AMP-Activated Protein Kinase α1 but Not α2 Catalytic Subunit Potentiates Myogenin Expression and Myogenesis

Abstract

fThe link between AMP-activated protein kinase (AMPK) and myogenesis remains poorly defined. AMPK has two catalytic ␣ subunits, ␣1 and ␣2. We postulated that AMPK promotes myogenesis in an isoform-specific manner. Primary myoblasts were prepared from AMPK knockout (KO) mice and AMPK conditional KO mice, and knockout of the ␣1 but not the ␣2 subunit resulted in downregulation of myogenin and reduced myogenesis. Myogenin expression and myogenesis were nearly abolished in the absence of both AMPK␣1 and AMPK␣2, while enhanced AMPK activity promoted myogenesis and myotube formation. The AMPK␣1-specific effect on myogenesis was likely due to the dominant expression of ␣1 in myoblasts. These results were confirmed in C2C12 cells. To further evaluate the necessity of the AMPK␣1 subunit for myogenesis in vivo, we prepared both DsRed AMPK␣1 knockout myoblasts and enhanced green fluorescent protein (EGFP) wild-type myoblasts, which were cotransplanted into tibialis anterior muscle. A number of green fluorescent muscle fibers were observed, showing the fusion of engrafted wildtype myoblasts with muscle fibers; on the other hand, very few or no red muscle fibers were observed, indicating the absence of myogenic capacity of AMPK␣1 knockout myoblasts. In summary, these results indicate that AMPK activity promotes myogenesis through a mechanism mediated by AMPK␣1. Skeletal muscle, which comprises about 40% of the body mass of adults, is the main peripheral tissue responsive to insulinstimulated uptake of glucose (1) and is critical in the development of type 2 diabetes (2). Proper myogenesis is critical for fetal muscle development (3-5) and postnatal muscle growth and regeneration, which relies heavily on the myogenic differentiation of satellite cells (6-8). In improper myogenesis and muscle regeneration, damaged muscle fibers are replaced with fibric tissue, leading to muscle atrophy and aging (9-11).AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme, composed of ␣, ␤, and ␥ subunits, which plays an important role in energy metabolism (12)(13)(14). In addition to its capacity to acutely regulate the activity of metabolic enzymes through phosphorylation, AMPK is increasingly recognized for its regulatory role in gene expression, cell differentiation, and tissue development (15, 16). The role of AMPK in muscle fiber atrophy has been well defined; a number of studies demonstrated that AMPK promotes muscle protein degradation and autophagy (17) and inhibits protein synthesis (18). To date, however, the role of AMPK in myogenesis (formation of muscle fibers) has been sparsely studied. Our previous studies showed that low AMPK activity due to obesity is correlated with attenuated myogenic differentiation during fetal muscle development (19-21). We further observed that AMPK promotes myogenin expression and myogenesis through phosphorylation of histone deacetylase 5 (HDAC5) (22), suggesting that AMPK has a critical role in myogenesis.The catalytic ␣ subunit of AMPK has two isoforms, ␣1 and ␣2, which display differential...

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

“…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.…”

“…Fluorescence-activated Cell Sorting-Tibialis anterior (TA) muscle was digested in DMEM with collagenase D and dispase II as previously described (24). Cells were blocked in antimouse CD16/CD32 antibody and then stained with anti-mouse CD45 PE-Cy7, anti-mouse TER119 PE-Cy7, anti-mouse CD31 PE-Cy7, anti-mouse Sca-1 APC-Cy7, and anti-mouse integrin ␣7 APC antibodies.…”

“…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.…”

AMPKα1 knockout enhances nociceptive behaviors and spinal glutamatergic synaptic activities via production of reactive oxygen species in the spinal dorsal horn

“…Mice carrying the homozygous floxed AMPKα1 allele (flanking exon 3 of the kinase [53]) were crossed with mice expressing the Cre recombinase under the control of the glial fibrillary acid protein promoter (GFAP) [51] to produce their offspring. Male offspring which carried the Cre-GFAP recombinase and were heterozygous for the AMPKα1 LoxP flanked allele were crossed with homozygous floxed AMPKα1 female mice to generate GFAP-AMPKα1 conditional knockout mice in the nervous system.…”

“…Studies of different cell types have shown that AMPKα1 and AMPKα2 play different roles in physiological and pathological processes [49-51]. In this study, to better define the molecular signaling pathways and synaptic mechanisms by which AMPKα1 controls the pain signaling system, we utilized the Cre-LoxP system to conditionally knockout the AMPKα1 gene in the nervous system of mice.…”