AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration*

Background: The mechanisms eliciting metabolic activation in satellite cells are unclear. Results: Noncanonical Sonic Hedgehog is activated following muscle injury, which activates AMPKα1 to induce Warburg-like glycolysis and promote satellite cell activation and proliferation. Conclusion: AMPKα1 is required for Warburg-like glycolysis in satellite cells, which promotes satellite cell activation and muscle regeneration. Significance: AMPK promotes satellite cell activation during muscle regeneration. Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.


Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPK␣1 is the dominant AMPK␣ isoform expressed in satellite cells, and AMPK␣1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPK␣1 knock-out. Taken together, AMPK␣1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.
Skeletal muscle is the main component in animal locomotion system. It is also the major tissue sustaining respiration and the primary peripheral tissue utilizing glucose and fatty acids, important in preventing obesity and type 2 diabetes (1-3). Skeletal muscle fibers are frequently damaged during exercise and because of physical trauma or diseases such as Duchenne muscular dystrophy (4,5). Efficient regeneration following muscle injury is critical for maintaining the normal physiological function of skeletal muscle. On the other hand, insufficient muscle regeneration replaces muscle fibers with fibrotic tissue and weakens the contractile function of muscle, which is a key etiological factor leading to progressive muscle weakness associated with aging and muscle dystrophic diseases (6 -8).
Despite the presence of multiple types of myogenic cells in skeletal muscle, satellite cells are the major postnatal myogenic cells indispensable for muscle regeneration (9). Satellite cells maintain in a quiescent stage and become activated when muscle regeneration process is triggered (10,11). Activated satellite cells proliferate to expand their population and undergo further myogenic differentiation orchestrated by sequential expression of myogenic regulatory factors, Myf5, MyoD, myogenin, and MRF4 (12).
impairs muscle regeneration, characterized by reduced satellite cell activation and muscle structure restoration.
Cell Culture-Satellite cells were resuspended in F-10 medium with 20% FBS, 1% antibiotic mixture and 5 ng/ml FGF2, and seeded on collagen-coated plates. Myogenic differentiation of satellite cells was induced by switching medium to DMEM supplemented with 2% horse serum and 1% antibiotic mixture. Nonmyogenic cells were cultured in DMEM with 10% FBS and 1% antibiotic mixture.
Satellite Cell Proliferation Essay-Satellite cells were isolated as previously described (25) with modifications. Muscle was removed from the hind limbs of 3-4-month-old mice. Muscle was cut into small pieces and digested in digestion buffer containing collagenase D and dispase II for about 30 min. Muscle slurry was passed through a 100-m cell strainer. Filtrate was centrifuged for 5 min at 350 ϫ g. Cell pellet was resuspended and cultured in F-10 medium with 20% FBS, 5 ng/ml FGF2, and 1% antibiotic mixture on collagen-coated plates. Satellite cells were enriched by preplating. Fast attaching nonmyogenic cells were also collected. Five thousand satellite cells were seeded in each well of 12 well plates. Cells were then trypsinized and counted at 1, 2, and 3 days after to determine the cell proliferation rate.
Single Muscle Fiber Culture-Single muscle fibers were isolated as previously described (26) with modification. The extensor digitorum longus muscle was removed from 1-month-old Pax7 Cre /AMPK␣1 fl/fl /tdtomato,EGFP and Pax7 Cre /tdtomato, EGFP mice that had been treated with tamoxifen. Extensor digitorum longus muscle was digested in digestion buffer containing collagenase D. Extensor digitorum longus muscle was then carefully flushed to release single muscle fibers. Intact single muscle fibers were then transferred to 24-well plates with one muscle fiber in each well and cultured in high glucose DMEM with 20% FBS, 5 ng/ml FGF2, 110 mg/ml sodium pyruvate, and 1% antibiotic mixture.
Glucose Uptake Test-Glucose uptake test was performed using glucose uptake cell base assay kit from Cayman (Ann Arbor, MI) following the manufacturer's protocol. The cells were seeded onto 96-well plates at a density of 1 ϫ 10 4 cells/ well. Cells were cultured with fluorescently labeled deoxyglucose analog, and fluorescence was detected using Synergy H1 hybrid reader (BioTek, Winooski, VT).
Real Time Quantitative PCR-Total RNA was extracted using TRIzol (Sigma) followed by DNase (New England Bio-Labs Inc., Ipswich, MA) treatment, and cDNA was synthesized using a reverse transcription kit (Bio-Rad). Real time PCR was carried out using CFX real time PCR detection system (Bio-Rad) with a SYBR Green real time PCR kit from Bio-Rad. After amplification, a melting curve (0.01°C/s) was used to confirm product purity, and agarose gel electrophoresis was performed to confirm that only a single product of the right size was amplified. Relative mRNA content was normalized to 18S rRNA content (24). Primer sequences and their respective PCR fragment lengths are listed below. Immunoblotting Analyses-Immunoblotting analysis was performed as previously described using an Odyssey Infrared Imaging System (LI-COR Biosciences) (27). Band density was normalized to ␤-tubulin content.
Immunocytochemical Staining-Cells grown on multiple well plates were fixed in cold methanol for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, blocked with 1% BSA, and incubated with primary antibodies at 4°C overnight. Cells were then stained with corresponding secondary antibodies (1:1,000) for 1 h. Images were taken using a EVOS microscope.
Immunohistochemical Staining-TA muscle was fixed in cold 4% paraformaldehyde and frozen in isopentane cooled in liquid nitrogen. Frozen tissue was sectioned (5-10 m thick). Sections were heated in citrate buffer for 20 min, blocked in 5% goat serum in TBS containing 0.3% Triton X-100, and stained with primary antibodies and corresponding fluorescent secondary antibodies. Sections were then mounted in a mounting medium containing DAPI (Vector Laboratories, Burlingame, CA).
Quantification of Satellite Cells and EMH ϩ Muscle Fibers-Pax7 ϩ cells with nuclei identified by DAPI staining were classified as satellite cells. For each TA muscle sample, the number of satellite cells and EMH ϩ muscle fibers on four randomly picked microscopic fields of each of three sections at different depths of the muscle were counted (four fields/section, three sections/muscle). Average numbers obtained from the three examined sections of each muscle sample were used as a biological replicate for comparative analysis.
Hemotoxylin Staining-TA muscle frozen sections were rinsed in PBS, stained with Gill's hemotoxylin, and counterstained with eosin Y following the manufacturer's protocol.
L-Lactate Assay-Ten thousand cells were seeded in each well of 96-well plates. 24 h after seeding, cell culture medium was collected and tested for lactate content using an L-lactate assay kit from Eton Bioscience, Inc. (San Diego, CA) following the manufacturer's instruction.
Oxygen Consumption Assay-200,000 cells were seeded in each well of 6-well plates. One day after seeding, cell culture medium was changed with fresh medium. Oxygen content in medium was measured after 30 min of incubation with Orion 3-Star Pus Dissolved Oxygen Meter (Thermo Scientific, Waltham, MA). Oxygen consumption was calculated from the difference between the oxygen content in medium after 30 min of incubation and the oxygen content in fresh medium.
Statistics-For all studies, at least three independent experiments were conducted. All data are expressed as means Ϯ S.D. The data were analyzed using the general linear model of SAS (SAS Inst. Inc., Cary, NC), and t test or Tukey range test was used to determine significance of differences among means. p Ͻ 0.05 was considered significant.

Expression of AMPK␣1 and AMPK␣2 during Skeletal Muscle
Regeneration-To test the role of AMPK␣1 in muscle regeneration, we first measured the expression of AMPK␣1 during the proliferation and differentiation of isolated satellite cells. AMPK␣1 expression profoundly increased (ϳ6-fold) during the activation and proliferation of satellite cells. After induction of myogenesis, the expression of AMPK␣1 dropped first, followed by a slight increase (Fig. 1A), which is consistent with a previous report showing AMPK␣1 activity in muscle increases during muscle regeneration, whereas the activity of AMPK␣2 remain unchanged (24,28). Moreover, AMPK␣1 expression was consistently and significantly higher than AMPK␣2 in quiescent, activated, and differentiating satellite cells (Fig. 1B). These data support our hypothesis that AMPK␣1 is important in activating satellite cell proliferation.
To test whether AMPK␣1 is critical for muscle regeneration following injury, we analyzed the expression of AMPK␣1 and AMPK␣2 in TA muscle injured by cardiotoxin (CTX) injection using immunohistochemical (IHC) staining after injury. AMPK␣2 was found to be expressed primarily in the cytoplasm of well differentiated myogenic cells and regenerating muscle fibers (Fig. 1C). In contrast, AMPK␣1 was found mainly in mononucleated cells including satellite cells (Fig. 1D). Moreover, we found that AMPK␣1 was also expressed in satellite cells in uninjured muscle (Fig. 1D). However, p-AMPK␣ was only detected in satellite cells after injury, indicating AMPK␣1 activation in satellite cells during muscle regeneration, which might be involved in satellite cell activation and proliferation (Fig. 1E). These data further suggest that AMPK␣1 has a regulatory role in satellite cell activation, whereas AMPK␣2 plays a major role in regulating metabolism in differentiated muscle fibers. Therefore, we focused further studies on the role of AMPK␣1 in satellite activation and proliferation.
AMPK␣1 Knock-out Reduces Satellite Cell Proliferation-To better understand the influence of AMPK␣1 KO on satellite cell activation and proliferation, we then tested the proliferation of satellite cells isolated from tamoxifen-treated Pax7 cre / AMPK fl/fl mice in which AMPK␣1 gene is specifically deleted in satellite cells. Satellite cell-specific AMPK␣1 KO was verified by different assays (Fig. 2, A-E). Indeed, AMPK␣1 KO satellite cells proliferated slower than WT myoblasts (Fig. 3A). Because isolated satellite cells lack the niche environment, we further prepared single muscle fibers from tamoxifen-treated Pax7 cre / tdomato,EGFP mice and Pax7 cre /AMPK␣1 fl/fl /tdomato,EGFP mice, in which satellite cells express membrane-located EGFP upon tamoxifen treatment accompanied with AMPK␣1 KO specifically in satellite cells. 48 h after muscle fiber isolation, satellite cells on muscle fibers were compared. Although 38 of 45 observed WT satellite cells had activated and started to proliferate, only 9 of 41 observed AMPK␣1 KO satellite cells showed sign of proliferation, which suggested less efficient satellite cell activation and proliferation because of AMPK␣1 KO (Fig. 3B).
Smoothened Agonist Promotes Satellite Proliferation through Activating AMPK-It has been recently reported that noncanonical Sonic Hedgehog (Shh) signaling promotes a Warburglike glycolysis in differentiated C2C12 myotubes (22). Therefore, we questioned whether this pathway is also present in undifferentiated myoblasts and satellite cells and whether AMPK␣1 has a mediatory role in eliciting Warburg-like glycolysis. We first tested the effect of Smoothened (Smo) agonist (SAG), an activator of Shh pathway, on AMPK activity in C2C12 myoblasts and WT satellite cells. C2C12 myoblasts and WT satellite cells were treated with 200 nM SAG for only 10 min to avoid the activation of canonical Shh signaling (22,29). In both C2C12 cells and WT satellite cells, 10 min of SAG treatment activated AMPK (Fig. 4, A and B). L-Lactate assay revealed  increased glycolysis rates in C2C12 cells and WT satellite cells in response to SAG treatment, which was absent in AMPK␣1 KO satellite cells (Fig. 4C).
In addition, SAG promoted the proliferation of both C2C12 cells and purified WT satellite cells but failed to promote proliferation of purified AMPK␣1 KO satellite cells (Fig. 4D), sug-gesting that the proliferative effects of SAG treatment on satellite cells require AMPK␣1. Skeletal muscle contains multiple cell types that interact with satellite cells and participate in muscle regeneration (30). To better understand the potential effect of SAG treatment on satellite cell activation and proliferation in the presence of other cell types, muscle tissue slurry from tamoxifen-treated Pax7 cre /tdomato,EGFP mice expressing EGFP in Pax7 ϩ cells was obtained by enzymatic digestion of muscle tissue and plated without sorting. 48 h later, all cells were harvested, and EGFP-positive satellite cells were quantified. We found that SAG treatment increased the number of EGFP ϩ satellite cells, further supporting the promotive effect of Shh signaling on satellite activation and proliferation (Fig. 4E).
Selective Activation of noncanonical Shh Promotes Satellite Cell Activation and Proliferation-To further test whether the observed effects of SAG on satellite cells was through noncanonical Shh signaling, WT primary myoblasts were treated with cyclopamine, a noncanonical Shh specific activator, which is also known to inhibit canonical Shh (22). 10 min of cyclopamine treatment successfully activated AMPK (Fig. 5A). Considering that noncanonical Shh signaling has relatively rapid response, we chose to only treat cells for 1 h with low doses of cyclopamine or SAG so that the impact of canonical Shh signaling on cell proliferation was minimized. Indeed, 1 h of SAG treatment did not change the expression of the two canonical Shh targets, Gli1 and Ptch1 (31,32), in primary myoblasts 24 h after treatment (Fig. 5B). Similarly, 1 h of cyclopamine treatment did not change the expression of Gli1 and only caused a slight reduction in Ptch1 expression 24 h after treatment (Fig.  5B). Therefore, fresh muscle tissue slurry from tamoxifentreated Pax7 cre /tdomato,EGFP mice and Pax7 cre /AMPK␣1 fl/fl / tdomato,EGFP mice with satellite cell-specific AMPK␣1 KO was treated with SAG and cyclopamine for 1 h in culture medium. Tissue slurry was then collected and cultured in SAG and cyclopamine-free medium. SAG and cycolopamine treatments were able to increase the number of EGFP ϩ satellite cells from Pax7 cre /tdomato,EGFP mice at 48 h after treatment but failed to do so to EGFP ϩ satellite cells without AMPK␣1 (isolated  from tamoxifen-treated Pax7 cre /AMPK␣1 fl/fl /tdomato,EGFP mice), indicating an AMPK␣1-dependent stimulatory effect of noncanonical Shh on satellite cell activation and proliferation (Fig. 5C). The same treatments were applied to purified satellite cells. We found that 1-h treatment of SAG and cyclopamine increased the number of purified WT myoblasts but not that of AMPK␣1 KO myoblasts, indicating a similar AMPK␣1-dependent stimulatory effect of noncanonical Shh on satellite cell proliferation (Fig. 5D). Moreover, glucose uptake of WT myoblasts was increased after 20 min of SAG or cyclopamine treatment, but no effect was observed for AMPK␣1 KO myoblasts (Fig. 5E). In addition, oxygen consumption was not changed in satellite cells treated with SAG or cyclopamine for 30 min, indicating a specific activation of glycolysis (Fig. 5F).
To assess the presence of canonical Shh and noncanonical Shh signaling during muscle regeneration, expression of Shh and Smo during muscle regeneration was analyzed. Surprisingly, a 30-fold increase in Smo expression was seen during the first day after muscle injury, showing profound activation of Shh signaling in the initiation of muscle regeneration, even though the expression gradually decreased in the following days (Fig. 5G). In contrast, no Shh expression was detected, which was not due to technical problems, because a high level of Shh expression was detected in day 13.5 embryo tissue, a positive control (Fig. 5H). In addition, the expression of two canonical Shh targets, Gli1 and Ptch1, dropped dramatically after injury and remained considerably lower afterward, indicating that noncanonical but not canonical Shh signaling was active in the initiation stage of muscle regeneration (Fig. 5I). The decreased Smo expression after 2 days post injury likely marked a completion of satellite cell activation and metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS) during myogenic differentiation; myogenic differentiation requires elevated OXPHOS and noncanonical Shh reduces OXPHOS (22,33).
Direct Activation of AMPK by AICAR Promoted Satellite Cell Activation-AICAR, a direct activator of AMPK, was used to test the effect of AMPK on satellite activation. 1 h of 250 M AICAR treatment successfully increased AMPK activity in isolated satellite cells (Fig. 6A). Cells were then isolated from whole muscle tissue of tamoxifen-treated Pax7 cre /tdomato,EGFP mice without sorting. 1 h of 250 M AICAR treatment after cell isolation also increased the number of GFP ϩ satellite 48 h after AICAR treatment (Fig. 6B). However, the same treatment failed to increase the number of GFP ϩ satellite cells isolated from tamoxifen-treated Pax7 cre /AMPK␣1 fl/fl /tdomato, EGFP mice, which indicates that direct activation of AMPK␣1 promotes satellite cell activation. Moreover, 20 min of AICAR treatment enhanced glucose uptake in WT satellite cells but not in AMPK␣1 satellite cells (Fig. 6C). Lactate content in culture medium of WT satellite cells increased by 24-h AICAR treatment, but no difference was found in AMPK␣1 KO satellite cells treated with AICAR, indicating an enhanced glycolysis in WT satellite cells mediated by enhanced AMPK␣1 activity (Fig.  6D). In addition, oxygen consumption was not altered in satellite cells treated with AICAR, clearly showing a specific effect of AICAR treatment on glycolysis (Fig. 6E).
Satellite Cell-specific KO of AMPK␣1 Attenuates Muscle Regeneration-To better understand the role of AMPK␣1 in the activation and proliferation of satellite cells during muscle regeneration while avoiding possible confounding effects of AMPK␣1 KO on cells other than satellite cells in muscle regeneration, we then employed a tamoxifen-inducible satellite cellspecific AMPK␣1 KO mouse model (Pax7 cre /AMPK␣1 fl/fl ). CTX was injected to the TA muscle of tamoxifen-treated AMPK␣1 fl/fl mice and Pax7 cre /AMPK␣1 fl/fl (Pax7 Cre / AMPK␣1 Ϫ/Ϫ ) mice, which showed similar degree of initial muscle damage (Fig. 7A). However, the number of Pax7 ϩ satellite cells and EMH ϩ muscle fibers was reduced in muscle from Pax7 Cre /AMPK␣1 Ϫ/Ϫ mice compared with AMPK␣1 fl/fl mice at 3 days post injury (Fig. 7, B and C). Because there was no difference in satellite cell density before injury, the reduced number of satellite cells and regenerating muscle fibers suggested attenuation of satellite cell activation and proliferation.
Moreover, at 3 days post injury, Pax7 ϩ satellite cells in both AMPK␣1 fl/fl mice and Pax7 Cre /AMPK␣1 Ϫ/Ϫ mice were proliferating as indicated by Ki67 expression (Fig. 8, A and C). However, although some MyoD ϩ myoblasts in AMPK␣1 fl/fl mice lost Ki67 expression indicating cell cycle arrest and initiation of myogenic differentiation, most MyoD ϩ myoblasts in Pax7 Cre / AMPK␣1 Ϫ/Ϫ mice remained positive for Ki67, indicating a delayed myogenic differentiation and a prolonged duration for proliferation (Fig. 8, B and C). Such a change was likely needed for compensating the impaired capacity of AMPK␣1 KO myoblasts to proliferate. These data clearly demonstrate that AMPK␣1 KO negatively affects satellite cell proliferation.

Discussion
Recent studies suggest the importance of metabolic transition in regulating the quiescence, activation, proliferation, and differentiation of stem cells (14,15,37). OXPHOS and glycolysis are two major metabolic pathways generating energy in mammalian cells (38). OXPHOS is more efficient in generating energy than glycolysis. However, OXPHOS requires ample oxygen supply and functional mitochondria to be accomplished, whereas glycolysis does not require oxygen and mainly occurs in cytoplasm (39). Many stem cells reside deep in tissue where access to oxygen is very limited (16,17). Moreover, most quiescent stem cells are relatively small with few immature mitochondria, indicating that glycolysis is a suitable metabolic pathway to supply energy (40 -42). In addition, glycolysis also provides intermediates needed for the synthesis of cellular components for stem cell self-renewal and proliferation such as nucleotides and lipids (43). On the other hand, overactive OXPHOS exposes stem cells to reactive oxygen species and in consequence increases the risk of DNA damage, inducing mutagenesis and impairing their stemness (15). Because of these advantages of glycolysis, stem cells mainly relay on glycolysis for energy generation (13,14). However, the relatively inefficient energy generation of glycolysis poses a challenge for stem cells to sustain multiple energy demanding processes associated with stem cell activation and proliferation.
Warburg glycolysis was first discovered in cancer cells (19,20). It is characterized by enhanced glycolysis even when oxygen is available. Because of the inefficient energy generation of glycolysis, Warburg glycolysis is associated with dramatically increased glucose uptake, which provides enough fuel to glycolysis to generate ample energy required for rapid and uncontrolled cell proliferation of cancer cells (44,45). Cancer cells and stem cells share similar characteristics including the ability to rapidly proliferate. Recently, a few studies reported that Warburg-like glycolysis is present in different types of stem cells and proliferating nontumor cells to support their energy demand during proliferation (21,46,47).
Satellite cells are adult stem cells residing in skeletal muscle (48,49). They stay in a quiescent state with low metabolic rate under normal conditions (10). However, their activation is rapidly triggered by muscle injury, followed by their proliferation and differentiation to mediate muscle regeneration (50). During the initial stage of muscle regeneration, satellite cells involve many rounds of cell proliferation before differentiation (50). Similar to other stem cells, quiescent satellite cells have immature mitochondria and a very low metabolic rate, and glycolysis supplies most energy for satellite cells (10,18). Robust mitochondria biogenesis and OXPHOS only start when myogenic differentiation is initiated (10,51). Therefore, there is a significant demand of energy to support the large biomass formation during satellite cell proliferation before myogenic differentiation. It was recently reported that activation and prolif- FIGURE 9. Schematic diagram shows the proposed mechanism for enhanced satellite cell activation and proliferation by noncanonical Shh via AMPK␣1 during muscle regeneration. Muscle injury triggers noncanonical Shh pathway in satellite cells, which activates AMPK␣1 and sequentially promotes a Warburg-like glycolysis supporting satellite cell activation and proliferation. When AMPK␣1 is absent in satellite cells, the proposed pathway is disrupted, resulting in reduced satellite cell activation and proliferation and the attenuated muscle regeneration. eration of satellite cells are associated with increased glycolytic metabolism (52). However, it remained unknown how satellite cells regulate their metabolism to support these processes. Although it has been shown that AMPK promotes Warburglike glycolysis in C2C12 myotubes, AMPK was shown to suppress Warburg-like glycolysis in certain cancer cells, suggesting the roles of AMPK in regulating metabolism might vary depending on cell types (22,53). Canonical Shh signaling is a well known signaling pathway promoting myogenic cell proliferation during prenatal myogenesis (54 -56). Our study revealed the critical role of a distinct noncanonical Shh signaling in muscle regeneration where it promotes satellite cell activation and proliferation during the initiation of muscle regeneration through enhancing glycolysis in satellite cells, a process mediated by AMPK␣1.
In summary, for the first time, to our knowledge, we showed that AMPK␣1 is important in muscle regeneration through mediating noncanonical Shh-triggered Warburg-like glycolysis in satellite cells, which is required for satellite activation and proliferation during muscle regeneration (Fig. 9). Because AMPK activity is attenuated because of a number of pathophysiological conditions such as obesity and type 2 diabetes, and drugs targeting AMPK are widely available as anti-diabetic drugs, our data suggest the possibility of applying these drugs to activate AMPK to facilitate muscle regeneration.