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Research Article

Higher AMPK activation in mouse oxidative compared with glycolytic muscle does not correlate with LKB1 or CaMKKβ expression

Published Online:17 Dec 2024https://doi.org/10.1152/ajpendo.00261.2024

Abstract

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AMP-activated protein kinase (AMPK) is an energy-sensing serine/threonine kinase involved in metabolic regulation. It is phosphorylated by the upstream liver kinase B1 (LKB1) or calcium/calmodulin-dependent kinase kinase 2 (CaMKKβ). In cultured cells, AMPK activation correlates with LKB1 activity. The phosphorylation activates AMPK, shifting metabolism toward catabolism and promoting mitogenesis. In muscles, inactivity reduces AMPK activation, shifting the phenotype of oxidative muscles toward a more glycolytic profile. Here, we compared the basal level of AMPK activation in glycolytic and oxidative muscles and analyzed whether this relates to LKB1 or CaMKKβ. Using Western blotting, we assessed AMPK expression and phosphorylation in soleus, gastrocnemius (GAST), extensor digitorum longus (EDL), and heart from C57BL6J mice. We also assessed LKB1 and CaMKKβ expression, and CaMKKβ activity in tissue homogenates. AMPK activation was higher in oxidative (soleus and heart) than in glycolytic muscles (gastrocnemius and EDL). This correlated with AMPK α1-isoform expression, but not LKB1 and CaMKKβ. LKB1 expression was sex dependent and lower in male than female muscles. CaMKKβ expression was very low in skeletal muscles and did not phosphorylate AMPK in muscle lysates. The higher AMPK activation in oxidative muscles is in line with the fact that activated AMPK maintains an oxidative phenotype. However, this could not be explained by LKB1 and CaMKKβ. These results suggest that the regulation of AMPK activation is more complex in muscle than in cultured cells. As AMPK has been proposed as a therapeutic target for several diseases, future research should consider AMPK isoform expression and localization, and energetic compartmentalization.

NEW & NOTEWORTHY It is important to understand how AMP-activated kinase, AMPK, is regulated, as it is a potential therapeutic target for several diseases. AMPK is activated by liver kinase B1, LKB1, and calcium/calmodulin-dependent kinase kinase 2, CaMKKβ. In cultured cells, AMPK activation correlates with LKB1 expression. In contrast, we show that AMPK-activation was higher in oxidative than glycolytic muscle, without correlating with LKB1 or CaMKKβ expression. Thus, AMPK regulation is more complex in highly compartmentalized muscle cells.

INTRODUCTION

The AMP-activated protein kinase (AMPK) is a ubiquitous serine/threonine protein kinase known as the master regulator of metabolism (1). Due to its enhancement of catabolism and inhibition of anabolism, activation of AMPK has been suggested as a therapeutic target for several diseases such as some types of cancer, tissue inflammation, and cardiovascular and metabolic diseases (2–10).

The phosphorylation of AMPK is the main regulator of its kinase activity (11). Therefore, the extent to which AMPK is phosphorylated is often used as a measure of its activation, and this is also used throughout the present paper.

The main AMPK kinase is liver kinase B1 (LKB1), which phosphorylates AMPK on Thr172. This is promoted by an increase in the concentrations of AMP or ADP, whose binding to AMPK changes its allosteric conformation, allowing its phosphorylation by LKB1 (12, 13). Conversely, ATP binding can prevent AMPK activation by obstructing AMP attachment and promoting its dephosphorylation by phosphatases (11, 14, 15). AMPK can also be activated by glucose deprivation, independent of AMP/ADP-levels, as the associated decline in fructose-1,6-bisphosphate promotes the aldolase-dependent activation of AMPK by LKB1 (16). Although it has been suggested that transforming growth factor β activated kinase-1 (TAK1) can activate AMPK directly (17, 18), others argue that TAK1 is required for the activation of AMPK by LKB1 (19). In cultured cells, the AMPK activity correlates with the LKB1 expression (13).

Alternatively, AMPK can be activated by an increase in Ca2+, which leads to its phosphorylation on Thr172 by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKKβ) (20–22). CaMKKβ is mainly expressed in the brain (23), and its role in muscle tissue is controversial (21, 22, 24).

LKB1 and CaMKKβ differ in potency depending on the isoform composition (25). AMPK is heterotrimeric and composed of a catalytic α subunit, a regulatory β subunit, and a γ subunit. Each of the subunits occurs as multiple isoforms (α1, α2, β1, β2, γ1, γ2, and γ3), allowing the formation of 12 different αβγ heterotrimer combinations. AMPKα1 seems to be more sensitive to Ca2+ signaling (21), whereas AMPKα2 is more likely to interact with LKB1 (26).

In muscle tissue, AMPK serves an important role in adjusting metabolism according to the needs, which in turn depends on mechanical performance. AMPK increases the cellular uptake of glucose and fatty acids by promoting the translocation of glucose transporter (GLUT)4 and CD36 transporters to the cell membrane (27–29). In addition, it enhances the oxidation of fatty acids through its inhibition of acetyl-CoA carboxylase (ACC) (30, 31). Finally, it stimulates mitogenesis through its phosphorylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) (32). It is involved in the regulation of muscle phenotype, as demonstrated in unloading studies, where inactivity is associated with a decline in AMPK activity and a decrease in oxidative enzymes (33–37). Thus, inactivity leads to a shift from oxidative to glycolytic phenotype.

The basal AMPK activation in different muscle types has, to the best of our knowledge, never been directly compared. The present study aimed to address this by assessing the expression and activation of AMPK in oxidative and glycolytic muscles. We expected a greater AMPK activation in oxidative muscles to maintain the phenotype, but we were uncertain whether this was regulated energetically or through Ca2+. Energetically, oxidative muscles are characterized by low and stable levels of ADP, whereas glycolytic muscles show greater fluctuations. The heart exhibits a stable ADP-concentration over a range of workloads when oxygen consumption increases (38). Among skeletal muscles, the oxidative soleus also demonstrates remarkable metabolic stability during increases in workload, whereas in glycolytic muscles such as the gastrocnemius (GAST), the levels of ADP and Pi increase significantly during exercise (39–41). In terms of Ca2+, oxidative muscles receive more continuous daily electrical stimulation (42), triggering Ca2+-efflux from the sarcoplasmic reticulum to the cytosol. Furthermore, although AMPKα2 is the most abundant isoform in skeletal muscle, the expression of the more Ca2+-sensitive AMPKα1 is higher in oxidative skeletal muscle (25, 43). While AMPKα2 represents 90% and 60%, AMPKα1 represents 10% and 40% of the AMPK expression in glycolytic and oxidative muscles, respectively (43). Thus, we speculated whether the mechanisms of AMPK activation differed between muscle types with energetic activation through LKB1 perhaps being more important in glycolytic muscle and Ca2+ activation through CaMKKβ being more important in oxidative muscle.

Using Western blotting, we compared AMPK expression and activation in four different muscles: the oxidative heart and soleus (SOL) muscles, the glycolytic extensor digitorum longus (EDL), and gastrocnemius (GAST). When we found that the AMPK activation was significantly higher in oxidative than glycolytic muscles, we assessed the expression of the upstream regulators, LKB1 and CaMKKβ, and the ability of CaMKKβ in tissue lysates to phosphorylate AMPK.

METHODS

Ethics

All animal procedures were carried out according to directive 2010/63/EU of the European Parliament and had been approved by the Project Authorization Committee for Animal Experiments in the Estonian Ministry of Rural Affairs.

Animals

Twenty-five C57BL6J mice, 11 males and 14 females, were kept and bred in the animal facility of Tallinn University of Technology, in a 12:12-h light-dark cycle, and an ambient temperature of 22–23°C with free access to food (V1534-000 rat/mouse maintenance from Ssniff Spezialdiäten GmbH, Germany) and water.

The mice were anesthetized with a ketamine/dexmedetomidine mixture (150 mg·kg−1 and 0.5 mg·kg−1, respectively) and received an injection of 250 U of heparin to prevent blood coagulation. When the toe-pinch reflex was absent, the animal was euthanized by cervical dislocation. The brain, heart, gastrocnemius, soleus, and EDL were excised and immediately placed in an ice-cold wash solution consisting of the following (in mM): 117 NaCl, 5.7 KCl, 1.5 KH2PO4, 4.4 NaHCO3, 1.7 MgCl2, 21 HEPES, 20 taurine, 11.7 glucose, and 10 2,3-butanedione monoxime (pH was adjusted to 7.4 with NaOH). After cleaning and weighing, the tissues for Western blot were snap frozen in liquid nitrogen and stored at −80°C.

Homogenization for Western Blotting

Frozen muscles were homogenized in a glass homogenizer at a concentration of 20 mg·mL−1 of extraction buffer (50 mM Tris, 150 nM NaCl, 2% SDS, 1 mM DTT) with protease and phosphate inhibitor cocktails (cOmplete Mini and PhosSTOP, respectively, both from Roche, Merck). The protein concentration was determined spectrophotometrically. Ten microliters of tissue extract were diluted 1:5 and incubated at 80°C for 30 min. After heating, the protein content was determined in a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). The protein content of each sample was measured at least three times, and the results were averaged. The absorption at 280 nm was background corrected by subtracting the absorption at 330 nm, and protein content was calculated using the molecular weight and extinction coefficient of BSA (66,400 g·mol−1 and 43,824 M−1·cm−1, respectively). The protein content of the tissue extract was corrected by the protein content of the extraction buffer.

Western Blotting

Twenty micrograms of protein (40 µg for AMPKα1) were loaded in a polyacrylamide SDS-page gel (4% stacking gel, 12% separating gel) and separated using a PowerPac HC power supply (Bio-Rad Laboratories), run at 60 V for ∼30 min and followed by 120 V for ∼1 h. Proteins were transferred onto a nitrocellulose blotting membrane (0.45 μm) using the Bio-Rad Turbo Transfer System. Membranes were stained with 0.1% Ponceau solution to stain the proteins and imaged in an Image Quant LAS 400 Imager for normalization. Then, membranes were washed with 0.1% Tween-20 (Tris-buffered saline-Tween 20, TBST) three times for 10 min and blocked with 5% milk in TBS-T solution for an hour on a roller machine at room temperature. Primary antibodies in 5% BSA TBS-T solution were incubated overnight on a roller machine at 4°C followed by three washes with TBS-T and a 1-h incubation of secondary antibody in 5% milk TBS-T solution. Membranes were washed three times for 10 min with TBS-T and incubated with Bio-Rad Clarity Western ECL substrate for 5 min. Finally, membranes were imaged with an Image Quant LAS 400 imager.

Phosphorylation of AMPK by CaMKKβ in Tissue Lysates

To assess the phosphorylation of AMPK by CaMKKβ in tissue lysates, we used a modification of the assay by Yurimoto et al. (44), where CaMKKβ was activated by CaCl2 and calmodulin (CaM) to phosphorylate a downstream target, in our case the mutant AMPKα2 K45R protein, which is catalytically inactive (45, 46) and does not inhibit CaMKKβ autonomous activity (47). This phosphorylation was then inhibited by STO-609, which is a specific inhibitor of CaMKK that can be used as an inhibitor of AMPK phosphorylation by CaMKKβ (20, 48). CaMKKβ has a three times lower IC50 than CaMKKα (49). This assay was first tested with recombinant CaMKKβ and brain lysate (see Supplemental Materials), before being performed with muscle lysate.

Recombinant Proteins

Recombinant Calmodulin bovine (CaM) was obtained from Sigma-Aldrich (C4874-0.5 mg). Recombinant rat AMPKα2 K45R mutant and rat CaMKKβ gene sequence were expressed in E. coli, as described in the Supplemental Materials.

The expression of CaMKKβ and AMPKα2 K45R was verified with specific antibodies (see Supplemental Figs. S4 and S5, respectively). Both proteins exhibited truncated versions or unspecific signals. We also verified that AMPKα2 K45R was not phosphorylated in E. coli (Supplemental Fig. S5).

Assay with Tissue Lysate

Frozen muscles and brain were lysed in nondenaturing extraction buffer [50 mM Tris, 150 nM NaCl, 1% Nonidet P40, 1 mM DTT, 1 mM EGTA, 1 mM EDTA, protease, and phosphate inhibitor cocktails (cOmpleteMini and PhosSTOP)]. Muscle and brain lysates were tested in 40 µL of kinase activity buffer (50 mM HEPES, 10 mM magnesium acetate, 2 mM DTT, 1 mM ATP, pH = 7.5) under different conditions:

Lysate: 5 µL of tissue lysate was added to the kinase activity buffer.

Lysate + AMPK: 5 µL of tissue lysate with 2 µL of purified rat AMPKα2 K45R.

Lysate + STO + AMPK: 5 µL of tissue lysate was incubated 10 min before with 5 µM STO-609 acetic acid (diluted in DMSO). After 10 min, 2 µL of rat AMPKα2 K45R was added.

Lysate + CaCl2 + CaM+ AMPK: 5 µL of tissue lysate was incubated with 1 µM of CaM and 2 mM calcium chloride to stimulate CaMKKβ activity. This was followed by the addition of 2 µL of rat AMPKα2 K45R.

Lysate + STO + CaCl2 + CaM+ AMPK: 5 µL of tissue lysate was incubated 10 min before with 5 µM STO-609 acetic acid. This was followed by 1 µM of CaM and 2 mM calcium chloride, and 2 µL of rat AMPKα2 K45R recombinant protein.

All preparations were incubated for 30 min at 30°C, and the reaction was stopped by the addition of 2% SDS (3.33 µL of 30% SDS). A total of 17.5 µL of each preparation was immediately mixed with 10 µL of SDS page buffer (90% 4× Laemmli and 10% β-mercaptoethanol) and loaded on a polyacrylamide gel (4% stacking gel, 12% separating gel). The western blot procedure used is the same as described earlier, and the p-AMPK antibody was used as described in Table 1.

Table 1. Antibodies and their dilutions used in this study

Protein of InterestCompanyCatalogue NumberRR-IDDilutionSecondary AntibodyDilution of Secondary
Total AMPKCell Signaling2532AB_3303311:1,000Anti-rabbit IgG 111-035-045 Jackson ImmunoResearch1:10,000
AMPKα1AbcamAb32047AB_7227641:5001:10,000
p-AMPK Thr172Cell Signaling2535AB_3312501:1,0001:10,000
LKB1Cell Signaling3047AB_21983271:1,0001:10,000
CaMKKBGeneTexGTX108305AB_19498181:5001:10,000

Data Analysis

Western blot images were analyzed using IOCBIO GEL software (50). For each sample, the antibody (AB) signal intensity was normalized to the Ponceau (P) signal intensity (ABsample/Psample), and this was normalized to the averaged ABRF/PRF of the reference samples called “RF” (two per gel). For Western blots of total AMPK, phosphorylated AMPK, AMPKα1, and LKB1, we used the same reference sample throughout. This was a mix of several muscle lysates (heart, gastrocnemius, EDL, and soleus) from both males and females. Using the same reference throughout allowed us to directly compare the results from different gels. The reference sample used to compare CaMKKβ gels was 10 µg of brain lysate from a single, male individual.

In Fig. 2, the total AMPK expression (T-AMPK) in each sample was related to that of the reference (RF) sample and was calculated as (T-AMPKsample/Psample)/(T-AMPKRF/PRF).

In Fig. 3, the AMPK activation was calculated as the ratio of phosphorylated AMPK (p-AMPK) to total AMPK (T-AMPK). The AMPK activation in each sample was related to that of the reference sample (RF) and was calculated as [(p-AMPKsample/Psample)/(p-AMPKRF/PRF)]/[(T-AMPKsample/Psample)/(T-AMPKRF/PRF)].

In Fig. 5, the ratio of AMPKα1 to total AMPK (T-AMPK) in each sample was related to that of the reference sample (RF) and was calculated as [(AMPKα1sample/Psample)/(AMPKα1RF/PRF)]/[(T-AMPKsample/Psample)/(T-AMPKRF/PRF)].

In Fig. 7, the LKB1 expression in each sample was related to that of the reference sample (RF) and was calculated as (LKB1sample/Psample)/(LKB1RF/PRF).

In Fig. 9, the CaMKKβ expression in each sample was related to that of the reference sample (RF) and was calculated as (CaMKKβsample/Psample)/(CaMKKβRF/PRF).

In Fig. 11, the amount of phosphorylated AMPK (p-AMPK) under different conditions was related to that in the lysate alone and was calculated as (p-AMPKcondition/Pcondition)/(p-AMPKlysate/Plysate).

Moreover, each primary antibody had been previously calibrated with the reference sample “RF” and different muscle samples at different protein and antibody concentrations to ensure that the antibody responded in a linear fashion to an increase in protein. This allowed us to be certain that we were below the maximal binding threshold. Each Western blot sample was measured at least twice (except AMPKα1 and CaMKKβ) and the average is shown as a single data point. Outliers were measured a third time or deleted from the dataset.

Kinase activity images were analyzed using ImageJ software. For the experiments with tissue lysates, the results from each condition, ABcondition/Pcondition, were further normalized to the result from the lysates alone, ABlysate/Plysate.

Statistics

The graphs are shown as box-and-whisker plots according to the Tukey notation and were made using R software.

Statistical analyses were performed in JASP software using Bayesian statistical tests (51). For the assessments of expression and phosphorylation, we first used Bayesian two-way ANOVA to test the effects of sex, muscle, and sex × muscle. Significance is shown using an asterisk, *. Then, Bayesian post hoc single comparisons were performed to assess differences between muscles (significance shown using daggers, †) and differences between sexes (significance shown using hashtags, #). Kinase activity gels were statistically analyzed with single Bayesian independent T tests between the conditions for each tissue type (brain, soleus, gastrocnemius, and heart), and significance is shown using daggers, †.

For statistical significance, we considered BF < 10 not significant (NS), 10 ≤ BF < 30 strong evidence (* or † or #), 30 ≤ BF < 100 very strong evidence (** or †† or ##), and BF ≥ 100 extremely strong evidence (*** or ††† or ###) as in Ref. 52.

RESULTS

Morphological Data

Table 2 shows the morphological characteristics of the mice used for the Western blot experiments. The body weight was significantly lower in females compared with males. Age, body length, and tibial length were not significantly different between males and females.

Table 2. Morphological characteristics of male and female mice used in this study

SexAge, daysBody Weight, gBody Length, cmTibial Length, cm
Females, n = 14232 ± 5524.7 ± 4.28.9 ± 0.62.16 ± 0.06
Males, n = 11226 ± 5033.4 ± 39.2 ± 0.32.25 ± 0.1
Bayesian T testNS###NSNS

Values are shown in means ± SE. Males and females were compared using a Bayesian T test. ###BF ≥ 100, extremely strong evidence.

AMPK Is More Activated in Oxidative Muscles

To determine whether the basal activation of AMPK differed between the muscle types, we assessed the levels of total and phosphorylated (Thr172) AMPK. An illustrative blot is shown in Fig. 1, but for the experiments, the samples were loaded in random order. The total AMPK expression was normalized to the overall protein staining by Ponceau (Fig. 1C).

Figure 1.

Figure 1.Total and phosphorylated AMP-activated protein kinase (AMPK) in extensor digitorum longus (EDL), gastrocnemius (GAST), heart, and soleus. Illustrative pictures showing the Western blots of total AMPK (A), p-AMPK Thr172 (B), and Ponceau protein staining (C). 20 µg of protein from each sample was loaded into each well.

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Total AMPK expression was similar in EDL, gastrocnemius, and heart, but it was lower in the soleus (Fig. 2).

Figure 2.

Figure 2.Total AMP-activated protein kinase (AMPK) expression in extensor digitorum longus (EDL), gastrocnemius, heart, and soleus. Soleus had a significantly lower AMPK expression than the other muscles. The total AMPK expression (T-AMPK) in each sample was normalized to that of the reference sample (mix of muscle lysates, calculation specified in materials and methods, Data Analysis). A two-way Bayesian ANOVA tested the impact of sex, muscles, and sex × muscles, and the results are shown in the gray box on the top left. The significant impact of muscle was followed up by T tests between muscle pairs, and these results are indicated with brackets. Significance notation: *ANOVA, and †T tests between muscles (including both subgroups Male and Female). †10 ≤ BF < 30, strong evidence; *** or ††† BF ≥ 100, extremely strong evidence. Each dot represents an average of at least two measurements for the same sample. n = 6 for each sex subgroup and n = 12 for each muscle group.

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AMPK activation was determined as the ratio of phosphorylated AMPK-Thr172 to total AMPK for each sample. The overall results are shown in Fig. 3 and demonstrate that AMPK activation was significantly higher in oxidative (soleus and heart) than in glycolytic muscles (EDL and gastrocnemius).

Figure 3.

Figure 3.AMPK activation in extensor digitorum longus (EDL), gastrocnemius, heart, and soleus. AMPK is more activated in the oxidative heart and soleus than in the glycolytic gastrocnemius and EDL. The AMPK activation [phosphorylated AMP-activated protein kinase (AMPK)/total AMPK] in each sample was normalized to that of the reference sample (mix of muscle lysates, calculation specified in materials and methods, Data Analysis). A two-way Bayesian ANOVA tested the impact of sex, muscles, and sex × muscles, and the results are shown in the gray box on the top left. The significant impact of muscle was followed up by T tests between muscle pairs, and these results are indicated with brackets. Significance notation: *ANOVA, and †T tests between muscles (including both subgroups Male and Female). *** or †††BF ≥ 100, extremely strong evidence. Each dot represents an average of at least two measurements for the same sample. n = 6 for each sex subgroup and n = 12 for each muscle group.

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AMPKα1 Isoform Is More Expressed in Oxidative Muscles

To evaluate the proportion of AMPKα1 within the different muscle types, we measured AMPKα1 expression and normalized the signal to the total AMPK expression. A representative picture is shown in Fig. 4, and the overall dataset is summarized in Fig. 5. The AMPKα1/total AMPK ratio was significantly higher in the oxidative than in the glycolytic muscles, in agreement with the findings of Treebak et al. (43). The relative expression of AMPKα2 was not assessed, but we expect the relative contributions of AMPKα1 and AMPKα2 to sum to unity, as also shown in Ref. 25.

Figure 4.

Figure 4.Representative picture of AMP-activated protein kinase (AMPK)α1 expression in extensor digitorum longus (EDL), gastrocnemius (GAST), heart, and soleus (SOL). RF is the reference sample (mix of muscle lysates). 40 µg of protein were loaded into each well. In these blots, there were nonspecific bands. We analyzed the band between the 50 and 75 kDa markers (shown with arrows), as AMPKα1 has a predicted weight of ∼63 kDa. In Supplemental Fig. S1, we show the same picture with enhanced contrast, where the analyzed bands are more visible. A: Western blot of AMPKα1 in different tissues. B: total AMPK expression in the same samples. C: Ponceau staining of protein on the AMPKα1 blot.

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Figure 5.

Figure 5.AMP-activated protein kinase (AMPK)α1/total (T)-AMPK in extensor digitorum longus (EDL), gastrocnemius, heart, and soleus. AMPKα1 is more abundant in oxidative than glycolytic muscles. The AMPKα1 expression in each sample was normalized to that of the reference sample (mix of muscle lysates, calculation specified in materials and methods, Data Analysis). A two-way Bayesian ANOVA tested the impact of sex, muscles, and sex × muscles, and the results are shown in the gray box on the top left. The significant impact of muscle was followed up by T tests between muscle pairs, and these results are indicated with brackets. Significance notation: *ANOVA, †T tests between muscles (including both subgroups Male and Female). †10 ≤ BF < 30, strong evidence; *** or ††† BF ≥ 100, extremely strong evidence. Each dot represents a single measurement of one sample. n = 6 for each sex subgroups and n = 12 for each muscle groups.

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LKB1 Expression Differs between Sexes but Cannot Explain the Muscle-Specific Differences in AMPK Activation

To explain the tissue-specific difference in AMPK activation, we looked into the expression of LKB1, which is considered the main activator of AMPK (12, 13). A representative picture of a Western blot for LKB1 is shown in Fig. 6, and the averaged results are shown in Fig. 7. Overall, the LKB1 expression was higher in the muscles of female compared with male mice (Bayesian two-way ANOVA, 30 ≤ BF < 100, very strong evidence). There was a significant effect of muscle type (Bayesian two-way ANOVA, 30 ≤ BF < 100, very strong evidence), as LKB1 expression was higher in the heart compared with gastrocnemius. However, LKB1 expression did not correlate with the AMPK activation (Supplemental Fig. S3).

Figure 6.

Figure 6.Representative picture of liver kinase B1 (LKB1) expression in extensor digitorum longus (EDL), gastrocnemius (GAST), heart, and soleus (SOL). RF is the reference sample (mix of muscle lysates). 20 µg of protein were loaded into each well. We analyzed the band just above the 50 kDa markers (shown with arrows), as LKB1 has a predicted weight of ∼55 kDa. In Supplemental Fig. S2, we show the same picture with enhanced contrast, where the analyzed bands are more visible. A: Western blot of LKB1 in different tissues. B: Ponceau protein staining from the same blot.

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Figure 7.

Figure 7.Liver kinase B1 (LKB1) expression in extensor digitorum longus (EDL), gastrocnemius, heart, and soleus. LKB1 expression was higher in muscles from female mice, and it was higher in the heart compared with gastrocnemius. The LKB1 expression in each sample was normalized to that of the reference sample (mix of muscle lysates, calculation specified in materials and methods, Data Analysis). A two-way Bayesian ANOVA tested the impact of sex, muscles, and sex × muscles, and the results are shown in the gray box on the top left. The significant impact of muscles and sex was followed up by T tests between muscle pairs, and T tests between males and females for each muscle, and these results are indicated with brackets. Significance notation: *ANOVA, †T tests between muscles (including both subgroups Male and Female), and #T tests of sex difference. † or #10 ≤ BF < 30, strong evidence; **30 ≤ BF < 100, very strong evidence. Each dot represents an average of at least two measurements for the same sample. n = 6 for each sex subgroups and n = 12 for each muscle groups.

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CaMKKβ Is Present in the Heart but Poorly Expressed in Skeletal Muscle

CaMKKβ is the second-most studied AMPK-kinase, activated by Ca2+ and CaM. We assessed the overall CaMKKβ expression in the different muscles and used the brain as a positive control. The raw Western Blot pictures (Fig. 8) and the summarized data (Fig. 9) showed that the expression of CaMKKβ was very low in all skeletal muscle types, but higher in the heart.

Figure 8.

Figure 8.Representative picture of calcium/calmodulin-dependent protein kinase kinase 2 (CaMKKβ) expression in extensor digitorum longus (EDL), gastrocnemius (GAST), heart, and soleus (SOL), with brain as a reference and positive control. 20 µg of protein were loaded for muscles samples and 10 µg for brain. CaMKKβ was found just below 75 kDa (shown with arrows) as in previous works (20, 47). A: Western blot of CaMKKβ expression in different tissues. B: Ponceau protein staining.

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Figure 9.

Figure 9.Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKKβ) expression in extensor digitorum longus (EDL), gastrocnemius, heart, and soleus. CaMKKβ is poorly expressed in skeletal muscles compared with heart. The CaMKKβ expression in each sample was normalized to that of the reference sample (brain lysate, calculation specified in materials and methods, Data Analysis). A two-way Bayesian ANOVA tested the impact of sex, muscles, and sex × muscles, and the results are shown in the gray box on the top left. The significant impact of muscle was followed up by T tests between muscle pairs, and these results are indicated with brackets. Significance notation: *ANOVA, †T test between muscles (including both subgroups Male and Female). *** or ††† BF ≥ 100, extremely strong evidence. Each dot represents a single measurement for one sample. n = 6 for each sex subgroups and n = 12 for each muscle groups.

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In Muscle Lysate, AMPK Phosphorylation Is Not Increased by CaMKKβ Stimulation, but by STO-609 Itself

We assessed whether AMPK could be phosphorylated through the Ca2+/CaM/CaMKKβ signaling pathway in muscle lysate. This assay was tested with recombinant CaMKKβ, where CaCl2 and CaM increased the amount of phosphorylated AMPK, and this was inhibited by STO-609, as expected (see Supplemental Figs. S6 and S7). The assay was also tested with brain lysate. In males, but not females, the addition of CaCl2 and CaM to stimulate CaMKKβ led to a modest, but significant increase in AMPK phosphorylation and this was reduced by the addition of STO-609. However, the effect of STO-609 was diminished by the fact that STO-609 alone tended to increase the amount of phosphorylated AMPK (see Supplemental Figs. S8 and S9).

We performed the same assay with lysate of different muscles: gastrocnemius, soleus, and heart. EDL was not included in this assay, as the muscles from one animal are too small to provide enough homogenate for all conditions. Furthermore, we assumed that the results from gastrocnemius were representative of the situation in glycolytic muscles. Representative Western blots of p-AMPK are shown in Fig. 10, and the summarized data are shown in Fig. 11. It is important to note that the data from all three muscles show the same overall pattern. In gastrocnemius, none of the effects were significant, because of the large spread of the data, but the trend is the same as in soleus and heart, and we will describe the data overall. The addition of AMPKα2 K45R to the lysate did not increase the amount of phosphorylated AMPK. As a control, we added STO to lysate and AMPK and were surprised to see that the addition of STO-609 increased AMPK phosphorylation, but this only reached significance in soleus and heart (in the latter only relative to lysate alone). A similar trend was observed with brain lysate (Supplemental Fig. S9). In contrast, the addition of CaM and CaCl2 to stimulate the CaMKKβ activity had no significant effect on AMPK phosphorylation relative to lysate alone or lysate and AMPK (Fig. 11).

Figure 10.

Figure 10.Phosphorylation of AMP-activated protein kinase (AMPK) in lysate of different muscles (gastrocnemius, soleus, and heart). A: representative Western blots of p-AMPK under the conditions shown below. B: Ponceau staining.

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Figure 11.

Figure 11.Phosphorylation of AMP-activated protein kinase (AMPK) under different conditions in lysate of gastrocnemius (A), soleus (B), and heart (C). Interestingly, AMPK phosphorylation was higher in the presence of 5 µM STO-609, but this effect only reached significance in soleus and heart and not under all conditions. The phosphorylation of AMPK under each condition was normalized to that of the lysate alone (calculation specified in materials and methods, Data Analysis). †10 ≤ BF < 30, strong evidence; ††30 ≤ BF < 100, very strong evidence; †††BF ≥ 100, extremely strong evidence. In total, 6–10 measurements were performed with muscle lysate from n = 5 animals (1 male and 4 females), and each dot represents a single measurement.

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As intracellular signaling in many cases depends on the colocalization of proteins involved in the signaling cascade, we speculated whether keeping the muscle structure and the relative position of CaMKKβ to AMPK would affect the results. To address that, we incubated intact soleus fibers with caffeine to stimulate Ca2+ release in the absence and presence of STO-609 and assessed the AMPK phosphorylation (Supplemental Fig. S10). We did not observe any effect of caffeine or STO-609 on AMPK phosphorylation.

Overall, our results suggest that CaMKKβ did not phosphorylate AMPK in any of the muscle homogenates and therefore cannot explain the differential AMPK activation in oxidative and glycolytic muscles (Figs. 1 and 3).

DISCUSSION

To the best of our knowledge, we show for the first time that the basal level of AMPK activation is muscle dependent and that a larger fraction of AMPK is phosphorylated in oxidative compared with glycolytic muscles. In addition, we confirmed that there is more AMPKα1 in oxidative compared with glycolytic muscles. The AMPK activation did not correlate with the expression of LKB1 or CaMKKβ. Our results point to that in muscle tissue, the regulation of AMPK phosphorylation is more complex than depending merely on the expression of the upstream kinases.

AMPK Activation Is Higher in Oxidative That in Glycolytic Muscles

AMPK, at the basal state, was much more activated in oxidative (soleus and heart) compared with glycolytic muscles (gastrocnemius and EDL; Fig. 3). This is consistent with its role as a regulator of muscle phenotype. AMPK activation is well known to induce changes in muscle phenotype toward a more slow, oxidative profile. When AMPK is activated, it phosphorylates peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), which is a transcription factor regulating gene expression. The phosphorylation of PGC-1α is necessary for many of the effects of AMPK activation, including the upregulation of PGC-1α expression (32), which increases the activity of oxidative enzymes and modifies the myosin heavy chain (MHC) composition (53, 54). Studies on AMPK knockout (KO) or knockdown models also highlighted its involvement in fiber-type transition from fast glycolytic toward a slow, oxidative phenotype (55–59). Inactivity, on the contrary, as studied by muscle unloading, greatly reduces electrical stimulation and ATP consumption (60). Overall, unloading leads to dephosphorylation of AMPK and a decrease in oxidative enzymes and PGC-1α expression in skeletal muscle (33–37). The greater AMPK activation in oxidative muscles (Fig. 3) highlights that continuous AMPK activation is needed to maintain the oxidative phenotype.

AMPK Activation Does Not Correlate with LKB1 Expression in Muscle Tissue

To elucidate the mechanism behind, we first measured the expression of LKB1, which is the main upstream kinase responsible for AMPK phosphorylation (13). Works carried out on LKB1-deficient mice show that disturbing LKB1 expression affects AMPK phosphorylation in all muscle types (26, 58, 61, 62).

Although LKB1 was significantly more expressed in the heart compared with gastrocnemius, there was no clear difference between the muscle types (oxidative vs. glycolytic) (Fig. 7), in agreement with another study (63). Thus, when comparing different muscle types, LKB1 expression did not correlate with AMPK activation (compare Figs. 3 and 7; Supplemental Fig. S3). This is different from the situation in cultured cells. When comparing different cell lines, Woods et al. (13) demonstrated a remarkable correlation between LKB1 and AMPK activity. Furthermore, in cultured adipocytes, the influence of sex hormones on the LKB1 expression translated into an effect on AMPK activation: dihydrotestosterone lowered the fraction of phosphorylated AMPK, and this effect was reversed by estrogen (64, 65). In contrast, the present results suggest that in muscle cells, the regulation of AMPK activation by LKB1 is more complex.

It is interesting that the LKB1 expression was higher in the muscles of female compared with male muscles (Fig. 7). It has been shown in adipocytes that LKB1 mRNA levels are increased by estrogen and decreased by testosterone and dihydrotestosterone (65). This effect is mediated by the androgen receptor and estrogen receptor α (ERα) (65). Androgen receptors are present in skeletal muscles (66), and although one study suggested that ERβ is the dominant ER in skeletal muscles (67), other studies have demonstrated that ERα is present in the nuclei of both mouse and human skeletal muscles (68, 69). This could explain the present results and suggest that sex hormones also influence LKB1 expression in muscle tissues (Fig. 7).

CaMKKβ Is Poorly Expressed in Skeletal Muscle and Does Not Seem to Play Any Role in AMPK Activation in Muscle Tissue

Another upstream kinase of AMPK is CaMKKβ, which is activated by Ca2+ and CaM. We speculated whether the AMPK activation in heart and soleus could be due to their tonic activity (42), leading to a chronic activation of the Ca2+/CaM/CaMKKβ/AMPK signaling pathway. As noted earlier, the AMPKα1 isoform seems to be more sensitive to Ca2+ signaling (21), and we found a higher expression of AMPKα1 in oxidative muscles (Fig. 5).

We assessed the CaMKKβ expression in heart, soleus, gastrocnemius, and EDL using brain lysate as a positive control and reference (Fig. 8). We observed CaMKKβ expression in the heart, albeit at lower levels than in the brain (Fig. 8). This is in contrast to some other studies showing that CaMKKβ is not significantly expressed in rat and mouse heart (70, 71). In the present study, the expression in the heart was higher than in any of the skeletal muscles, where only faint bands were detected (Figs. 8 and 9). The low CaMKKβ expression in skeletal muscles is in agreement with the literature (71).

To assess the function of CaMKKβ, we conducted a CaMKKβ activity assay in which we assessed its phosphorylation of recombinant AMPKα2 K45R, which is catalytically inactive (45). This kinase activity assay worked with recombinant CaMKKβ (Supplemental Figs. S6 and S7), in agreement with the works of others (20, 72), and in brain lysate, although here, the effects were sex-dependent (Supplemental Figs. S8 and S9). In brain lysate, the addition of STO-609 alone also increased AMPK phosphorylation. This could explain why, in male brain lysates, STO-609 reduced, but did not abolish the effect of Ca2+ and CaM (Supplemental Fig. S9).

In the skeletal muscle lysates, the addition of CaCl2 and CaM had no effect on AMPK phosphorylation (Figs. 10 and 11), suggesting that CaMKKβ is not responsible for the basal tissue-specific AMPK activation. This is in agreement with the very low expression of CaMKKβ in soleus, gastrocnemius, and EDL (Figs. 8 and 9). The physiological importance of CaMKKβ as an activator of AMPK in skeletal muscle is controversial. A few studies have found that CaMKKβ can increase AMPK activity in skeletal muscle (21, 22, 73), although these studies did not find a direct effect on AMPK phosphorylation (in agreement with the present results), but observed significant effects on AMPK downstream targets such as acetyl-CoA carboxylase (ACC) or glucose uptake. However, a recent study demonstrated that contraction-induced AMPK phosphorylation and glucose uptake are similar in EDL and soleus from wild-type (WT) and CaMKKβ KO mice (24), and others have also been skeptical about the role of CaMKKβ in skeletal muscle (74). This is in agreement with the present results.

The heart differed from the skeletal muscles in having a greater expression of CaMKKβ (Figs. 8 and 9). However, CaMKKβ expression in the heart is controversial. As noted earlier, some have found only trace levels (70, 71). However, others have shown clearly visible CaMKKβ expression in the heart and suggested that its phosphorylation of AMPK is important during cardiac ischemia and pressure overload (75, 76). CaMKKβ is also the main activator of CaMKI and CaMKIV (77), which are involved in cardiac hypertrophy (78). Thus, CaMKKβ has the potential to play an important role in the heart in stressful situations. Despite the higher CaMKKβ expression in the heart, our results from the kinase activity assay suggested that CaMKKβ neither phosphorylated AMPK in heart nor in skeletal muscles (Figs. 10 and 11).

STO-609 Moderately Increases Phosphorylated AMPK in Tissue Lysates

The addition of STO-609 led to a moderate increase in phosphorylated AMPK. This effect was only observed in the tissue lysates (Fig. 11 and Supplemental Fig. S9) and not with recombinant CaMKKβ (Supplemental Fig. S7). It was not significant in brain or gastrocnemius lysates (Supplemental Fig. S9 and Fig. 11), and only under some conditions in soleus and heart lysates (Fig. 11). At present, we are unable to explain this. STO-609 is known to be a strong inhibitor of CaMKKβ (49), which is also confirmed by our experiments (Supplemental Fig. S7). Although some works have highlighted its potency to inhibit other kinases (24, 79, 80), it is unclear how STO-609 could lead to an increase in AMPK phosphorylation.

Limitations of the Study

As the phosphorylation of AMPK by LKB1 depends on the concentrations of AMP and ADP, we speculated whether these varied between the muscles. We tried to measure the concentrations of ATP, ADP, and AMP in heart, soleus, and gastrocnemius muscles using perchloric acid extraction and HPLC/MS, which is widely used for these kinds of measurements (81–83). However, in our hands, there was a degradation of ATP even when extracting with high concentrations of acid. This degradation manifested itself as unphysiologically high concentrations of ADP. In muscle, the overall ATP and ADP concentrations are in the mM and µM range, respectively. However, in our experiments, the peaks of ATP and ADP were of similar magnitude (results not shown), indicating that ATP was broken down to ADP, or reflected the bound fraction of ADP that would not play a role in regulation of AMPK in vivo. Furthermore, assuming the equilibrium of adenylate kinase, the concentration of AMP is estimated to be 10–100 times lower than that of ADP (84). Considering that the samples are diluted upon perchloric acid extraction and neutralization, the AMP concentrations would be below the limit of quantification of the available equipment. Therefore, we did not proceed with these experiments.

The literature on AMP and ADP concentrations in different muscles is inconclusive. Knowing the concentrations of ATP, phosphocreatine, creatine, and pH from NMR measurements and assuming equilibrium of the creatine kinase reaction, the intracellular ADP concentration has been estimated to be around 50 µM in the working heart (85) and to increase from 20 µM at rest to peak at ∼170 µM during high-intensity exercise in gastrocnemius (86). However, these values are from separate studies. Another study calculated the overall concentrations of ADP and AMP from NMR and HPLC measurements and found no difference between mouse soleus and EDL (87).

The overall concentrations of AMP and ADP in different muscles may be inadequate to explain the muscle-specific AMPK activation because muscle cells are highly compartmentalized. Energetic compartmentalization has been demonstrated in cardiomyocytes, (88–92), and a recent study also pointed to energetic compartmentalization near cytosolic adenylate kinase, the main generator of AMP (93). This suggests that in some intracellular compartments, AMP and ADP concentrations are significantly different from the overall concentrations. Recent works by others have also highlighted the compartmentalization of AMPK activation at specific subcellular locations such as the mitochondria (94–97). Thus, it is conceivable that some AMPK is associated with compartments whose ADP and/or AMP concentrations are different from the overall cytosolic levels. In this respect, it is interesting that the higher AMPK activation coincides with a larger fraction of the α1 isoform in oxidative muscles (Fig. 5). However, further studies are needed to address whether the greater AMPK phosphorylation in oxidative muscles is specific to isoforms and/or intracellular localization.

Conclusions

The present study demonstrates that AMPK is more activated in oxidative muscle (heart and soleus) compared with glycolytic muscle (gastrocnemius and EDL), highlighting that a high AMPK activity is required to maintain the oxidative phenotype. Oxidative muscles also had a greater expression of the AMPK α1 isoform. In contrast to other cell types, AMPK activation did not correlate with the expression of the main AMPK upstream kinases, LKB1 and CaMKKβ, in muscle tissue. We hypothesize that the regulation of AMPK activation is more complex in muscle tissues because of their highly compartmentalized intracellular environment.

DATA AVAILABILITY

All data supporting the results are included in the figures or as Supplemental Material. The datasets generated during the current study are available from the corresponding author upon reasonable request. The analysis tools used in this paper are referred to in the Methods.

SUPPLEMENTAL MATERIAL

Supplemental Figs. S1–S10: https://www.doi.org/10.6084/m9.figshare.27210642.

GRANTS

This work was supported by the Estonian Research Council under Grant No. PRG1127.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R. Bernasconi and R. Birkedal conceived and designed research; R. Bernasconi, K.S., A.S., K.Z., and M.K. performed experiments; R. Bernasconi analyzed data; R. Bernasconi and R. Birkedal interpreted results of experiments; R. Bernasconi prepared figures; R. Bernasconi and R. Birkedal drafted manuscript; R. Bernasconi and R. Birkedal edited and revised manuscript; R. Bernasconi, K.S., A.S., K.Z., M.K., T.L., M.V., and R. Birkedal approved final version of manuscript.

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