Chronic treatment of old mice with AICAR reverses age‐related changes in exercise performance and skeletal muscle gene expression

Abstract

Sarcopenia refers to the decline in muscle mass and function that occurs with advancing age. It is driven by alterations in multiple cellular processes. AMP‐activated protein kinase (AMPK) is a cellular energy sensor that opposes many age‐related changes, making it an attractive target for the treatment of sarcopenia. This study aimed to test the effect of chronic treatment of old mice with the AMPK‐activating prodrug, AICAR, on treadmill running capacity and muscle mass, force production, gene expression, and intracellular markers relevant to sarcopenia. Old (23 months) mice were tested for treadmill running capacity, then randomly assigned to receive daily treatment with AICAR (OA; 300 to 500 mg/kg, delivered via subcutaneous injection) or an equivalent volume of saline vehicle (OS) for 31 days. Young (5 months) saline‐treated mice (YS) served as controls. Treadmill posttesting was performed after 24 days, and the mice were euthanized after 31 days of treatment. Extensor digitorum longus (EDL) muscles were tested for force generation and RNA sequencing, RT‐PCR, and western blotting were performed on quadricep muscles. Treadmill running capacity declined from pre‐ to posttesting by 24.5% in OS mice. This decline was not observed in YS or OA mice. Quadricep weight was ~8% higher, and tetanic force production by the EDL muscle increased by 26.4% in OA versus OS. These phenotypic improvements with AICAR treatment were accompanied by changes in gene expression in OA/YS versus OS muscles consistent with the “rejuvenation” of gene ontologies associated with connective tissue, neurodegenerative disease, Akt signaling, and mitochondrial function, among others. AICAR increased the mitochondrial markers cytochrome C by ~33%, and citrate synthase by ~22%. Serum insulin‐like growth factor‐1 levels increased, and Akt phosphorylation tended (p = 0.07) to increase with AICAR treatment. Although protein levels of the mTORC1 signaling pathway intermediate, rpS6, were higher in OA versus OS muscles, the phosphorylation of mTORC1 pathway intermediates was unaffected. On the other hand, gene expression of the muscle‐specific ubiquitin ligases Mafbx and Murf1 were reduced with AICAR treatment. AICAR treatment mildly increased/preserved muscle mass and force production and prevented a decline in treadmill running performance in old mice. These effects were associated with altered skeletal muscle gene and protein expression, suggesting improved mitochondrial content and metabolic signaling (particularly through Akt) as contributing factors to the observed phenotypic effects. Our findings support further development of AMPK‐activating drugs as a therapeutic strategy for improving age‐related organismal dysfunction and sarcopenia.

Daily injections of the AMP mimetic, 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) for 1 month in old mice activates AMP‐activated protein kinase (AMPK), increases mitochondrial enzyme content and activity, and restores expression of many genes to youthful levels while reducing the expression of the muscle atrogenes muscle atrophy F‐box (MAFBx) and muscle RING‐finger protein‐1 (MuRF1). These AICAR‐driven cellular changes are accompanied by increased quadricep muscle mass and ex vivo EDL force production, along with the prevention of a decline in treadmill running performance over the treatment period, suggesting that AMP mimetics and AMPK activators are likely beneficial in treating age‐related dysfunction.

1. INTRODUCTION

Sarcopenia refers to the age‐related loss of skeletal muscle mass and strength commonly occurring in advanced age. It can increase the risk of injury, impede recovery, lead to more frequent hospitalization, decrease independence, diminish quality of life, and increase mortality. ¹

Like other aspects of the general decline in organismal function in old age, sarcopenia may be driven by as many as twelve general alterations in cellular function, commonly referred to as the “Hallmarks of Aging”. ² These alterations include mitochondrial dysfunction, compromised autophagy, loss of proteostasis, inflammation, dysregulated nutrient‐sensing, and altered paracrine/hormonal communication, among other changes. Identifying therapeutic targets that can attenuate these age‐related changes is of enormous interest in basic geroscience. AMP‐activated protein kinase (AMPK) has long been considered one such target since AMPK activation is overwhelmingly positive in promoting healthy cell function under most conditions. Indeed, in skeletal muscle, many of the detrimental hallmarks of aging are opposed by the actions of AMPK. For instance, AMPK activity promotes mitochondrial biogenesis and autophagy, downregulates inflammatory signaling and oxidative stress, and improves glucose uptake in skeletal muscle. ³ On the other hand, mouse models in which AMPK activation is impaired lead to a muscle and organismal premature aging phenotype. ⁴ , ⁵ This, along with the findings that AMPK activation is often compromised in aged muscle in rodents ⁶ , ⁷ and humans, ⁸ , ⁹ provides a compelling rationale for AMPK as a pharmacological target in age‐related muscle dysfunction.

Canonical activation of AMPK occurs when the cell's energy balance is disturbed such that AMP levels in the cells increase relative to that of ATP. Generally, this happens during increased ATP breakdown, such as in skeletal muscle during exercise. In response to the elevated utilization of ATP, AMPK phosphorylates many targets, promoting cell processes that enhance the ability of the cell to produce ATP while inhibiting energetically expensive processes. ¹⁰ AICAR (5‐aminoimidazole‐4‐carboxamide ribonucleoside) is a prodrug used extensively over the previous three decades as a tool compound to activate AMPK. It does this as it is taken up into the cell and then phosphorylated to form ZMP (5‐aminoimidazole‐4‐carboxamide ribonucleotide), which mimics elevated AMP levels without disturbing energy balance.

Chronic activation of AMPK with AICAR has previously been shown to improve treadmill running capacity and metabolic gene expression in young mice, ¹¹ and shown to improve oxidative capacity and induce fiber‐type switching in young rat muscle. ¹² Our lab has previously shown that AMPK activation by AICAR is similar in young and old skeletal muscle in rats. ¹³ Nevertheless, AICAR‐induced glucose uptake is mildly attenuated in very old (26–27 months) mice. ¹⁴ Still, AICAR appears to have beneficial effects in aged animals. For example, 10 weeks of AICAR treatment in aged mice reduced fibrosis and improved muscle regeneration after injury, similar to the effect of exercise training. ¹⁵ Furthermore, 2 weeks of AICAR treatment improved open‐field locomotor activity and motor coordination, measured via rotorod testing. ¹⁶ On the other hand, while AICAR improved treadmill running performance in aged (20 months) myostatin knockout mice, it did not affect treadmill running time in aged wild‐type mice. ¹⁷

Given the somewhat disparate findings on AICAR's effects on physical performance in old mice, we hypothesized that longer‐term (1 month) AICAR treatment would improve treadmill running capacity, muscle force production, and rejuvenate gene expression in skeletal muscle. We found that even in this relatively short‐term treatment period, AICAR prevented a decline in treadmill running capacity, increased ex vivo force production, reversed aspects of age‐related changes in gene expression, and suppressed expression of atrophy‐related genes. Our results demonstrate the potential for AMPK activation as a therapeutic strategy for improving muscle function in sarcopenia.

2. METHODS

2.1. Animal Care

Brigham Young University's Institutional Animal Care and Use Committee approved all experimental procedures. Young (Y; 5‐month‐old at the start of the study) and old (O; 23‐month‐old at the start of the study) male C57Bl/6 mice were housed in a temperature‐controlled (20–21°C) environment with a 12 h:12 h light–dark cycle and fed standard chow and water ad libitum.

2.2. Acute AICAR treatment study

Y and O mice (n = 4 mice/condition) were randomly assigned to be subcutaneously injected with AICAR dissolved in saline (500 mg/kg body weight) or an equivalent volume of saline without AICAR. Forty minutes after injection, the mice were anesthetized by isoflurane inhalation sufficient to achieve surgical anesthetic depth. At 60 min postinjection, the white portion of the quadricep muscle was removed and snap‐frozen in liquid nitrogen, then stored at −90°C until further analysis.

2.3. Chronic AICAR treatment study

Treadmill running capacity was determined (as described below) in Y and O mice, after which they were randomly assigned to be treated with daily subcutaneous injections of saline (YS = Y, saline‐treated; OS = O, saline‐treated) or, for the O mice, AICAR (OA = O, AICAR‐treated) for 31 days (n = 9–13 mice/group). The dosage and time frame were chosen based on previous in vivo work in young mice where AICAR improved running performance, and to increase the treatment time versus previous work where AICAR had no effect on treadmill running in old mice. ¹¹ Preliminary experiments indicated that the full desired AICAR dose (0.5 mg/g BW) led to severe lethargy in some of the mice (presumably due to hypoglycemia); therefore, the dosage was incrementally increased from 300 mg/kg BW for the first week to 400 mg/kg BW the second week, and 500 mg/kg BW for the remaining 17 days. This approach was well‐tolerated by the mice. Treadmill running capacity was determined again after 24 days of treatment. Eight days later (to allow for “washout” of treadmill running effects before tissue collection), a day after the final AICAR treatment, the mice were anesthetized by isoflurane inhalation sufficient to achieve surgical anesthetic depth, and tissues were harvested from the animals.

2.4. Treadmill Testing Protocol

Mice were first acclimated to the treadmill by setting their cages on it for 5–10 min, allowing them to explore the treadmill for 5 min without it running, then walking on a flat grade for 5 min at 10 m/min. This was performed twice before testing. Treadmill testing was performed at an environmental temperature of 16°C. The mice (4–6 at a time) ran on a 6‐lane treadmill on a 7% grade at 12 m/min for 3 min, followed by 16 m/min for 3 min, then 20 m/min until exhaustion (defined as unresponsiveness to prodding with a soft brush at the back of the treadmill for five consecutive seconds). The change in treadmill running capacity was determined by expressing the posttest running time relative to the pretest running time. The technician performing the treadmill testing was blinded to the mouse's treatment condition.

2.5. Measurement of maximal tetanic force

EDL muscles were removed from the mice and attached to a servomotor (300B Dual‐Mode Lever System; Aurora Scientific). The muscle was bathed in Ringer's solution (137 mM NaCl, 24 mM HaHCO₃, 11 mM D‐glucose, 5 mM KCl, 2 mM CaCl₂, 1 mM NaH₂PO₄.H₂0, 1 mM MgSO₄, pH 7.4; aerated with 95% O₂/CO₂ at 37°C) and continuously aerated with 95% O₂/CO₂ at 37°C within a jacketed tissue bath. The muscle was allowed to equilibrate for 10 min. Optimal length was determined from a resting tension of 0.2–0.5 mN at 100 V. Force frequency relationship and maximal tetanic force was determined using a train duration of 500 msec at 10, 20, 40, 80, 100, 150, 200, and 250 Hz (S88X Grass Stimulator; Astro‐Med Inc.), after which the muscle was stimulated for 5 min at 150 Hz with a train frequency of 0.2/s and train duration of 150 ms to determine the rate of fatigue. Five additional trains were elicited to determine muscle recovery after resting for 5 min. Due to a clerical error, EDL length measurements were lost. Therefore, the maximal tetanic force measures are presented as N/mg EDL weight rather than as specific force since the cross‐sectional area cannot be estimated without muscle length.

2.6. Tissue homogenization

Tissues were pulverized under liquid nitrogen using a ceramic mortar and pestle, then ground‐glass homogenized in 19 volumes of homogenization buffer (50 mM Tris–HCl, pH 7.4; 250 mM mannitol; 50 mM NaF; 5 mM Sodium Pyrophosphate; 1 mM EDTA; 1 mM EGTA; 1% Triton X‐100; 50 mM b‐glycerophosphate; 1 mM sodium orthovanadate; 1 mM DTT; 1 mM benzamidine; 0.1 mM phenylmethane sulfonyl fluoride; 5 ug/mL soybean trypsin inhibitor). The homogenates were subjected to three freeze–thaw cycles and then centrifuged at 10,000 × g for 10 min. Clarified supernatants were analyzed for protein content using the DC Protein Assay (Biorad Laboratories).

2.7. Western Blotting and Immunodetection

Quadricep homogenates or cell lysates were diluted in sample loading buffer (125 mM Tris HCl, pH 6.8; 20% glycerol, 4% SDS, 5% b‐mercaptoethanol, and 0.01% bromophenol blue), then electrophoresed through Tris–HCl gels (Biorad Criterion System). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes, and equal protein loading was verified by ponceau staining. After blocking with 5% nonfat dry milk, the membranes were probed for specific proteins via immunodetection (see Table S1 for the list of antibodies used). Appropriate fluorophore‐conjugated secondary antibodies were applied, and then the fluorescent signal was captured using the Odyssey DLx imaging system (LI‐COR Biosciences). Band intensity was quantified using automated background subtraction using ImageStudio Lite software (LI‐COR Biosciences).

2.8. AMPK Activity Assay

AMPK activity was determined by detecting the incorporation of radiolabeled phosphate from ATP into SAMS peptide (HHMRSAMSGLHLVKRR‐OH) by AMPK α1 and α2 subunits from the white quadriceps muscle. Forty microliter of white quadricep homogenate was incubated overnight with protein‐G Sepharose and AMPK α1 or α2 antibodies at 4°C. The antibodies were commercially prepared (Affinity Bioreagents, Golden, CO) and affinity‐purified against the peptides TSPPDSFLDDHHLTR (AMPK‐α1) and MDDSAMHIPPGLKPH (AMPK‐α2). After centrifugation, the pellet was washed with ice‐cold immunoprecipitation buffer (50 mM Tris–HCl, 150 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM benzamidine, 0.1 nN PMSF, 5 μg/mL soybean trypsin inhibitor, pH 7.4) plus an additional 1 M NaCl. The pellet was then washed with lysate assay buffer (62.5 mM Na HEPES, 62.5 mM NaCl, 62.5 mM NaF, 6.25 mM sodium pyrophosphate, 1.25 mM EDTA, 1.25 mM EGTA, 1 mM DTT, 1 mM benzamidine, 0.1 mM PMSF, 5 μg/mL soybean trypsin inhibitor, pH 7.4), resuspended in HEPES‐brij buffer (25 mM HEPES, 0.02% Brij, 1 mM DTT, pH 7.4), and aliquoted to microcentrifuge tubes. The kinase reaction was initiated by adding 15 mL of working assay cocktail (including 0.2 mM SAMS peptide, 0.2 mM AMP, and radiolabeled ATP) to the immunoprecipitated AMPK, then incubating them for 10 min while shaking at 30°C. Fifteen milliliter of the reaction mix was transferred to a piece of P81 filter paper, washed five times with 1% phosphoric acid, washed with ddH₂O, then acetone, and allowed to dry. Radioactivity was measured by scintillation counting, and AMPK activity was expressed as picomoles of phosphate incorporated into SAMS peptide per gram tissue per minute.

2.9. Citrate Synthase Activity Assay

0.025 mL of chronic AICAR homogenates were diluted into 1.225 mL of 100 mM Tris, pH 8.0 and vortexed gently. The following reagents were added to a 1 mL quartz cuvette: 0.60 mL of 100 mM Tris, pH 8.0; 0.10 mL of 3.0 mM Acetyl‐CoA; 0.10 mL of 1.0 mM DTNB, and 0.10 mL of the diluted homogenate. The cuvette was mixed by inversion and placed in a spectrophotometer at 30°C for 7 min. The change in optical density (O.D.) for 3 min at 1 min intervals (4 readings) at 412 nm was recorded. The reaction was started by adding 0.10 mL of 5 mM oxaloacetate and mixed by inversion several times. The change in O.D. for 3 min at 1 min intervals (4 readings) was recorded. Calculations were determined by the chane in O.D. per minute and the amount of tissue in the reaction cuvette.

2.10. RNA isolation

Quadricep muscle was ground to powder in a liquid nitrogen‐cooled mortar and pestle, then homogenized in Trizol reagent. RNA was isolated from the Trizol homogenates with a Direct‐zol RNA MiniPrep kit (Zymo Research #R2062).

2.11. RNA sequencing and analysis

The integrity of a subset (n = 4, randomly selected from the treatment groups) of quadricep RNA samples was assessed (TapeStation, Agilent Genomics), with a cutoff of RNA integrity number (RIN) ≥ 7.0, then sequenced with Poly A selection and HiSeq 50 Cycle Single‐Read sequencing (35 million reads per sample), with 50 bp reads by the DNA Sequencing Center at Brigham Young University. Assessment of raw RNA‐seq data quality was performed using FastQC version 0.11.9. Transcript quantification was performed with kallisto version 0.46.1 using the Ensembl Mus musculus version 104 transcriptome as the reference. After importing into R using tximeta 1.16.1 package, the resulting abundance estimates were summarized to gene‐level counts. The DESeq2 package was used to perform differential gene expression analysis in R. Genes with an adjusted p‐value (FDR) < 0.05 were considered differentially expressed.

The differentially expressed genes were clustered based on their co‐expression patterns using hierarchical clustering with Pearson correlation and complete linkage. The optimal number of clusters was determined using the cutree function in R, resulting in four clusters for the OA versus OS comparison and two for the OA + YS versus OS comparison.

Functional enrichment analysis of the DEGs was performed using the ClusterProfiler version 4.10.1 package. Over‐representation analysis was conducted against Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and Medical Subject Headings (MESH) databases to identify significantly enriched biological pathways and diseases. The results were visualized using ggplot2, with dot sizes representing the number of enriched genes and bar plots showing the percentage of enriched genes in each pathway.

2.11.1. RT‐PCR

cDNA synthesis from the RNA template was performed using iScript Reverse Transcription Supermix (Bio‐Rad Laboratories #1708841) following the manufacturer's instructions. Real‐time PCR amplification was performed using iTaq Universal SYBR Green Supermix (Bio‐Rad Laboratories #1725121), following the manufacturer's instructions on a CFX96 real‐time detections system (Bio‐Rad Laboratories), using primer sets listed in Table S2. Gene expression was calculated for all groups versus the control group (Young Saline‐treated) using the 2‐ΔΔCT method and β‐actin as the reference gene.

2.11.2. ELISA Assay

Insulin‐like growth factor‐1 levels were measured using a commercial ELISA kit (R&D Systems #MG100) following the manufacturer's instructions.

2.12. Statistics

Factorial and one‐way ANOVA analyses were performed as appropriate with GraphPad Prism statistical software. Fisher's LSD posthoc testing was applied where significant differences (p ≤ 0.05) were identified by factorial or simple ANOVA testing, as appropriate. Two‐group comparisons were performed using a t‐test in Microsoft Excel software. Data is reported as mean ± SE.

3. RESULTS

3.1. AICAR activates AMPK similarly in young adult and old muscles

Most, ¹³ , ¹⁸ , ¹⁹ but not all, ⁹ studies indicate that AICAR effectively activates AMPK in aged rodent skeletal muscle. To verify AICAR's effect in old muscle, we assessed AMPK activation in muscles from Y and O mice 1 h after subcutaneous injection of AICAR (0.5 mg/kg BW). We found that basal AMPK phosphorylation (Figure 1A,B) and ACC phosphorylation (Figure 1A,C), as well as in vitro AMPK activity (Figure 1D–1E), were not different between Y and O muscles. Furthermore, AICAR increased phosphorylation of AMPK (Figure 1A,B) and ACC (Figure 1A,C) and in vitro AMPK α1 (Figure 1D) and α2 (Figure 1E) activity similarly in Y and O mice.

FIGURE 1.

AICAR effectively activates AMPK in old skeletal muscle. Quadricep muscle was removed from Young (5 months) and Old (24 months) mice 1 h after a subcutaneous injection of AICAR (n = 4/group) and assayed for indicators of AMPK activity. (A) Representative blots for phospho‐AMPK (p‐AMPK) and phospho‐ACC (p‐ACC), and representative Ponceau stain showing equal protein loading across conditions for western blotting experiments. (B) Levels of phosphorylated AMPK. (C) Levels of phosphorylated ACC (a classic AMPK target). (D & E) Activity of immunoprecipitated AMPKα1 and AMPK α2 subunits, respectively. Data was analyzed by factorial ANOVA (age × treatment). Main effects were observed in each instance for AICAR treatment, regardless of age. No interaction or main effect for age for any of the measures was observed.

3.2. Chronic AICAR treatment improves muscle force production and maintains treadmill running endurance in old mice

Narkar et al. ¹¹ previously demonstrated that chronic AICAR treatment enhanced treadmill running capacity in young mice. To determine if chronic AICAR treatment has a similar effect in old mice, Y and O mice were injected with either saline or 500 mg/kg of AICAR daily for 31 days (Figure 2A). The mice were tested for treadmill running capacity before and after 24 days of treatment, and muscle mass and function were analyzed posttreatment.

FIGURE 2.

Chronic AICAR treatment increases quadriceps weight and force production and preserves treadmill running capacity in old mice. (A) After treadmill pretesting, young adult (Y; 5 months) and old (O; 24 months) mice received daily saline (YS, OS) or AICAR (OA) injections for 31 days. After 24 days of treatment, the mice were tested again for treadmill running capacity. Mice were euthanized, and tissue was harvested after 31 days of treatment. Body weight (B), quadricep weight (C), and gastrocnemius weight (D) were measured. Treadmill run time, expressed as the % change from pre‐ to posttesting, is shown in (E). The EDL muscle's maximal ex vivo tetanic force is shown in (F). N = 10–13 mice per group. ANOVA post hoc p‐values for YS versus OS and OA versus OS are indicated on the graphs.

Although O mice weighed more than their young counterparts (as expected), AICAR treatment did not affect body weight (Figure 2B), but the quadricep mass was significantly increased by ~8% in the OA versus OS mice (Figure 2C). Gastrocnemius mass also tended to increase in OA versus OS mice, though this difference was not significant (p = 0.15 Figure 2D). Tibialis anterior muscle mass was not affected by AICAR.

At pretesting, the average running time to fatigue was not significantly different between young and old mice (29.9 ± 3.1 vs. 25.1 ± 2.8 min, respectively). However, running capacity declined from pre‐ to posttesting in the OS (75.5 ± 9.7% of pretest run time) but not in the YS (112.9 ± 9.7%) or OA mice (119.0 ± 10.5%; Figure 2E).

Normalized maximal force production was not different in YS versus OS EDL muscles but was significantly increased by 26.4% in OA versus OS muscles (18.2 ± 0.9 vs. 14.4 ± 1.1 N/mg; Figure 2F). Rate of fatigue and recovery in stimulated EDL muscles was not affected by AICAR treatment (data not shown).

3.3. Chronic AICAR treatment alters gene expression in old muscles

RNA sequencing was performed to find genes whose expression is altered by chronic AICAR, and that might contribute to the improvement in run time and muscle force production in the old mice. We first compared gene expression between OA versus OS muscles, between which we identified 143 differentially expressed genes (Table S3). When organized into a heatmap (Figure 3A) including the expression of these genes in muscles from YS mice, we observed the following clusters: 55 genes whose expression is reduced in OS muscle but restored to youthful levels with AICAR (Figure 3B, cluster 1), 37 genes whose expression was not affected by age, but increased in OA muscles (Figure 3B, cluster 2), 40 genes whose expression was not affected by age, but was reduced in OA muscles (Figure 3B, cluster 3), and 11 genes whose expression was increased in OS versus YS muscles and restored to youthful levels in OA muscles (Figure 3B, cluster 4). Figure 3C shows a volcano plot demonstrating genes significantly up‐ and down‐regulated in OA versus OS muscles, with labeling of selected potential genes of interest. Figure 3D shows the differentially expressed genes organized by overrepresentation analysis using GO, KEGG, Reactome, and MESH databases, with 25 selected gene sets of particular relevance to aging biology included on the graph (out of the 315 total significantly enriched gene sets; 106 GO, 29 KEGG, 35 Reactome, 145 MESH).

FIGURE 3.

Chronic AICAR treatment reverses age‐related changes in gene expression in skeletal muscle of old mice. (A) Heatmap displaying the expression levels of 143 differentially expressed genes (DEGs) identified in the comparison between Old AICAR‐treated (OA) and Old Saline‐treated (OS) quadricep muscles. Expression values are normalized and represented as Z‐scores. The samples are grouped by treatment (Young Saline [YS], Old Saline [OS], and Old AICAR [OA]). (B) Volcano plot illustrating the log2 fold change versus −log10 (q‐value) for all tested genes in the comparison between Old AICAR and Old Saline mice. Genes without an adjusted p‐value and those annotated as “pseudogene,” “predicted,” or “RIKEN” were excluded from the plot. Significant genes are highlighted based on q‐value <0.05. (C) Clustering of the 143 DEGs into four groups based on their co‐expression patterns. Hierarchical clustering with Pearson correlation and complete linkage was used. The relative expression (Z‐score) of each cluster is shown across the YS, OS, and OA groups. (D) Over‐representation analysis of the 143 DEGs using the clusterProfiler package from BioConductor, performed against the Gene Ontology (GO), KEGG, Reactome, and MESH databases. The bar plot shows the percentage of enriched genes in various biological pathways and diseases, with dot sizes representing the number of enriched genes.

3.4. Chronic AICAR treatment rejuvenates the expression of 84 genes in old muscles

To specifically identify genes whose expression is “rejuvenated” by chronic AICAR treatment, we next compared gene expression between OS muscles and combined YS + OA muscles. Although the second principal component (PC2) in our PCA accounts for only 13% of the total variance, the OA samples show a trend toward clustering separately from saline‐treated old samples and closer to YS samples on this axis (Figure 4A). Combined with gene‐level analyses, this partial convergence suggests that AICAR treatment may help counteract some aging‐related transcriptional changes, warranting further investigation. We identified 84 differentially expressed genes in OS versus YS + OA muscles (Figure 4B, Table S4), with 50 being downregulated and 34 being upregulated in OS versus YS muscle and restored to youthful levels in OA muscles (Figure 4D). Individual genes of relevance to aging muscle biology are indicated on the volcano plot in Figure 4C. Figure 4E shows the differentially expressed genes organized by overrepresentation analysis using GO, KEGG, Reactome, and MESH databases, with 32 selected gene sets of particular relevance to aging biology included on the graph (out of the 350 total significantly enriched gene sets; 68 GO, 25 KEGG, 26 Reactome, 231 MESH).

FIGURE 4.

Differential gene expression analysis in old mice treated with chronic AICAR combined with young saline (YS) compared to old saline‐treated mice. (A) Principal Component Analysis (PCA) plot showing the variance in gene expression profiles among YS, Old Saline (OS), and Old AICAR + Young Saline (OA) treated quadricep muscles. The first two principal components (PC1 and PC2) capture 17% and 13% of the variance, respectively. (B) Heatmap displaying the expression levels of 84 differentially expressed genes (DEGs) identified in the comparison between Old AICAR + Young Saline (OA) and OS mice. Expression values are normalized and represented as Z‐scores. The samples are grouped by treatment (Young Saline [YS], Old Saline [OS], and Old AICAR + Young Saline [OA]). (C) Volcano plot illustrating the log2 fold change versus −log10(q‐value) for all tested genes in the comparison between Old AICAR + Young Saline and OS mice. Genes without an adjusted p‐value and those annotated as “pseudogene,” “predicted,” or “RIKEN” were excluded from the plot. Significant genes are highlighted based on q‐value <0.05. (D) Clustering of the 84 DEGs into two groups based on their co‐expression patterns. Hierarchical clustering with Pearson correlation and complete linkage was used. The relative expression (Z‐score) of each cluster is shown across the YS, OS, and Old AICAR + Young Saline (OA) groups. (E) Over‐representation analysis of the 84 DEGs using the clusterProfiler package from BioConductor, performed against the Gene Ontology (GO), KEGG, Reactome, and MESH databases. The bar plot shows the percentage of enriched genes in various biological pathways and diseases, with dot sizes representing the number of enriched genes.

3.5. Markers of mitochondrial biogenesis and content are increased by chronic AICAR treatment in old muscles

The sequencing study indicated that several mitochondria‐related gene sets were affected by chronic AICAR treatment. Therefore, we assessed several markers of mitochondrial biogenesis and content by RT‐PCR and western blotting. Expression of Ppargc1a, the gene for the master mitochondrial transcriptional coactivator, PGC‐1α, was not significantly affected by age or AICAR (Figure 5A). However, PGC‐1α protein levels were 137% and 225% higher in OS and OA muscles, respectively, versus YS muscles, but AICAR had no significant effect on PGC‐1α levels (Figure 5B,D). Cytochrome C levels (a marker of mitochondrial content) were not affected by age but were increased by ~33% with AICAR treatment in old muscles (Figure 5C,D). Citrate synthase activity, another marker of mitochondrial content, was ~22% higher in OS versus YS muscles, and further increased by ~14% in OA versus OS muscles (Figure 5E). Gene expression of osteocrin (Ostn), which encodes a myokine that putatively drives mitochondrial biogenesis, ²⁰ was elevated in OS versus YS muscles, and further increased by AICAR treatment in old muscles (Figure 5F). Finally, the expression of Mss51, which encodes a mitochondrially localized protein that negatively regulates mitochondrial processes such as beta‐oxidation and oxidative phosphorylation, ²¹ was elevated in OS versus YS muscles by ~203% but restored to YS levels by AICAR treatment (Figure 5G).

FIGURE 5.

Chronic AICAR treatment affects mitochondrial marker content in old muscle. (A) Ppargc1a gene expression, (B) PGC‐1α protein expression; (C) Cytochrome C protein content; (D) Representative blots for PGC‐1α and cytochrome C, and representative Ponceau S stain showing even protein loading across conditions; (E) Citrate synthase activity, (F) Ostn gene expression, and (G) Mss51 gene expression in quadricep muscle from mice treated for 31 days with AICAR. n = 8 mice/group. One‐way ANOVA and Fisher's LSD posthoc test p‐values are shown.

3.6. Chronic AICAR treatment suppresses the expression of the muscle atrophy‐related genes Mafbx and Murf1

To determine whether altered anabolic or catabolic signaling could underlie the preserved muscle mass we observed in the chronic AICAR‐treated mice, we measured the phosphorylation of Akt and downstream targets of the mechanistic target of rapamycin complex (mTORC1) signaling pathway. AICAR tended to increase levels of phospho‐Akt (p = 0.0661; Figure 6A) but did not significantly affect total Akt protein levels (Figure 6B). There were no significant differences in levels of phospho‐4EBP1 (Figure 6C), total 4EBP1 (Figure 6D), or phospho‐rpS6 (Figure 6E) with AICAR, although it did increase total levels of rpS6 (Figure 6F). Levels of insulin‐like growth factor (IGF‐1) in the gastrocnemius tended (p = 0.07) to be elevated in O mice and were significantly elevated by AICAR treatment (Figure 6G). The Mafbx (Atrogin‐1) and Murf1 genes encode E3 ubiquitin ligases that promote muscle atrophy. Murf1 (Figure 6I), but not Mafbx (Figure 6H) expression, was elevated in OS versus YS muscles, and AICAR treatment reduced the expression of both.

FIGURE 6.

Chronic AICAR treatment increases muscle IGF‐1 signaling and decreases muscle atrophy gene levels in old mice. (A) phosphorylated‐Akt (T308) levels, measured by western blot (WB); (B) total Akt protein levels, measured by WB; (C) phosphorylated‐eIF4E binding protein 1 (4EBP1; T37/46) levels, measured by WB; (D) Total 4EBP1 protein levels, measured by WB; (E) phosphorylated ribosomal protein S6 (rpS6; S235/236) levels, measured by WB; (F) total rpS6 protein levels, measured by WB; (G) gastrocnemius (gastroc) IGF‐1 concentration, measured by ELISA on muscle lysates (n = 7); (H) Mafbx mRNA, measured by RT‐PCR; (I) Murf1 mRNA levels, measured by RT‐PCR. N = 8/group unless otherwise indicated. One‐way ANOVA and Fisher's LSD posthoc test p‐values are shown.

4. DISCUSSION

Age‐related dysfunction, including that associated with sarcopenia, occurs due to changes in multiple cellular processes that have been referred to as the “hallmarks of aging”. ² Interestingly, AMPK activity has been shown to oppose many of these hallmarks by promoting healthy cellular function through increased autophagy ²² and mitochondrial biogenesis, ²³ reduced cell senescence, ²⁴ and suppression of inflammatory signaling, ²⁵ among other effects. Furthermore, basal AMPK activity and activation by some forms of muscle contraction are impaired in aged muscles, ⁶ , ⁷ , ⁸ , ⁹ , ¹⁸ suggesting that restoration of AMPK activity would be beneficial. Therefore, we hypothesized that chronic treatment of old mice with the AMPK‐activating prodrug AICAR would produce therapeutic benefits for sarcopenia in aged mice. Indeed, we found that 24–31 days of AICAR treatment improved the sarcopenic phenotype by mildly increasing muscle mass and force production, maintaining treadmill running capacity and increasing mitochondrial content while inhibiting markers of muscle degradation.

Like most, ¹³ , ¹⁸ , ¹⁹ but not all, ⁹ previous work, we found that acute AICAR treatment increases AMPK phosphorylation and activity of the α1 and α2 subunit in aged muscle, similar to that seen in young muscle. Chronic (up to 31 days) AICAR treatment did not affect body weight but increased (or preserved) quadricep muscle mass versus OS mice. A similar nonsignificant trend was observed for the gastrocnemius muscle. This is consistent with findings in other muscle‐wasting conditions, such as cancer cachexia, where chronic AICAR treatment preserves muscle mass. ²⁶ , ²⁷ However, this effect is somewhat counterintuitive since acute AMPK activation has long been known to inhibit anabolic signaling through mTORC1 and promote catabolism by increasing the expression of muscle‐specific ubiquitin ligases. ²⁸ , ²⁹ , ³⁰ , ³¹ One potential explanation for this paradox is that while acute AMPK activation promotes temporary muscle catabolism, it may subsequently protect the cell against catabolic insult (e.g., by its well‐established effects improving mitochondrial content and function and/or attenuating inflammatory signaling), thereby promoting a more anabolic basal state during recovery from AMPK stimulation. Recent findings also demonstrate that anabolic signaling through Akt and mTORC1 may be elevated 3 h after withdrawal of AICAR, ³² suggesting that the acute inhibitory effects of AICAR on protein synthesis may be reversed during recovery from AMPK activation.

However, when we examined markers of muscle anabolism and catabolism in the AICAR‐treated mice, we found that basal (48 h after the final AICAR injection) phosphorylation of Akt and mTORC1 pathway components were not significantly affected by age and AICAR treatment did not have a major effect on Akt or mTORC1 pathway phosphorylation in the OA compared to the OS mice at the time points tested here. Interestingly, however, ribosomal protein S6 total protein content and serum IGF‐1 levels were elevated in aged OA mice. These findings may indicate an increased content of ribosomal machinery and increased potential for protein translation under anabolic stimulation. Testing whether prolonged AICAR treatment enhances the muscle's response to anabolic stimuli would be interesting.

Previous work indicates that the muscle‐specific “atrogenes”, Mafbx and Murf1, are elevated in old muscle and contribute to the sarcopenic phenotype. ³³ , ³⁴ In this study, we found that Murf1 but not Mafbx RNA content was higher in the OS‐treated mice compared to the Y mice. Furthermore, we found that AICAR decreased RNA levels for both Mafbx and Murf1, suggesting suppression of protein degradation in AICAR‐treated muscles. Again, this is interesting because acute AICAR treatment has previously been shown to increase the expression of Mafbx and Murf1 in C2C12 myotubes and mice. ³⁰ , ³⁵ While there is limited evidence of chronic AICAR treatment effects on Mafbx and Murf1, our findings contrast with work in a Huntington's Disease model, where chronic AICAR increased atrogene expression in muscle. ³⁶ It is not clear in that study at what time after the last AICAR injection the muscles were harvested, so this discrepancy may be due to the timing after AICAR treatment or to the specific condition of the muscles being studied. Regardless, the decrease in Mafbx and Murf1 expression corresponds with the trending (p = 0.066) elevation of phosphorylated Akt and significant elevation in serum IGF‐1, which are known repressors of ubiquitin ligase expression and supports the idea that the adaptation to chronic AMPK activation in muscle may result in a less catabolic basal state.

As noted previously, AICAR has been shown to improve treadmill running capacity in young mice, ¹¹ but a previous study reported no change in treadmill running capacity in old mice with 14 days of chronic AICAR treatment. ¹⁷ Likewise, chronic AICAR treatment in a muscular dystrophy model does not improve treadmill running capacity, but it improves functional aerobic capacity. ³⁷ In agreement with those studies, we did not find that AICAR significantly increased running capacity in OA mice from pre‐ to post‐testing. However, we did find that AICAR prevented the expected decline in running capacity observed in the OS mice from pre‐ to posttesting in the OA mice. Additionally, AICAR treatment increased the maximum tetanic force production by EDL muscles from the old mice. These findings are consistent with Kobio et al., ¹⁶ who showed improved motor function in 23‐month‐old female mice after 14 days of AICAR treatment, and suggest that AMPK activation is effective in attenuating the effects of sarcopenia, both in protecting from atrophy as well as functional decline. Although we did not measure ambulatory activity in these mice, AICAR is known to increase basal ambulation in rats. ³⁸ This increase in basal activity may contribute to the effects we observed in this study.

To provide additional mechanistic insights into this protection, we assessed changes in gene expression by RNA sequencing. AMPK is well known to regulate gene transcription, particularly that supporting mitochondrial biogenesis, at least in part by increasing the content of the mitochondrial master regulator, PGC‐1α, ³⁹ and AICAR is well known to increase gene expression for mitochondrial and mitochondria‐related genes. ¹⁶ , ⁴⁰ Interestingly, we did not observe increased PGC‐1α protein content in OA versus OS muscles in our study. Still, consistent with previous work, ¹⁶ mitochondria‐related gene ontologies (e.g. oxidative phosphorylation, mirochondrial protein‐containing complex, respiratory chain complex) were among those most upregulated by AICAR treatment (Figure 3), and in gene sets where AICAR restored gene expression levels toward youthful levels (Figure 4). Western blotting and RT‐PCR confirmed that multiple markers of mitochondrial content, including citrate synthase and cytochrome C, were elevated in OA versus OS muscles.

Two specific transcriptional targets altered by AICAR treatment are of particular interest in relation to mitochondrial adaptation and warrant future research. Osteocrin (Ostn), also known as musclin, is an activity‐stimulated gene that improves muscular endurance through a calcium‐dependent activation of Akt that increases mitochondrial biogenesis. ²⁰ AICAR treatment increased Ostn gene content in the old muscle, which could be an additional mechanism by which AMPK activation promotes mitochondrial biogenesis. Secondly, Mss51 (also known as Zmynd17) is a novel cellular metabolism regulator found in skeletal muscle that is upregulated in aging muscle. ⁴¹ Mss51, a downstream target of the TGF‐β family (including myostatin), inhibits mitochondrial respiration and impairs glucose metabolism. ⁴² , ⁴³ It has also been shown that elevated Mss51 is associated with muscular dystrophy, and its deletion improves endurance in the Mdx muscular dystrophy mouse model. ⁴⁴ We confirmed by RT‐PCR that the Mss51 gene was elevated in our sarcopenic mouse model and, importantly, that it was reduced to youthful levels by AICAR treatment.

Interestingly, PGC‐1α protein content (but not gene expression) and citrate synthase activity, a commonly used marker of mitochondrial content, were elevated in the OS versus YS quadricep muscles. While this agrees with some previous work showing decreased citrate synthase activity and mitochondrial number in aged rodent muscle, ⁴⁵ , ⁴⁶ mitochondrial content is generally decreased in old‐age, ⁴⁷ and a decline in mitochondrial function has been demonstrated consistently, regardless of mitochondrial content. ⁴⁵ , ⁴⁶ , ⁴⁷ Since mitochondrial dysfunction is a key hallmark of aging, a putative increase in presumably “fresh” and functional mitochondria suggested by our findings may underlie at least part of the improved function observed in our study.

In addition to the effects of AICAR on mitochondria‐related gene expression, AICAR treatment restored other gene‐ontologies with well‐known roles in aging biology. For example, the top gene‐sets “rejeuvenated” by AICAR were related to regulation of collagen, fibrosis and extracellular matrix biology (e.g., collagen formation, collagen degradation, nonintegrin membrane‐ecm interactions, connective tissue development), which is consistent with previous work showing that AICAR decreases fibrosis (measured by Masson staining) in aged muscle, ¹⁵ and AMPK's general protective effects against fibrosis in other tissues. ⁴⁸ AICAR also significantly affected broader genesets relating to a number of neuromuscular disease conditions (e.g., amyotrophic lateral sclerosis, muscular dystrophies, sarcopenia, wasting syndrome, Parkinson disease, muscle weakness), which potentially underlie the improvements in function that we observed in our mice.

In conclusion, this study demonstrated functional improvement in organismal and muscle function after 31 days of AICAR treatment in aged mice. It should be remembered, however, that as an AMP analog, AICAR has effects apart from AMPK activation, and off‐target effects are likely to contribute to some degree. Additionally, AICAR has poor oral bioavailability and pharmacokinetics and is not a good therapeutic option for humans. Therefore, studies using more specific AMPK activators will be important. Nonetheless, our findings support pharmacological AMPK activation as a beneficial approach for treating age‐related muscle and organismal dysfunction.

AUTHOR CONTRIBUTIONS

S.H. Wilcox and D.M. Thomson planned the research; S.H. Wilcox, S.D. Dray, D.M. Rasch, A.H. Smith, K.D. Brodowski and D.M. Thomson performed the experiments; J. Calixto and J.T. Hill performed the RNA sequencing analysis; S.H. Wilcox, S.D. Dray, D.M. Rasch, A.H. Smith, K.D. Brodowski and D.M. Thomson analyzed all other data; S.H. Wilcox, J. Calixto and D.M. Thomson wrote the paper.

CONFLICT OF INTEREST STATEMENT

David Thomson has received sponsored research funding from Biolexis Therapeutics and Skylark Bioscience relating to AMPK‐activating drugs.

Supporting information

Table S1.

Table S2.

Table S3.

Table S4.

Wilcox SH, Calixto J, Dray SD, et al. Chronic treatment of old mice with AICAR reverses age‐related changes in exercise performance and skeletal muscle gene expression. FASEB BioAdvances. 2025;7:e1491. doi: 10.1096/fba.2024-00252

DATA AVAILABILITY STATEMENT

The RNA sequencing data produced in this study is available from the NCBI/SRA database at http://www.ncbi.nlm.nih.gov/bioproject/1184792. Other raw data is available from the corresponding author upon reasonable request.