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Am J Physiol Endocrinol Metab 293: E1572-E1579, 2007. First published October 9, 2007; doi:10.1152/ajpendo.00371.2007
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LKB1 and the regulation of malonyl-CoA and fatty acid oxidation in muscle

D. M. Thomson, J. D. Brown, N. Fillmore, B. M. Condon, H-J. Kim, J. R. Barrow, and W. W. Winder

Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah

Submitted 14 June 2007 ; accepted in final form 3 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
5'-AMP-activated protein kinase (AMPK), by way of its inhibition of acetyl-CoA carboxylase (ACC), plays an important role in regulating malonyl-CoA levels and the rate of fatty acid oxidation in skeletal and cardiac muscle. In these tissues, LKB1 is the major AMPK kinase and is therefore critical for AMPK activation. The purpose of this study was to determine how the lack of muscle LKB1 would affect malonyl-CoA levels and/or fatty-acid oxidation. Comparing wild-type (WT) and skeletal/cardiac muscle-specific LKB1 knockout (KO) mice, we found that the 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR)-stimulated decrease in malonyl-CoA levels in WT heart and quadriceps muscles was entirely dependent on the presence of LKB1, as was the AICAR-induced increase in fatty-acid oxidation in EDL muscles in vitro, since these responses were not observed in KO mice. Likewise, the decrease in malonyl-CoA levels after muscle contraction was attenuated in KO gastrocnemius muscles, suggesting that LKB1 plays an important role in promoting the inhibition of ACC, likely by activation of AMPK. However, since ACC phosphorylation still increased and malonyl-CoA levels decreased in KO muscles (albeit not to the levels observed in WT mice), whereas AMPK phosphorylation was entirely unresponsive, LKB1/AMPK signaling cannot be considered the sole mechanism for inhibiting ACC during and after muscle activity. Regardless, our results suggest that LKB1 is an important regulator of malonyl-CoA levels and fatty acid oxidation in skeletal muscle.

5'-AMP-activated protein kinase; acetyl-coenzyne A carboxylase; 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; electric stimulation; muscle contraction

5'-AMP-ACTIVATED PROTEIN KINASE (AMPK) plays an important role in the cellular response to decreased energy charge. Its activation, which in skeletal muscle is dependent on phosphorylation by its upstream kinase, LKB1 (when complexed with STRAD and MO25) (6, 8, 14, 26, 42), occurs under conditions in which intracellular ATP levels decline and AMP levels increase (7, 9). When thus activated, AMPK acts to enhance the rate of catabolic, ATP-generating processes and to inhibit anabolic, ATP-consuming processes, thereby helping to maintain energy balance (13, 17, 19, 32, 39, 43).

One of the first targets identified for AMPK was acetyl-CoA carboxylase (ACC), which catalyzes the production of malonyl-CoA from acetyl-CoA (9, 36, 40). ACC2 is the major isoform expressed in skeletal muscle (5, 33). Upon phosphorylation of ACC2 by AMPK at Ser221, the production of malonyl-CoA by ACC2 is inhibited (40, 41). In skeletal muscle, the primary role of malonyl-CoA is to decrease the rate of long-chain fatty acid oxidation by inhibiting carnitine palmitoyltransferase-1 (15). This step is widely recognized as rate limiting in fatty acid oxidation (15, 21), and in this way AMPK is thought to play a key role in increasing the catabolism of fat when cellular energy begins to decline.

For instance, during contraction of rodent (4, 9, 34–36) and human (3, 20) skeletal muscle, AMPK and ACC phosphorylation are increased concurrently with a decline in malonyl-CoA concentration and an increase in fatty acid oxidation (9, 20, 36). Pharmacological treatment with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; an AMPK activator) leads to similar changes (11, 17, 18), suggesting that AMPK plays an important role in regulating malonyl-CoA levels and the rate of fatty acid oxidation during exercise. However, since both exercise and AICAR treatment may also affect other cellular processes independently of AMPK, these results do not eliminate the possibility that one or both of these stimuli may affect malonyl-CoA levels and fatty acid oxidation by mechanisms other than through the activation of AMPK signaling.

We (31) and others (12, 23, 24) have reported data regarding the effect of muscle-specific LKB1 knockout (KO) on skeletal and cardiac muscle. Markers of mitochondrial content in both types of muscle are decreased in KO mice (31), but it is not known what effect this has on fat metabolism. Koh et al. (12) reported that intramuscular triglyceride content is elevated in KO muscles. This finding could result from increased fatty acid uptake or from decreased fatty acid oxidation by the muscle. As the major AMPK kinase in skeletal and cardiac muscle, LKB1 is likely an important player in the regulation of malonyl-CoA levels and the promotion of fatty-acid oxidation in those tissues. However, LKB1 also phosphorylates several other proteins, the function of which are not well-characterized (2), and so its overall impact regarding fatty acid oxidation cannot be assumed. Therefore, in an effort to better understand the role of LKB1 and AMPK in the regulation of malonyl-CoA and fatty-acid oxidation, we tested the hypothesis that the muscle-specific deletion of LKB1 would 1) increase basal malonyl-CoA levels and decrease basal fatty-acid oxidation, 2) prevent the AICAR-induced decrease in malonyl-CoA and increase in fatty-acid oxidation, and 3) prevent the contraction-induced decrease in malonyl-CoA concentration.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Generation of muscle specific LKB1 KO mice was performed as previously described (31). Briefly, we cross-bred homozygous LKB1 conditional mice in which the LKB1 gene is flanked by loxP sites (kindly provided by R. DePinho and N. Bardeesy, Dana-Farber Cancer Institute, Boston, MA) with transgenic mice heterozygous for muscle-creatine kinase (MCK)-Cre in which Cre recombinase is continuously expressed in cardiac and skeletal muscle under the creatine kinase promoter (provided by C. R. Kahn, Joslin Diabetes Center, Boston, MA). In those offspring expressing MCK-Cre, the muscle-specific expression of Cre leads to recombination of the loxP sites and deletion of the LKB1 gene at approximately day 17 of development. Presence of the floxed LKB1 and the MCK-Cre genes was assessed in our mice by PCR ear-snip analysis as described previously (31). The lack of LKB1 protein in each muscle was verified by Western blotting (data not shown). KO and wild-type (WT; FVB strain) mice were bred and housed in a temperature-controlled (21–22°C) room with a 12:12-h light-dark cycle and free access to water and food. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Brigham Young University.

AICAR administration. In the morning, WT and KO mice [n= 8 (3 male + 5 female)/group] were subcutaneously injected with AICAR (0.5 mg/g body wt; Sigma Chemical, St. Louis, MO) or an equivalent volume of saline. The mice were anesthetized via intraperitoneal injection of pentobarbital sodium (0.08 mg/g body wt), and tissues were harvested 1 h after the injection of AICAR or saline. Extensor digitorum longus (EDL) and soleus (SOL) muscles were utilized for in vitro measurement of fatty acid oxidation. Quadriceps (QUAD), gastrocnemius (GAST), and tibialis anterior (TA) muscles were frozen between metal tongs at the temperature of liquid nitrogen and stored at –95°C until analysis for malonyl-CoA concentration assessment and/or Western blotting.

Measurement of fatty acid oxidation. Once removed from the hindlimb, EDL and SOL muscles were immediately weighed and then preincubated for 0.5 h at 37°C in 20-ml scintillation vials containing 1 ml of incubation medium (2% bovine serum albumin, 0.5 mM palmitate, 5 mM glucose, and 0.2 mU/ml insulin in Krebs-Henseleit buffer) under 95% O2-5% CO2. The incubation medium for muscles from AICAR-injected animals also contained 2 mM AICAR. EDL muscles from both hindlimbs were incubated together to increase the amount of total tissue analyzed. After preincubation, the muscles were transferred to fresh vials containing incubation medium plus 0.2 µCi/ml [1-14C]palmitic acid (MP Biomedicals, Salon, OH), flushed with oxygen, and incubated for 90 min at 37°C. After the incubation period, 0.75 ml of the incubation medium was transferred to a fresh scintillation vial containing 0.75 ml of 10% perchloric acid. A filter paper wick was glued to the cap and saturated with 1 M NaOH immediately before addition of the medium. The vials were immediately capped and incubated for 1 h at room temperature to allow absorption of 14CO2 onto the wick. The wick was then transferred to a fresh scintillation vial, Ecolite (ICN, Irvine, CA) was added, and radioactivity was assessed by scintillation counting. 14C that was unaccounted for due to TCA cycle isotopic exchange was accounted for by homogenizing the muscles immediately after incubation in 0.375 ml of 1:2 chloroform-methanol. Chloroform (0.25 ml) was then added, followed by rehomogenization. The homogenates were centrifuged at 2,000 g for 10 min. The supernatant was transferred to a clean glass tube, and 0.5 ml of 1:2 chloroform-methanol was then added to the pellet, which was then vortexed and recentrifuged. The supernatant was combined with the previous supernatant. Distilled water (1.0 ml) was added, and this was shaken for 10 min and then centrifuged for 10 min. Next, 0.5 ml of the upper aqueous phase was transferred to a scintillation vial and mixed with Ecolite. All vials were kept in the dark overnight before quantification of radioactivity by scintillation counting.

Electric stimulation. Twenty minutes after injection of mice with pentobarbital sodium, control muscles were removed from the right hindlimb of the mice. The GAST muscle was frozen between liquid nitrogen-cooled steel blocks, whereas plantaris and EDL muscles were frozen in isopentane cooled to the temperature of liquid nitrogen. After control muscles were removed, the left sciatic nerve was isolated just proximal to the point of trifurcation and stimulated for 5 min (frequency of 2/s, 10-ms duration, 2.5 V). Muscles from the stimulated limb were then removed and frozen.

Measurement of malonyl-CoA. The distal ~25 mg of the GAST (stimulated animals) and QUAD (AICAR/saline-treated animals) muscles were cut away and saved for Western blotting. The remaining tissue was homogenized in 6% perchloric acid, neutralized, and analyzed for malonyl-CoA according to the methods of McGarry et al. (16).

Western blotting and immunodetection. Muscles were homogenized in 19 volumes of homogenization buffer (50 mM Tris·HCl, 250 mM mannitol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM DTT, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml soybean trypsin inhibitor; pH 7.4). Homogenates were clarified by centrifugation at 5,000 g and 4°C. Homogenates were then diluted 1:1 in loading buffer and run out on 5% (phospho-ACC, total ACC) or 7.5% (phospho-AMPK) Tris·HCl gels (Bio-Rad Criterion System, Hercules, CA.). Proteins were then transferred to polyvinylidene difluoride membranes, which were then stained with Ponceau S to ensure even transfer and protein loading across lanes, washed in Tris-buffered saline plus Tween 20 (TBST), and blocked for 1 h at room temperature in 5% nonfat dry milk in TBST. Phospho-ACC and phospho-AMPK antibodies (Cell Signaling Technology, Beverly, MA) were diluted 1:4,000 in 1% BSA dissolved in TBST. Streptavidin-horseradish peroxidase conjugate (GE Healthcare Biosciences, Piscataway, NJ) was diluted 1:4,000 in TBST for the detection of total ACC levels. Membranes were probed overnight at 4°C with their respective primary antibodies. After primary antibody incubation, the membranes were washed in TBST and incubated for 1 h at room temperature in horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in 1% nonfat dry milk in TBST, except for ACC, which requires no second antibody. Membranes were again washed in TBST and then covered with enhanced chemiluminescence Western blotting detection reagent (GE Healthcare Biosciences) for 1 min. HRP activity was then detected using autoradiographic film (Classic Blue Sensitive; Midwest Scientific, St. Louis, MO). Relative band intensity was quantified using the Spot Denso function of AlphaEaseFC software (Alpha Innotech, San Leandro, CA).

Phosphorylation/activation of AMPK by AMP and ZMP. Recombinant {alpha}2β2{gamma}2-AMPK (rAMPK) was prepared as described previously (29, 30). Recombinant AMPK (4 µg) was incubated with and without 0.1 µg of recombinant LKB1-STRAD-MO25 (Upstate-Millipore, Billerica, MA) in 25 µl of medium containing 40 mM HEPES, 80 mM NaCl, 8% glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, and 0.2 mM ATP, pH 7.0, for 60 min at 30°C. Activity of the phosphorylated and nonphosphorylated rAMPK was determined in the presence and absence of 0.2 mM AMP and 0.2 mM ZMP in the same reaction mix indicated above. SAMS peptide (0.2 mM) was used as the phosphate acceptor in this assay. The reaction was initiated by addition of either phosphorylated or nonphosphorylated AMPK. After a 10-min incubation at 30°C, an aliquot of the reaction mix was spotted on a 1-cm2 P81 filter paper. The labeled ATP was washed out by six washes of 1% phosphoric acid. The papers were immersed briefly in acetone, dried, and added to scintillation vials. After addition of Ecolite scintillation cocktail, radioactivity was determined in a liquid scintillation counter.

Effect of AMP on phosphorylation of rAMPK at Thr172 by rLKB1-STRAD-MO25. Recombinant AMPK was incubated for 20 min at 30°C in the phosphorylation medium shown above in the presence and absence of 0.2 mM AMP. At the end of the 20-min incubation, 25 µl of 2x sample buffer containing 60 mg/ml were added, followed by electrophoresis and Western blotting using an antibody against Thr172 of the {alpha}-subunit of AMPK (Upstate-Millipore).

Statistics. Data are means ± SE. Means were compared using the NCSS statistical program (Kaysville, UT). The comparison between WT and KO for the percent decrease in malonyl-CoA with stimulation, body weights, and muscle weights were made using Student's t-test. All other means were compared using factorial ANOVA and Fisher's least significant difference post hoc test. In the case of malonyl-CoA concentration of QUAD muscles from AICAR- and saline-treated mice, greatly unequal variance was observed between groups, which appeared to undermine our ability to statistically detect the apparent decrease in malonyl-CoA concentration with AICAR treatment in WT muscles. Therefore, we performed a square root transformation of that data before factorial ANOVA analysis, which eliminated the inequality of variance between groups. To verify our conclusions in this case, we applied the conservative Bonferroni adjustment to multiple t-test comparisons of the untransformed data (with the significance level set to 0.0125 for 4 comparisons), which gave identical statistical results compared with the square root-transformed ANOVA.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Body and muscle weights. Body weight was similar between KO and WT mice (Table 1). GAST muscles from KO mice weighed slightly (~8%) but significantly less than those from WT mice. Likewise, TA muscles tended (P = 0.10) to weigh less in KO mice than in WT mice, but this difference was not significant. Protein concentration was not different between genotypes in the GAST or TA muscles. Heart weight, on the other hand, was ~9% greater in KO mice compared with WT counterparts, whereas protein concentration in KO hearts (12.6 ± 0.36 mg protein/g muscle) was ~10% lower than in WT hearts (14.1 ± 0.36 mg/g muscle; data not shown). With heart weight and protein concentration taken together, protein content per heart was not different, despite the increased weight in KO hearts.


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  		Table 1. Body and muscle weight

 
Effect of AICAR and muscle contraction on AMPK and ACC phosphorylation. As expected, 5 min of electrically-stimulated muscle contraction increased AMPK activity in the GAST muscles of WT mice as indicated by an increase in the phosphorylation of AMPK itself and that of its downstream target, Ser221 of ACC2, the predominant muscle ACC isoform (Fig. 1, A and B). An injection of 0.5 mg/g body wt AICAR failed to significantly increase the amount of phosphorylated AMPK in the QUAD or heart muscle but strongly stimulated the phosphorylation of AMPK in the TA and GAST muscles and increased the phosphorylation of ACC2 in all muscle types analyzed (Table 2).


Figure 1
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  		Fig. 1. A: phospho-AMP-activated protein kinase (p-AMPK) in resting (REST) and stimulated (STIM) gastrocnemius muscles from wild-type (WT) and muscle-specific LKB1-knockout (KO) mice. B: phospho-acetyl-CoA carboxylase-2 (p-ACC2) in REST and STIM gastrocnemius muscles from WT and KO mice. Values are means ± SE (n = 8 mice/group for WT and 7 mice/group for KO). *P < 0.05, significant difference compared with corresponding REST muscle. #P < 0.05, significant difference compared with corresponding WT muscle.

 

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  		Table 2. AMPK and ACC phosphorylation after saline or AICAR injection

 
Similar to our previous findings (31), as well as those of others (12, 23, 24), AMPK phosphorylation was essentially eliminated in the QUAD, GAST, TA, and heart muscles of KO mice (Table 2; Fig. 1). ACC phosphorylation was also greatly reduced in control KO muscles. Total ACC protein content was not different in the QUAD (Table 2) and GAST muscles (data not shown), but was slightly lower in the heart, of KO mice (Table 2). AICAR injection had no effect on AMPK or ACC phosphorylation in the heart, TA, GAST, or QUAD muscles of KO mice (Table 2), confirming previous findings (12, 23) that the AMPK-activating effects of AICAR are entirely LKB1 dependent. Interestingly, although contraction failed to phosphorylate AMPK in KO GAST muscles, it led to substantial phosphorylation of ACC2, although not to the level observed in WT muscles (Fig. 1).

Effect of AICAR and muscle contraction on malonyl-CoA concentration. Electrical stimulation of the sciatic nerve led to a significant decrease in malonyl-CoA concentration in both WT and KO GAST muscles (Fig. 2). However, the decrease was of a significantly greater magnitude in WT (67.7% decrease) than in KO muscles (49.8% decrease).


Figure 2
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  		Fig. 2. Percent decrease for malonyl-CoA levels in electrically stimulated gastrocnemius muscles compared with contralateral resting muscles in WT and muscle-specific LKB1 KO mice. Values are means ± SE (n = 8 mice/group for WT and 7 mice/group for KO). *P < 0.05, significant difference compared with WT.

 
In the QUAD (Fig. 3A) and heart (Fig. 3B) from WT mice, malonyl-CoA concentration was decreased 1 h after AICAR injection, as expected. Malonyl-CoA concentrations were not different between saline-injected WT and KO muscles. However, upon AICAR injection, malonyl-CoA concentration in muscles from KO mice increased significantly, opposite to the AICAR effect observed in WT muscles (Fig. 3).


Figure 3
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  		Fig. 3. Malonyl-CoA levels in saline- and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR)-treated quadriceps (A) and heart muscle (B) from WT and muscle-specific LKB1 KO mice. Values are means ± SE (n = 9 mice/group). *P < 0.05, significant difference compared with corresponding saline-treated muscle. #P < 0.05, significant difference compared with corresponding WT muscle.

 
Effect of AICAR incubation on fatty acid oxidation. Baseline fatty acid oxidation was higher in the SOL vs. EDL muscles, but was not different between WT and KO muscles (Fig. 4). However, the inclusion of AICAR in the incubation medium led to a significant (~85%) increase for fatty acid oxidation in WT but not KO EDL muscles (Fig. 4B). fatty acid oxidation was not altered by AICAR treatment in SOL muscles from either genotype (Fig. 4A).


Figure 4
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  		Fig. 4. Rate of palmitate oxidation in control and AICAR-treated soleus (A) and extensor digitorum longus (EDL; B) muscles from WT and muscle-specific LKB1 KO mice. Values are means ± SE (n = 6 mice/group). *P < 0.05, significant difference compared with corresponding control muscle. #P < 0.05, significant difference compared with corresponding WT muscle.

 
Effect of AMP and ZMP on AMPK activity. To determine whether the activity of unphosphorylated AMPK can be increased by AICAR, we performed an AMPK activity assay using phosphorylated and nonphosphorylated rAMPK in the presence or absence of ZMP (which is derived from AICAR and mimics the effect of AMP on AMPK) and AMP (Fig. 5A). Nonphosphorylated rAMPK was not activated by AMP or ZMP, whereas phosphorylated AMPK, as expected, was activated similarly by both AMP and ZMP.


Figure 5
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  		Fig. 5. A: activity of phosphorylated and nonphosphorylated recombinant AMPK (rAMPK) in the presence and absence of AMP and ZMP (n = 8 reactions per condition). ND, not detected. B: phosphorylation of rAMPK by LKB1-STRAD-MO25 in the presence and absence of AMP (n = 7 reactions per condition). Values are means ± SE. *P < 0.05, significant difference compared with assay run in the absence of AMP or ZMP.

 
We also sought to determine whether phosphorylation of AMPK by LKB1-STRAD-MO25 is enhanced by the presence of AMP. As shown in Fig. 5B, the presence of AMP did not increase the phosphorylation of rAMPK by LKB1-STRAD-MO25.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In these studies we investigated the role of LKB1 in regulating muscle malonyl-CoA levels and fatty acid oxidation. Using a muscle-specific LKB1 KO mouse, we observed the following main findings: 1) in vivo AICAR-stimulated reductions in skeletal and cardiac muscle malonyl-CoA concentration are entirely dependent on the presence of LKB1; 2) the in vitro AICAR-stimulated increase in fatty-acid oxidation in EDL muscles is also dependent on the presence of LKB1; and 3) the contraction-induced decrease in malonyl-CoA is only partially dependent on the presence of LKB1.

In WT heart and QUAD muscles, malonyl-CoA levels were decreased an hour after AICAR injection, which is in line with previous findings in skeletal muscle from our laboratory (17) as well as others (11, 22). This effect in WT hearts is, to our knowledge, a novel finding. Baseline malonyl-CoA levels were not affected by KO compared with WT muscles, suggesting that LKB1 may not be required for the maintenance of fat oxidation at rest, which was supported by our in vitro finding of basal fat oxidation in KO muscles. We hypothesized that malonyl-CoA levels would not be affected by AICAR treatment in KO muscles, because of the lack of AMPK activation. However, we were surprised to find that malonyl-CoA levels in KO muscles did respond to AICAR, but increased instead of decreased, as was observed in WT muscles. The mechanism for this increase in malonyl-CoA with AICAR is not known. One possibility is that AICAR, in the absence of activated AMPK (as in our KO muscles), led to increased ACC activity. This could occur if the activity of another kinase (i.e., PKA) was modified by AICAR treatment and led to a covalent change in ACC activity. Likewise, allosteric activation of ACC might have occurred if glycolysis were stimulated by AICAR treatment. This is a plausible explanation, because ZMP, which is intracellularly derived from AICAR, mimics AMP and could thus activate both PFK-1 (the rate-limiting enzyme in glycolysis) and phosphorylase kinase (important in the regulation of glycogenolysis). If glycolytic flux were thus elevated by AICAR, then acetyl-CoA and citrate levels might be expected to rise, and both of these could lead to increased production of malonyl-CoA by activation of ACC. Another possibility is that the activity of malonyl-CoA decarboxylase (MCD), which catalyzes the conversion of malonyl-CoA to acetyl-CoA (opposite the action of ACC), was increased by AICAR in the KO mice.

Similar to the effect of AICAR, contraction of the GAST muscles in WT mice led to a decrease in malonyl-CoA concentration. Contrary to our hypothesis, however, malonyl-CoA also decreased significantly in KO muscles, although not to the same extent as in WT muscles. This suggests, then, that while LKB1 signaling contributes to the decrease in malonyl-CoA concentration with contraction, it is not absolutely necessary for the response. This finding may reflect redundancy in signaling to acetyl-CoA carboxylase, a scenario that is consistent with our finding that ACCβ phosphorylation at Ser221 increased significantly in KO as well as in WT GAST muscles after contraction. In another study we observed a similar trend, although it was not significant (31). This raises the possibility that one or more kinases other than AMPK are capable of phosphorylating ACC at this site. Alternatively, phosphatases that normally act on ACC may have been inactivated with contraction in KO muscle, thereby leading to increased ACC phosphorylation. Regardless, our results suggest that LKB1 is necessary for a maximal decrease in malonyl-CoA levels.

Similar to previous reports (17, 27) and consistent with the decline in malonyl-CoA levels observed after AICAR injection, fatty acid oxidation increased in WT EDL muscles after 1 h of in vitro AICAR incubation. This effect was likely due to activation of AMPK by LKB1, since no such increase occurred in KO muscles. One might have expected fatty acid oxidation to decrease in KO muscles with AICAR incubation, since malonyl-CoA concentrations were elevated in the QUAD muscles after AICAR injection, but this was not the case. We have previously reported that half-maximum inhibition of fat oxidation by malonyl-CoA occurs at ~0.6 nmol/g and that maximal inhibition occurs at a malonyl-CoA concentration of ~2 nmol/g (37). Therefore, inhibition of fat oxidation by malonyl-CoA is likely maximal under basal conditions, and further increasing the concentration of malonyl-CoA would not produce further inhibition. Although we did not measure fatty acid oxidation during muscle contraction, the fact that malonyl-CoA levels did not decrease as much in KO as in WT GAST muscles with electrical stimulation suggests that in KO mice, fat oxidation may contribute less to metabolic needs during muscle activity. This may explain, at least partially, the previously observed elevation in intramuscular triglycerides in KO muscles (12), as well as our finding that wheel-running activity in the KO mice was substantially reduced compared with that in WT mice (31).

In our WT mice, phosphorylation of AMPK did not increase significantly 1 h after AICAR injection in the QUAD or heart. Like others using the same AICAR dosage (10), we did observe an increase in phospho-AMPK in the GAST, as well as a robust increase in the TA muscles. The variability in phosphorylation response among the various muscles is not surprising, since others have shown AICAR sensitivity to be less in red compared with white tissues (1). Furthermore, chronic activation of AMPK in a muscle has been shown to dampen its responsiveness to AICAR (38). As weight-bearing muscles, the GAST and QUAD muscles would likely undergo a degree of chronic AMPK activation and training relative to the non-weight-bearing TA muscle in these mice, which are very active, and this would also likely contribute to the attenuated AICAR responsiveness compared with the TA muscle. Despite the absence of an increase in phospho-AMPK concentration, it is apparent that in vivo AMPK activation occurred with AICAR in the WT QUAD muscles, because phosphorylation of its target, ACC, increased. This is possible because AMPK is regulated both by covalent means (via phosphorylation) and allosterically. AMP (and AICAR-derived ZMP) promotes an increase in the phosphorylation of AMPK but also allosterically activates whatever phosphorylated AMPK is present. Since the increase in ACC phosphorylation did not occur in KO muscles, it is very likely that this was due in WT muscles to the allosteric activation of AMPK, which would not be represented by the Western blot.

The lack of AMPK activation by AICAR in the KO muscles suggests that the allosteric activation effect of AMP/ZMP is only applicable to phosphorylated AMPK. That is, AMP/ZMP are unable to enhance the activity of unphosphorylated AMPK, as was found in KO muscle. We also observed that the in vitro activation of AMPK by AMP and ZMP only occurred with rAMPK that had been phosphorylated. We also found that the phosphorylation of rAMPK by LKB1 was not enhanced by the presence of AMP, which is in agreement with recent reports indicating that the ability of AMP to increase AMPK phosphorylation is regulated at the level of AMPK phosphatases instead of kinases (25, 28).

We found that when expressed relative to body weight, GAST muscles were significantly smaller in KO than in WT mice, and a similar, nonsignificant relationship was observed for the TA muscle. Given the role of LKB1 as a tumor suppressor and that of AMPK in suppressing protein synthesis, it might have been hypothesized that KO muscles would have increased in size. How LKB1 signaling may contribute to the maintenance of muscle mass has not been established, but since we previously reported that voluntary wheel-running activity is lower in KO mice, it may be that the lower muscle size is due to lower overall activity levels, and thus less loading, of the muscles.

Although we found that hearts from KO mice were larger than those from WT mice, protein concentration in the KO hearts was lower than in WT hearts such that the total protein weight per heart was not different between genotypes. This suggests that the increase in heart weight may be due to elevated glycogen levels, or perhaps edema. Our findings are in contrast to those of Sakamoto et al. (24), who reported smaller hearts and similar skeletal muscle size in their KO mice compared with WT mice. The reason for this discrepancy is not readily apparent but could be related to differences in their knockout model compared with ours. For instance, floxing of the LKB1 gene (without expression of the Cre recombinase) in their mice led to decreased expression of LKB1 in a variety of muscle and nonmuscle tissues, including the liver, whereas in our mice, LKB1 expression was maintained in the liver (where Cre is not expressed) (31). Thus, although our mice are similar in many regards, systemic alterations in LKB1 signaling in their mice demonstrate that the two constructs are not identical.

In conclusion, we found that the AICAR-stimulated decline in malonyl-CoA levels is LKB1/AMPK dependent and that the lack of muscle LKB1 results in an increase in malonyl-CoA with AICAR injection. The AICAR-stimulated increase in fatty acid oxidation is also LKB1 dependent. A maximal decline in malonyl-CoA levels after muscle contraction appears to be LKB1 dependent, although other mechanisms clearly play a role. These findings demonstrate the importance of LKB1 in the metabolism of fat in muscle.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants R01 AR-51928 and R01 AR-41438.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. W. Winder, 545 WIDB, Dept. of Physiology and Developmental Biology, Brigham Young Univ., Provo, UT 84602 (e-mail: william_winder{at}byu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Ai H, Ihlemann J, Hellsten Y, Lauritzen HP, Hardie DG, Galbo H, Ploug T. Effect of fiber type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat muscle. Am J Physiol Endocrinol Metab 282: E1291–E1300, 2002.[Abstract/Free Full Text]
  2. Alessi DR, Sakamoto K, Bayascas JR. LKB1-dependent signaling pathways. Annu Rev Biochem 75: 137–163, 2006.[CrossRef][Web of Science][Medline]
  3. Dean D, Daugaard JR, Young ME, Saha A, Vavvas D, Asp S, Kiens B, Kim KH, Witters L, Richter EA, Ruderman N. Exercise diminishes the activity of acetyl-CoA carboxylase in human muscle. Diabetes 49: 1295–1300, 2000.[Abstract]
  4. Duan C, Winder WW. Nerve stimulation decreases malonyl-CoA in skeletal muscle. J Appl Physiol 72: 901–904, 1992.[Abstract/Free Full Text]
  5. Ha J, Lee JK, Kim KS, Witters LA, Kim KH. Cloning of human acetyl-CoA carboxylase-β and its unique features. Proc Natl Acad Sci USA 93: 11466–11470, 1996.[Abstract/Free Full Text]
  6. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, Hardie DG. Complexes between the LKB1 tumor suppressor, STRAD{alpha}/β and MO25{alpha}/β are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2: 28, 2003.[CrossRef][Medline]
  7. Hawley SA, Selbert MA, Goldstein EG, Edelman AM, Carling D, Hardie DG. 5'-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J Biol Chem 270: 27186–27191, 1995.[Abstract/Free Full Text]
  8. Hong SP, Leiper FC, Woods A, Carling D, Carlson M. Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci USA 100: 8839–8843, 2003.[Abstract/Free Full Text]
  9. Hutber CA, Hardie DG, Winder WW. Electrical stimulation inactivates muscle acetyl-CoA carboxylase and increases AMP-activated protein kinase. Am J Physiol Endocrinol Metab 272: E262–E266, 1997.[Abstract/Free Full Text]
  10. Jorgensen SB, Treebak JT, Viollet B, Schjerling P, Vaulont S, Wojtaszewski JF, Richter EA. Role of AMPK{alpha}2 in basal, training-, and AICAR-induced GLUT4, hexokinase II, and mitochondrial protein expression in mouse muscle. Am J Physiol Endocrinol Metab 292: E331–E339, 2007.[Abstract/Free Full Text]
  11. Kaushik VK, Young ME, Dean DJ, Kurowski TG, Saha AK, Ruderman NB. Regulation of fatty acid oxidation and glucose metabolism in rat soleus muscle: effects of AICAR. Am J Physiol Endocrinol Metab 281: E335–E340, 2001.[Abstract/Free Full Text]
  12. Koh HJ, Arnolds DE, Fujii N, Tran TT, Rogers MJ, Jessen N, Li Y, Liew CW, Ho RC, Hirshman MF, Kulkarni RN, Kahn CR, Goodyear LJ. Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3. Mol Cell Biol 26: 8217–8227, 2006.[Abstract/Free Full Text]
  13. Lee WJ, Kim M, Park HS, Kim HS, Jeon MJ, Oh KS, Koh EH, Won JC, Kim MS, Oh GT, Yoon M, Lee KU, Park JY. AMPK activation increases fatty acid oxidation in skeletal muscle by activating PPAR{alpha} and PGC-1. Biochem Biophys Res Commun 340: 291–295, 2006.[CrossRef][Web of Science][Medline]
  14. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, Alessi DR. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23: 833–843, 2004.[CrossRef][Web of Science][Medline]
  15. McGarry JD, Brown NF. The mitochondrial carnitine palmitoyltransferase system. From concept to molecular analysis. Eur J Biochem 244: 1–14, 1997.[Web of Science][Medline]
  16. McGarry JD, Stark MJ, Foster DW. Hepatic malonyl-CoA levels of fed, fasted and diabetic rats as measured using a simple radioisotopic assay. J Biol Chem 253: 8291–8293, 1978.[Abstract/Free Full Text]
  17. Merrill GF, Kurth EJ, Hardie DG, Winder WW. AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol Endocrinol Metab 273: E1107–E1112, 1997.[Abstract/Free Full Text]
  18. Merrill GF, Kurth EJ, Rasmussen BB, Winder WW. Influence of malonyl-CoA and palmitate concentration on rate of palmitate oxidation in rat muscle. J Appl Physiol 85: 1909–1914, 1998.[Abstract/Free Full Text]
  19. Muoio DM, Seefeld K, Witters LA, Coleman RA. AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target. Biochem J 338: 783–791, 1999.[CrossRef][Web of Science][Medline]
  20. Roepstorff C, Halberg N, Hillig T, Saha AK, Ruderman NB, Wojtaszewski JF, Richter EA, Kiens B. Malonyl-CoA and carnitine in regulation of fat oxidation in human skeletal muscle during exercise. Am J Physiol Endocrinol Metab 288: E133–E142, 2005.[Abstract/Free Full Text]
  21. Ruderman NB, Saha AK, Vavvas D, Witters LA. Malonyl-CoA, fuel sensing, and insulin resistance. Am J Physiol Endocrinol Metab 276: E1–E18, 1999.[Abstract/Free Full Text]
  22. Saha AK, Schwarsin AJ, Roduit R, Masse F, Kaushik V, Tornheim K, Prentki M, Ruderman NB. Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside. J Biol Chem 275: 24279–24283, 2000.[Abstract/Free Full Text]
  23. Sakamoto K, McCarthy A, Smith D, Green KA, Grahame Hardie D, Ashworth A, Alessi DR. Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction. EMBO J 24: 1810–1820, 2005.[CrossRef][Web of Science][Medline]
  24. Sakamoto K, Zarrinpashneh E, Budas GR, Pouleur AC, Dutta A, Prescott AR, Vanoverschelde JL, Ashworth A, Jovanovic A, Alessi DR, Bertrand L. Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPK{alpha}2 but not AMPK{alpha}1. Am J Physiol Endocrinol Metab 290: E780–E788, 2006.[Abstract/Free Full Text]
  25. Sanders MJ, Grondin PO, Hegarty BD, Snowden MA, Carling D. Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade. Biochem J 403: 139–148, 2007.[CrossRef][Web of Science][Medline]
  26. 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 USA 101: 3329–3335, 2004.[Abstract/Free Full Text]
  27. Smith AC, Bruce CR, Dyck DJ. AMP kinase activation with AICAR simultaneously increases fatty acid and glucose oxidation in resting rat soleus muscle. J Physiol 565: 537–546, 2005.[Abstract/Free Full Text]
  28. Suter M, Riek U, Tuerk R, Schlattner U, Wallimann T, Neumann D. Dissecting the role of 5'-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase. J Biol Chem 281: 32207–32216, 2006.[Abstract/Free Full Text]
  29. Taylor EB, Ellingson WJ, Lamb JD, Chesser DG, Compton CL, Winder WW. Evidence against regulation of AMP-activated protein kinase and LKB1/STRAD/MO25 activity by creatine phosphate. Am J Physiol Endocrinol Metab 290: E661–E669, 2006.[Abstract/Free Full Text]
  30. Taylor EB, Ellingson WJ, Lamb JD, Chesser DG, Winder WW. Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated protein kinase at threonine-172 by LKB1/STRAD/MO25. Am J Physiol Endocrinol Metab 288: E1055–E1061, 2005.[Abstract/Free Full Text]
  31. Thomson DM, Porter BB, Tall JH, Kim HJ, Barrow JR, Winder WW. Skeletal muscle and heart LKB1 deficiency causes decreased voluntary running and reduced muscle mitochondrial marker enzyme expression in mice. Am J Physiol Endocrinol Metab 292: E196–E202, 2007.[Abstract/Free Full Text]
  32. Towler MC, Hardie DG. AMP-activated protein kinase in metabolic control and insulin signaling. Circ Res 100: 328–341, 2007.[Abstract/Free Full Text]
  33. Trumble GE, Smith MA, Winder WW. Purification and characterization of rat skeletal muscle acetyl-CoA carboxylase. Eur J Biochem 231: 192–198, 1995.[Web of Science][Medline]
  34. Winder WW, Arogyasami J, Barton RJ, Elayan IM, Vehrs PR. Muscle malonyl-CoA decreases during exercise. J Appl Physiol 67: 2230–2233, 1989.[Abstract/Free Full Text]
  35. Winder WW, Arogyasami J, Elayan IM, Cartmill D. Time course of exercise-induced decline in malonyl-CoA in different muscle types. Am J Physiol Endocrinol Metab 259: E266–E271, 1990.[Abstract/Free Full Text]
  36. Winder WW, Hardie DG. Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am J Physiol Endocrinol Metab 270: E299–E304, 1996.[Abstract/Free Full Text]
  37. Winder WW, Holmes BF. Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated. J Appl Physiol 89: 2430–2437, 2000.[Abstract/Free Full Text]
  38. Winder WW, Holmes BF, Rubink DS, Jensen EB, Chen M, Holloszy JO. Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle. J Appl Physiol 88: 2219–2226, 2000.[Abstract/Free Full Text]
  39. Winder WW, Taylor EB, Thomson DM. Role of AMP-activated protein kinase in the molecular adaptation to endurance exercise. Med Sci Sports Exerc 38: 1945–1949, 2006.
  40. Winder WW, Wilson HA, Hardie DG, Rasmussen BB, Hutber CA, Call GB, Clayton RD, Conley LM, Yoon S, Zhou B. Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A. J Appl Physiol 82: 219–225, 1997.[Abstract/Free Full Text]
  41. Witters LA, Kemp BE. Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase. J Biol Chem 267: 2864–2867, 1992.[Abstract/Free Full Text]
  42. 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: 2004–2008, 2003.[CrossRef][Web of Science][Medline]
  43. Yoon MJ, Lee GY, Chung JJ, Ahn YH, Hong SH, Kim JB. Adiponectin increases fatty acid oxidation in skeletal muscle cells by sequential activation of AMP-activated protein kinase, p38 mitogen-activated protein kinase, and peroxisome proliferator-activated receptor alpha. Diabetes 55: 2562–2570, 2006.[Abstract/Free Full Text]



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