Highlights
- •AMP deaminase (AMPD) decreases ATP without activating AMPK or its substrates.
- •AMPD alters the intracellular metabolome similar to atrophic muscle.
- •AMPD slows mitochondria synthesis and oxygen consumption similar to atrophic muscle.
- •Metabolome shift is independent of metabolic genes and precede mitochondria changes.
Abstract
Background
Skeletal muscle atrophy, whether caused by chronic disease, acute critical illness,
disuse or aging, is characterized by tissue-specific decrease in oxidative capacity
and broad alterations in metabolism that contribute to functional decline. However,
the underlying mechanisms responsible for these metabolic changes are largely unknown.
One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3:
AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP).
Given the central role of AdN in signaling mitochondrial gene expression and directly
regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would
be sufficient to alter their metabolic phenotype similar to that of atrophic muscle.
Methods
AMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles
via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles
were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted
measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics,
and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression.
Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the
AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity
were measured.
Results
TA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls
(−25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly
decrease ATP concentrations (−16%), increase IMP, and increase efflux of IMP catabolites
into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes
were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted
decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such,
pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3
at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular
metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The
most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and
ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced
pAMPK/AMPK (−24%), phosphorylation of AMPK substrates (−14%), and PGC-1α protein (−22%).
Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates
(−55%), basal ATP synthase-dependent (−13%), and maximal uncoupled oxygen consumption
(−15%).
Conclusions
Increased expression of AMPD3 significantly decreased mitochondrial protein synthesis
rates and broadly altered cellular metabolites in a manner similar to that of atrophic
muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK
signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism
is not dependent on reductions in oxidative capacity, but may be consequence of increased
AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism
that alters the metabolic phenotype of skeletal muscle during atrophy and could be
a target to improve muscle function during muscle wasting.
Keywords
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Article info
Publication history
Published online: August 12, 2021
Accepted:
August 10,
2021
Received:
March 30,
2021
Identification
Copyright
© 2021 Elsevier Inc. All rights reserved.
