sciences, Univ. of Southern California, 3560 Watt Way, PED 107, Los Angeles, CA ( turcotte@usc.edu). EVIDENCE SUGGESTS THAT MULTIPLE cellular signals regulate the changes in substrate metabolism with muscle contraction, including the AMP-activated protein kinase (AMPK) signaling cascade (2, 34, 35, 52). During states of low energy, such as muscle contraction or exercise, it is widely accepted that liver kinase B1 (LKB1) acts as an upstream AMPK kinase that phosphorylates and activates AMPK and initiates a multitude of signaling events to maintain energy homeostasis (39 41). Although strong evidence indicates that the LKB1-AMPK cascade is a key signaling pathway that regulates changes in glucose uptake, fatty acid (FA) uptake, and FA oxidation in contracting skeletal muscle (16, 25, 54, 56), data also show that AMPK may not be the sole signal mediating contractioninduced changes in glucose uptake, FA uptake, and FA oxidation (35, 36, 56). Along with AMPK, mounting data suggest that increases in intracellular Ca 2 concentration ([Ca 2 ]) may play a key role in the regulation of glucose uptake, FA uptake, and FA oxidation in skeletal muscle and that this may occur in part via AMPK activation (3, 28, 51, 53, 56, 57). There is evidence that Ca 2 /calmodulin-dependent protein kinase (CaMK) kinase (CaMKK) activates AMPK in vitro in cells, whole skeletal muscle, and yeast (17, 20, 21, 55), and we recently showed that activation of CaMKK and CaMKII leads to activation of AMPK in perfused rat hindlimbs (1, 35). Additionally, we showed that inhibition of CaMKK or CaMKII reduces glucose uptake, FA uptake, and FA oxidation (1, 35). However, it is unclear whether Ca 2 signaling relies extensively on the activation of AMPK to mediate its metabolic effects or whether there are Ca 2 -dependent AMPK-independent regulators of glucose uptake, FA uptake, and FA oxidation. Furthermore, it is unclear whether AMPK acts upstream of alternative signaling pathways that include extracellular signal-regulated kinases 1 and 2 (ERK1/2) in the regulation of contraction-induced substrate metabolism. A recent study found that AMPK does not lie upstream of ERK1/2 following muscle contraction (11); however, another study measured an increase in ERK1/2 phosphorylation in AMPK transgenic mouse skeletal muscle following treadmill exercise (26). To study the role of AMPK 2 in the regulation of glucose uptake, FA uptake, and FA oxidation in skeletal muscle during increased intracellular [Ca 2 ] induced by caffeine treatment and muscle contraction, we employed a transgenic mouse model possessing a dominant-negative (DN) 2 -isoform of AMPK in skeletal muscle and heart muscle (32). Multiple studies have sought to determine the importance of AMPK in the regulation of glucose and FA metabolism. However, studies utilizing transgenic mouse models where AMPK is either inactive or knocked out reveal conflicting results (24 26, 30). Some studies suggest that AMPK is necessary in the regulation of glucose and FA metabolism (24, 25), whereas other studies show that AMPK is not necessary to measure increases in substrate metabolism during exercise or other metabolic challenges (26, 30). Therefore, the purpose of this study was to determine 1) whether a caffeine-induced increase in intracellular [Ca 2 ] regulates muscle glucose uptake, FA uptake, and FA oxidation solely via AMPK 2 activation and 2) whether the 2 -subunit of AMPK is essential to increase glucose uptake, FA uptake, and FA oxidation in contracting skeletal muscle in a hindlimb perfusion system. Although the perfusion system is not as physiologically relevant as tracer studies in live animals, it allowed us to measure glucose uptake, FA uptake, and FA oxidation at various time points and provided an in situ model /11 Copyright 2011 the American Physiological Society 125

2 126 SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION We hypothesized that AMPK 2 is necessary to measure increases in glucose and FA uptake, as well as FA oxidation, in skeletal muscle during a caffeine-induced increase in intracellular [Ca 2 ]. We further hypothesized that AMPK 2 plays a role in fuel use in contracting skeletal muscle but that it is not the sole signal involved in the aforementioned regulation. MATERIALS AND METHODS Animal preparation. Male C57BL/6 mice ( g body wt, 2 3 mo old) expressing an AMPK 2 DN transgene (kindly provided by M. J. Birnbaum, University of Pennsylvania, Philadelphia, PA) in cardiac and skeletal muscle (32) and their wild-type (WT) littermates were kept on a 12:12-h light-dark cycle; standard chow and water were provided ad libitum. Animals were randomly divided into three experimental groups: rest (n 11), caffeine treatment (n 10), and muscle contraction (n 11). All procedures for the present study were approved by the Institutional Animal Care and Use Committee at the University of Southern California. Hindlimb perfusion. On the day of the experiment, mice were anesthetized by an intraperitoneal injection of ketamine-xylazine cocktail (20 mg/kg body wt). Surgical preparation for the hindlimb perfusion was performed as previously described (38, 49, 50). Before placement of the perfusion catheters, heparin (15 IU) was injected into the inferior vena cava. The mice were then euthanized with an intracardial injection of pentobarbital sodium (0.4 mg/g body wt), and catheters were immediately inserted into the descending aorta and ascending vena cava. The hindlimbs were then washed extensively with saline and placed in a perfusion apparatus for the equilibration (20 min) and experimental perfusion (20 min) periods. The perfusion system employed in this experiment was a reperfusion (36, 38). The perfusate consisted of Krebs-Henseleit solution, 5% BSA (Millipore, Billerica, MA), 550 M albumin-bound palmitate (4:1), 24 Ci of albumin-bound [1-14 C]palmitate, 6 mm glucose, and 3 mm caffeine (Sigma, St. Louis, MO) dissolved in Krebs-Henseleit buffer in the caffeine-treated groups. The perfusate was kept at 37 C and was continuously gassed with 95% O 2-5% CO 2. Perfusion flow rate was maintained at 1.5 ml/min for all groups (average ml min 1 g perfused muscle 1 ). Arterial ph levels were between 7.20 and 7.65, and arterial PO 2 and PCO 2 were and mmhg, respectively. Perfusion pressures were not affected by any of the experimental conditions and averaged , , and mmhg in the resting, caffeine-treated, and muscle contraction groups, respectively (P 0.05). After the 20-min equilibration period, arterial and venous samples were taken at 5, 10, 15, and 20 min of the experimental period for further analysis. At the end of the experimental period (20 min), the gastrocnemius-soleus-plantaris (GSP) muscle groups of both legs were freeze-clamped in situ with precooled aluminum clamps, removed, and stored in liquid N 2 for further analysis. Muscle contraction protocol. Force production measurements were made in anesthetized WT and DN mice to determine whether AMPK 2 deficiency would affect (10) or not affect (29) force production with this protocol (13, 14). The right tibiopatellar ligament was stabilized for the recording of force production, and a modular chart recorder (Cole Parmer, Vernon Hills, IL) was used to measure the tension developed by the GSP muscle group during the 20-min muscle stimulation protocol. The length of the GSP muscle group was adjusted at the initiation of electrical stimulation to obtain maximal active tension, and the GSP muscle group was connected to a Grass stimulator (model S48, Grass Telefactor, West Warwick, RI) to induce isometric muscle contractions via delivery of 15-V trains of 100 Hz lasting 50 ms with impulse duration of 1 ms with 30 trains/min. This moderate-intensity protocol has been shown to maximize FA metabolism (34). Perfusate sample analyses. Perfusate samples collected during the perfusion were analyzed to determine FA, glucose, and lactate concentrations, as well as radioactive [ 14 C]FA and 14 CO 2 contents. A nonesterified FA kit (NEFA HC kit, WAKO Chemicals, Richmond, VA) was used to measure perfusate FA concentrations spectrophotometrically. A YSI-1500 analyzer (Yellow Springs Instruments, Yellow Springs, OH) was used to measure glucose and lactate concentrations in the collected perfusate samples. Glycerol concentration was measured using a glycerol detection reagent (Sigma). Perfusate [ 14 C]FA and 14 CO 2 radioactivities were measured as previously described (46, 48, 49). An ABL-5 analyzer (Radiometer America, Westlake, OH) was used to determine PCO 2,PO 2, and ph. Muscle sample preparation. For Western blot analysis, frozen GSP muscle samples (40 mg) were powdered under liquid N 2 and homogenized in 500 l of ice-cold RIPA buffer, as previously described (34, 36). The total cell homogenate was then transferred to a microcentrifuge tube and vortexed frequently for 1 h, whereupon the samples were centrifuged at 4,500 g at 4 C for 1 h. For immunoprecipitation procedures, 90 mg of powdered muscle samples were homogenized in HEPES buffer and centrifuged at 15,000 g for 5 min. Supernatants (200 g) were incubated with antibodies for AMPK 1 or AMPK 2 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4 C with gentle agitation (35). After the incubation, protein A/G-agarose (Santa Cruz Biotechnology) was added to the tubes, which were gently agitated overnight (4 C). The immunoprecipitates were collected by centrifugation. Pellets were washed with PBS, and the final pellets were resuspended in sucrose homogenizing buffer and stored at 80 C until analysis. Protein concentrations were determined with the Bradford protein assay (Bio-Rad, Hercules, CA). Muscle glycogen content was determined as glucose residues after hydrolysis of the muscle samples in 1 M HCl (100 C, 2 h) and corrected for free glucose in the sample (44). For ADP measurements, frozen GSP muscle samples (10 mg) were powdered under liquid N 2 and homogenized as described above. The total cell homogenates were used to measure ADP levels using a colorimetric assay kit and the manufacturer s instructions (Abcam, Cambridge, MA). For intramuscular triglyceride (IMTG) extraction procedures, the Folch method was utilized as previously described (7, 42). Briefly, 100 mg of muscle sample were homogenized in 2:1 chloroform-methanol and incubated overnight (4 C) with gentle rotation. NaCl (0.9%) was then added, and samples were spun at 2,000 g (10 min). The bottom layer was aspirated, evaporated under N 2 gas, and resuspended in a 1% Triton X-100-PBS solution. The samples were read using a colorimetric triglyceride (TG) detection reagent (Thermo Scientific, Fremont, CA). Western blot analysis. Approximately 20 g of protein from the total cell homogenate preparations were separated on a 10% gel via SDS-PAGE. Proteins were transferred onto Immobilon-P polyvinylidene difluoride membranes and blocked with 5% BSA in Tween- Tris-buffered saline for 1 h. The membrane was incubated (4 C) in 5% BSA in Tween-Tris-buffered saline with antibodies (1:1,000) against phosphorylated (Ser 79 ) acetyl-coa carboxylase (ACC), total ACC (Cell Signaling, Danvers, MA), phosphorylated (Thr 177 ) CaMKI, or total CaMKI (Santa Cruz Biotechnology); phosporylated ERK1/2, total ERK1/2, or total AS160 (Cell Signaling, Danvers, MA); or carnitine palmitoyltransferase 1 (CPT1), CD36, peroxisome proliferator-activated receptor- coactivator 1 (PGC1 ), or silent mating-type information 2 homolog 1 (SIRT1; Santa Cruz Biotechnology). After overnight incubation, the membranes were probed with a secondary antibody (anti-rabbit IgG; 1:25,000 dilution) raised in goats (Pierce, Rockford, IL). Blots were then washed and subjected to enhanced chemiluminescence (Pierce). Band density was quantified using Scion Image (National Institutes of Health, Bethesda, MD). All bands analyzed from the perfusion studies were compared with the band obtained from the resting WT mice. The bands analyzed for basal protein parameters were compared with a single hindlimb muscle from an age-matched WT mouse. A Ponceau S total protein stain (Sigma) was used on the membranes as a loading control.

3 SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION 127 Activity assays. AMPK 1 and AMPK 2 activities were measured using [ 32 P]ATP incorporation into SAMS peptide (Upstate Signaling, Lake Placid, NY), as described elsewhere (36). Briefly, immunoprecipitates were added to an assay cocktail containing [ 32 P]ATP and SAMS peptide. After incubation, an aliquot was spotted onto a piece of Whatman filter paper, and all paper samples were washed with phosphoric acid and then with acetone. Sample papers were analyzed for radioactivity in a Packard scintillation counter, and counts were used to calculate phosphotransferase activity. Calculations and statistics. Palmitate delivery, fractional and total palmitate uptake, and percent and total palmitate oxidation were calculated as described previously in detail (46, 49). Acetate correction factors (49) were used to correct percent and total FA oxidation for label fixation. The specific activity for palmitate in the arterial samples was not different between groups and did not vary over time, averaging , , and Ci/mmol for the resting, caffeine-treated, and muscle contraction groups, respectively (P 0.05). O 2 uptake, glucose uptake, and lactate release were calculated as described elsewhere (49). All uptake and release rates are expressed per gram of perfused hindlimb muscles of both legs, which we determined to be 7% of total body weight for WT and DN mice. The contribution of muscle TG to oxidative metabolism was calculated on the basis of mean O 2 consumption of 2.03 l O 2/g fat and l O 2/g carbohydrate (8, 45). Time effects for glucose, lactate, glycerol, and FA concentrations and FA kinetic data were analyzed using a two-way ANOVA with repeated measures for each experimental condition (resting, caffeine-treated, and muscle contraction). If there were no significant differences in the values measured after 5, 10, 15, or 20 min of perfusion, mean values were used for subsequent analysis. The effects of experimental condition (resting, caffeine-treated, and muscle contraction) and genotype (WT vs. DN) were the two factors analyzed using a two-way ANOVA (Statview 5.0). The Tukey-Kramer test for post hoc multiple comparisons was performed when appropriate. A significance level of 0.05 was chosen for all statistical methods. RESULTS Effects of DN transgene on basal protein expression and force production. In the resting condition, the GSP muscle group was used to determine whether expression of the DN transgene led to differences in expression of key proteins implicated in the regulation of substrate use in skeletal muscle. There were no alterations in protein content of SIRT1, PGC1, ACC, CPT1, CD36, ERK1/2, CaMKI, or AS160 between WT and DN mice (Table 1, Fig. 1). Force production by the GSP Fig. 1. Effect of AMP-activated protein kinase- 2 (AMPK 2) dominantnegative (DN) transgene on skeletal muscle protein expression of multiple signaling intermediates in rest groups from a mixed gastrocnemius-soleusplantaris muscle preparation. Western blots represent total protein content of silent mating-type information 2 homolog 1 (SIRT1), peroxisome proliferator-activated receptor- coactivator 1 (PCG1 ), acetyl-coa carboxylase (ACC), carnitine palmitoyltransferase 1 (CPT1), CD36, ERK1/2, Ca 2 /calmodulin-dependent protein kinase I (CaMKI), and Akt substrate of 160 kda (AS160). Bands analyzed for basal protein parameters were compared with a single hindlimb muscle from an age-matched wild-type (WT) mouse. muscle group was not different (P 0.05) between the WT and DN mice (Fig. 2). Force was not produced or recorded in the resting or caffeine-treated group. Perfusion characteristics. To verify physiological perfusion conditions, O 2 uptake was measured during the experimental perfusion period. There were no differences in O 2 uptake over time in any of the experimental groups (P 0.05). Caffeine had no effect on O 2 uptake compared with rates measured in the resting group (P 0.05; Table 2). Muscle contraction resulted in an increase in O 2 uptake compared with rest and Table 1. Effect of AMPK 2 DN transgene on muscle protein expression WT SIRT PGC ACC CPT CD ERK1/ CaMKI AS Values (means SE) are expressed as percentage of control of a single wild-type (WT) hindlimb muscle. Western blotting was performed using homogenate samples prepared from hindlimbs of WT (n 5) or AMPactivated protein kinase (AMPK)- 2 dominant-negative (DN; n 5) mice. SIRT1, silent mating-type information 2 homolog 1; PGC1, peroxisome proliferator-activated receptor- coactivator 1 ; ACC, acetyl-coa carboxylase; CPT, carnitine palmitoyltransferase; CAMKI, Ca 2 /calmodulin-dependent protein kinase I; AS160, Akt substrate of 160-kDa. DN Fig. 2. Effect of AMPK 2 DN transgene on force production over the 20-min contraction period. Values are means SE (n 3 in each group). MC, muscle contraction.

4 128 SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION Table 2. Effect of AMPK 2 DN transgene on hindlimb perfusion characteristics during rest, caffeine treatment, or muscle contraction Rest Caffeine MC WT (n 5) DN (n 6) WT (n 5) DN (n 5) WT (n 6) DN (n 5) O 2 uptake, mol g 1 h * * FA concentration, mol/l FA delivery, nmol min 1 g Glucose concentration, mmol/l Lactate release, mol g 1 h * * ADP, mol/g Values are means SE; n, number of mice. WT or AMPK 2 DN mice were perfused during rest, caffeine treatment, or moderate-intensity muscle contraction (MC). *P 0.05 vs. respective rest group. P 0.05 vs. caffeine group. caffeine treatment (P 0.05). For all experimental conditions, O 2 uptake was not significantly different between WT and DN mice (P 0.05). Palmitate concentration and delivery were not significantly different at any time point in any of the groups, as dictated by the protocol (P 0.05; Table 2). In WT and DN mice, muscle ADP levels were not affected (P 0.05) by caffeine treatment or muscle contraction. Similarly, the DN transgene had no effect on muscle ADP levels (P 0.05; Table 2). Substrate exchange across the hindlimb. Lactate release was increased (178%) during muscle contraction (P 0.05; Table 2). The DN transgene did not affect lactate release in any of the groups (P 0.05). Arterial perfusate glucose concentration did not vary over time in any of the groups, and mean glucose concentration was not different between any of the groups (P 0.05; Table 2). Mean glucose uptake ( mol h 1 g 1 ) was significantly increased (P 0.05; Fig. 3C) by caffeine treatment (168%), and the DN transgene blunted the caffeineinduced increase in glucose uptake by 34% (P 0.05; Fig. Fig. 3. Effect of AMPK 2 DN transgene on time effects for glucose uptake in muscle contraction (A) and caffeine treatment (B) groups and mean glucose uptake (C) and glycogen content (D) in perfused mouse hindlimbs during caffeine (Caff) treatment or moderate-intensity muscle contraction. R, rest. Values are means SE (n 5 for R WT, Caff WT, Caff DN, and MC DN; n 6 for R DN and MC WT). *P 0.05 vs. respective rest group; #P 0.05 vs. respective WT group; &P 0.05 vs. rest WT; ##P 0.05 vs. caffeine groups; **P 0.05 vs. MC DN group; P 0.05 vs. 5 min; ***P 0.05 vs. 10 min. 3C). During muscle contraction, time-dependent differences were uncovered between the WT and DN mice. Within 5 min of initiation of the muscle contraction protocol, glucose uptake had increased by 156% in the WT mice, and this elevated glucose uptake rate was maintained throughout the rest of the contraction period (P 0.05; Fig. 3A). In contrast, glucose uptake rates were not elevated in the DN mice until the last 5 min (200% increase vs. resting DN mice at the same time point) of muscle contraction (P 0.05; Fig. 3A). At rest and during caffeine treatment, glucose uptake did not vary over time (Fig. 3B). At rest, muscle glycogen content was 29% lower in the DN than WT group (P 0.05; Fig. 3D). In the WT mice, caffeine treatment and muscle contraction resulted in a 33 38% reduction in muscle glycogen content (P 0.05; Fig. 3D). In the DN mice, muscle glycogen content was not affected by caffeine treatment or muscle contraction (P 0.05). During muscle contraction, glucose uptake, expressed as percentage of its respective rest group, was lower in the DN than WT mice at 5, 10, and 15, but not 20, min (P 0.05) and

5 SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION 129 tended to increase (P 0.089) over time in the DN mice (Fig. 5A). Palmitate metabolism. At rest and during caffeine treatment, palmitate uptake (nmol min 1 g 1 ) did not vary over time (P 0.05; Fig. 4, A and B). Mean palmitate uptake was significantly increased (P 0.05; Fig. 4E) by caffeine treatment (54%), but there were no DN effects. As shown for glucose uptake, time-dependent differences were found between the WT and DN mice during muscle contraction. Within 5 min of initiation of the muscle contraction protocol, palmitate uptake had increased 76% and 54% in the WT and DN mice, respectively, over their resting counterparts, and palmitate uptake remained elevated in the WT mice (P 0.05; Fig. 4A). However, the contraction-induced elevation in palmitate uptake had dropped off after 10 min in the DN mice, and palmitate uptake was not different from the resting condition for the remaining 10 min of the muscle contraction protocol (P 0.05; Fig. 4A). During muscle contraction, palmitate uptake, expressed as percentage of its respective rest group, was lower in the DN than WT mice at 20 min (P 0.05; Fig. 5B). In WT mice, mean palmitate oxidation (nmol min 1 g 1 ; Fig. 4F) increased by 180% with caffeine treatment and 260% with muscle contraction. In the resting condition, palmitate oxidation was higher (120%) in the DN than WT group (P 0.05; Fig. 4F). Unlike glucose uptake and palmitate uptake, no time-dependent effects were found in the caffeine or muscle contraction condition (P 0.05; Fig. 4, C and D). Mean contraction-mediated palmitate oxidation was blunted in the Fig. 4. Effect of AMPK 2 DN transgene on time effects for palmitate uptake in muscle contraction (A) and caffeine treatment (B) groups, time effects for palmitate oxidation in muscle contraction (C) and caffeine treatment (D) groups, and mean palmitate uptake (E), mean palmitate oxidation (F), mean glycerol release (G), and triglyceride (TG) content expressed as percent decrease compared with average respective rest group (H) in perfused mouse hindlimbs during caffeine treatment or moderate-intensity muscle contraction. Values are means SE (n 5 for rest WT, caffeine WT, caffeine DN, and MC DN; n 6 for rest DN and MC WT). *P 0.05 vs. respective rest group; #P 0.05 vs. respective WT group; &P 0.05 caffeine effect; **P 0.05 vs. MC DN group; P 0.05 vs. 5 min.

6 130 SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION Fig. 5. Effect of AMPK 2 DN transgene on glucose uptake (A) and palmitate uptake (B). Values are means SE (n 6 for MC WT; n 5 for MC DN). #P 0.05 vs. WT group. DN mice and not significantly different from the resting condition (P 0.05; Fig. 4F). Mean glycerol release throughout the perfusion period was not significantly different in any of the perfusion conditions; however, during rest and muscle contraction, glycerol release was higher in the DN groups than their respective WT groups (P 0.05; Fig. 4G). Mean muscle TG content was not different between the WT and DN groups during the resting condition (P 0.05, mol/g). After muscle contraction, TG content was lower in WT and DN mice. However, the DN group had 39% less IMTG, while the WT group had 20% less IMTG, during muscle contraction (P 0.05; Fig. 4H). Neither caffeine nor genotype had an effect on muscle TG content. Enzyme activities. As expected, AMPK 2 activity (pmol min 1 g 1 ) was decreased by the DN transgene (P 0.05; Fig. 6A). Caffeine treatment and muscle contraction increased (P 0.05; Fig. 6A) AMPK 2 activity by 81% and 161%, respectively, but did not affect (P 0.05) AMPK 1 activity (Fig. 6B). The DN transgene completely prevented the increase in AMPK 2 activity with muscle contraction and caffeine treatment (Fig. 6A) but did not affect (P 0.05) AMPK 1 activity (Fig. 6B). Total protein expression of ACC was not affected by caffeine treatment or muscle contraction, and there was no effect of the DN transgene (P 0.05; Fig. 6C). In the WT mice, caffeine treatment Fig. 6. Effect of AMPK 2 DN transgene on AMPK 2 activity (A), AMPK 1 activity (B), and ACC phosphorylation (C) in perfused mouse hindlimbs during caffeine treatment or moderate-intensity muscle contraction. Activity values were calculated per gram of muscle present in assay preparation (AMPK) or per microgram of protein present in assay preparation. Preparations are from the mixed gastrocnemius-soleus-plantaris muscle group. p-acc (Ser 79 ), phosphorylated (Ser 79 ) ACC. Values are means SE (n 5 in each group). *P 0.05 vs. rest WT group; #P 0.05 vs. respective WT group.

7 and muscle contraction increased (P 0.05) phosphorylation of ACC by 32% and 64%, respectively (Fig. 6C). Consistent with the results obtained for AMPK 2 activity, the DN transgene prevented the increase in phosphorylated ACC with caffeine treatment and muscle contraction. In the rest condition, phosphorylated ACC was 11% lower in the DN than WT mice. Activation of signaling intermediates. Total protein expression of CaMKI and ERK1/2 was not different between any of the groups (P 0.05), and there was no effect of the DN transgene for any of the proteins (Fig. 7). Phosphorylation (Thr 177 ) of CaMKI was increased by 150% and 104% with caffeine treatment and muscle contraction, respectively (P 0.05; Fig. 7A). ERK1/2 phosphorylation was not affected by caffeine treatment (P 0.05; Fig. 7B) but was increased 38% by muscle contraction (P 0.05; Fig. 7B). Fig. 7. Effect of AMPK 2 DN transgene on CaMKI (A) and ERK1/2 (B) protein expression and phosphorylation state in perfused mouse hindlimbs during caffeine treatment or moderate-intensity muscle contraction. Preparations are from the mixed gastrocnemius-soleus-plantaris muscle group. CaMKI-p (Thr 177 ), phosphorylated (Thr 177 ) CaMKI; ERK1/2-p, phosphorylated ERK1/2. Values are means SE (n 5 in each group). *P 0.05 vs. respective rest group. SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION DISCUSSION 131 Our data provide further evidence for the involvement of AMPK 2 in the regulation of substrate use during muscle contraction. Additionally, the results of this study suggest that while AMPK 2 is activated by caffeine-induced increases in intracellular [Ca 2 ], this activation is not essential to observe an increase in substrate uptake and FA oxidation during caffeine treatment. Additionally, these data indicate a role for AMPK-independent Ca 2 -dependent signaling in the regulation of substrate metabolism in skeletal muscle. However, AMPK 2 appears to be necessary to record normal timedependent changes in FA and glucose uptake in contracting mouse skeletal muscle, and these findings are not in agreement with previous studies that showed that AMPK activation is not time-dependent (27, 36). Indeed, one of the more interesting findings of this study is that AMPK 2 deficiency led to a reciprocal switch in FA and glucose uptake during the 20-min contraction protocol. Interestingly, during resting perfusion conditions, FA oxidation rates were elevated in mice that possessed the AMPK 2 DN transgene. These results were surprising to us, since AMPK 2 has been repeatedly shown to be an upstream regulator of ACC activation, which in turn has been shown to reduce malonyl-coa content, resulting in a rise in FA oxidation rate (37, 52). We hypothesized that low AMPK 2 activity measured in the DN mice would result in lower or equivalent resting FA oxidation rates, as well as lower ACC phosphorylation, than in WT mice. In line with our hypothesis, we measured a decrease in ACC phosphorylation during the resting condition in the DN mice, which is consistent with previous findings (26), but this decrease in ACC phosphorylation was not accompanied by a decrease in FA oxidation. This is in agreement with recent reports demonstrating that ACC may not be the only regulator of FA oxidation (33). We attempted to determine if the DN mice had increased expression of proteins known to be associated with the regulation of FA oxidation in skeletal muscle, such as CD36, CPT1, and PGC1, which may have accounted for the increase in oxidation at rest. However, no differences in protein content were observed. Because we could not isolate mitochondrial membrane fractions on top of nuclear fractions from the small amount of hindlimb muscles that we had, we could not determine whether CD36 protein content was altered in mitochondrial membranes. Indeed, it has been suggested by some (18, 19), but not others (23), that mitochondrial membrane CD36 content is an important factor in the regulation of FA oxidation. Because of the methodological limitations described above, we cannot contribute data to this debate. Given that AMPK 2 is known to regulate gene expression (15), the deficiency in AMPK 2 activity associated with our model was likely associated with alterations in the expression of genes involved in metabolic regulation in DN mice. This conclusion is consistent with the possibility that multiple redundant pathways are responsible for regulating substrate metabolism in skeletal muscle. It has been shown by us and others that caffeine-induced increases in Ca 2 signaling regulate substrate metabolism, possibly via AMPK activation (1, 6, 22, 35). Indeed, we have shown that when CaMKII or CaMKK was inhibited during caffeine stimulation, there were subsequent decreases in AMPK 2 activity along with decreases in the rates of FA

8 132 SUBSTRATE METABOLISM DURING MUSCLE CONTRACTION uptake, glucose uptake, and FA oxidation (1, 35). In line with these previous results, we measured an increase in AMPK 2 activity in caffeine-treated WT mice in this study. Because caffeine-induced AMPK 2 activity was reduced in the DN mice, we expected to measure coincident decreases in FA uptake and oxidation with caffeine treatment. However, there were no measurable differences between the DN and WT mice in FA metabolism during caffeine treatment. In contrast, caffeine-induced glucose uptake was blunted in the DN mice. Although glucose uptake was lower with caffeine treatment in the DN mice, there continued to be elevated glucose uptake rates over resting conditions. This increase in glucose uptake supports the notion that glucose uptake is regulated by Ca 2 signaling intermediates that are independent of AMPK activation (22, 53). Our results further suggest that AMPK 2 activation may be more critical to the regulation of glucose metabolism during states of increased intracellular [Ca 2 ] induced by caffeine than to the regulation of FA metabolism in skeletal muscle. It has also been postulated by some that AMPK activation is associated with increased expression of a downstream target of the CaMKK pathway, such as CaMKIV, during low energy states (58). However, we did not measure any effect of the DN transgene on the expression or phosphorylation state of CaMKI, an additional downstream target of CaMKK (5, 43), in any of the experimental conditions. These data suggest that CaMKK signaling either lies upstream of AMPK 2 or is not affected by low AMPK 2 activity. While no caffeine-induced force production was observed in our study, the increase in Ca 2 release from the sarcoplasmic reticulum induced by caffeine treatment may have been associated with a small twitch potentiation that was unrecordable with our system. We consider this possibility to be unlikely, because caffeine-induced twitch potentiation and force production have not been measured at concentration 3.5 mm in mixed skeletal muscle (9, 57). It should be noted that there was variation in the concentration of FA in the perfusate preparation. Unlike glucose and pyruvate, for example, controlling for albumin-bound FA concentration in perfusate is challenging. This is due mostly to the fact that cold and radioactive FA (in this study, palmitate) is bound to albumin in separate batches before they are introduced to the perfusion medium on the day of the experiment. However, given that FA availability can affect FA uptake under some, but not all, physiological conditions (45, 46), we wanted to ensure that the variability in palmitate concentration was not associated with our calculated values for palmitate uptake. Correlational analysis of palmitate concentration vs. palmitate uptake resulted in a low association (R , P 0.05; data not shown), suggesting that the variation in the palmitate concentration did not affect our palmitate uptake measurements. Taken together, our results suggest that caffeine-induced AMPK 2 activation is important in the regulation of glucose uptake but is not required to measure increases in FA uptake and oxidation in skeletal muscle during caffeine treatment. Because significant metabolic shifts were observed during muscle contraction in the DN mice, our data suggest that appropriate AMPK 2 activation is necessary to measure a typical response in glucose uptake, FA uptake, and FA oxidation during muscle contraction. These conclusions are consistent with previous studies in which AMPK 2 was shown to be a necessary signal in the regulation of substrate metabolism during muscle contraction or treadmill exercise (24, 25). However, our data are not consistent with some additional reports in which low AMPK 2 activity did not affect muscle glucose clearance during treadmill exercise (26), glucose uptake in contracting extensor digitorum longus muscle, which is a mixed-fiber type muscle (10), or FA oxidation in isolated muscle during muscle contraction or following treadmill exercise (4, 10, 25, 29, 30). Possible explanations for these differences include the fact that the exercise mode and the fiber types were different between studies. In the current study, we utilized an in situ electrical stimulation protocol and measured substrate uptake and FA oxidation during moderate-intensity muscle contraction in a mixed-muscle preparation. In contrast, Miura et al. (30) measured FA oxidation in incubated muscle strips following a low-intensity treadmill exercise protocol. As such, their data might be more appropriately viewed as postexercise data. Similarly, it is generally recognized that incubated muscle strips, such as those used by Dzamko et al. (4), lack proper circulation, and, as such, the response of these preparations is not always similar to that of perfused muscle. On the other hand, low AMPK 2 activity did not affect muscle glucose clearance during treadmill exercise in chronically cannulated mice whose muscle maintains functional circulation and appropriate neural input (26). Most importantly, our data are different from results reported by others, because the perfused hindlimb model allowed us to measure substrate uptake and FA oxidation at different times during the muscle contraction protocol. Time-dependent analysis revealed that decreased AMPK 2 activity impaired the ability of the contracting muscle to increase glucose uptake during the early phase of the stimulation protocol and to maintain higher rates of FA uptake throughout the stimulation protocol. However, as shown in isolated muscle strips (10), our glucose uptake rates were not affected by low AMPK 2 activity during the last 5 min of the stimulation protocol. This comparison demonstrates that the timing of the measurements is important when studying cellular signaling during muscle contraction. Consistent with previous data in the same mouse model (31, 32), DN mice had lower resting muscle glycogen content and did not utilize glycogen to the same extent as the WT mice during muscle contraction. However, this is not consistent with recent reports suggesting similar or higher rates of glycogen utilization during treadmill exercise in muscle of mice with low AMPK 2 activity (24, 26). Given that glycogen use and mean glucose uptake and FA oxidation were lower in the DN mice during muscle contraction, alternate fuel sources must have been used to maintain force production. Consistent with this notion, the glycerol and TG data show that contracting muscle in DN mice relied more heavily on intramuscular TG as a fuel source. Evidence shows that AMPK phosphorylates and inhibits hormone-sensitive lipase at Ser 565 in skeletal muscle and that this is associated with a reduced rate of lipolysis (12). Given the absence of AMPK 2 activation in the DN mice, our results agree with these other data and are consistent with an increase in the lipolytic rate and a greater reliance on muscle TG as a fuel. To ascertain that the measured TG breakdown would be sufficient as a fuel source to supply alternate fuel in the DN mice, we estimated the contribution of muscle TG to total oxidative metabolism in DN mice. Given an average O 2 uptake of 10.3 mol g 1 h 1 in the DN mice, our calculations estimate that 1 mol of muscle TG would have been needed to

9 completely fulfill the energy needs of the contracting muscle during 20 min of perfusion. Our measured TG breakdown could therefore have been sufficient as a fuel source for this type of muscle contraction protocol. These calculations are in line with other data from our laboratory (45) and show that when other fuel supplies are limited, skeletal muscle can efficiently utilize muscle TG. Low AMPK 2 activity did not affect the phosphorylation state of other signaling intermediates known to be implicated in the regulation of substrate use in skeletal muscle. Consistent with our previous results and those of others (26, 35, 47), ERK1/2 phosphorylation was increased during muscle contraction but was not affected by the DN transgene. Overall, our results suggest that AMPK 2 activation is not necessary to observe a rise in CaMKI and ERK1/2 phosphorylation during moderate-intensity muscle contraction. However, this conclusion is limited by the fact that the phosphorylation state of these signaling intermediates was not measured at the 5- or 10-min time point. The aim of future studies will be to determine whether time-dependent differences in the activation state of the aforementioned signaling intermediates might explain the changes in substrate use observed in DN mice. In summary, our results suggest that AMPK 2 activation is a necessary component of metabolic signaling during muscle contraction, especially as it relates to time-dependent changes in the regulation of contraction-induced metabolism in perfused muscle. Most notably, our data show that low muscle AMPK 2 activity is associated with a switch in FA and glucose uptake during muscle contraction and a greater dependency on muscle TG as a fuel source. These time-dependent data reinforce the notion that differences in exercise mode, fiber type, and sampling time may partially explain contradictory results between studies. Future studies will also examine the contribution of alternate signaling intermediates in the regulation of fuel use at various time points during muscle contraction. Our data in DN mice also provide evidence for the notion that caffeine-induced Ca 2 signaling regulates substrate use in an AMPK-independent manner in skeletal muscle. Overall, our data suggest that while AMPK 2 is a critical signaling intermediate in the regulation of substrate utilization, it is not the sole signal responsible for regulating skeletal muscle metabolism. ACKNOWLEDGMENTS The authors thank Emily Kallen, Matthew Burkhard, and Nadia El-Fakih for valuable technical expertise. GRANTS This study was supported by grants from the University of Southern California Women in Science and Engineering Program and by fellowships from the Integrative and Evolutionary Biology Program and the Gold Family Foundation. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. REFERENCES 1. 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