Ampk alpha2 T172 activation dictates exercise performance and energy transduction in skeletal muscle.

AMPKα2 T172A knock-in mice, but not AMPKα1 T172A knock-in mice, showed increased fat-to-lean mass ratio.

• CRISPR-Cas9 was used to generate nonactivatable Ampkα knock-in (KI) mice with mutation of threonine-172 to alanine (T172A).
• This approach was designed to circumvent limitations of previous genetic interventions that disrupt protein stoichiometry.
• Only Ampkα2 KI mice, not Ampkα1 KI mice, demonstrated this body composition phenotype.
• The T172A mutation renders the kinase nonactivatable by preventing phosphorylation at the activation loop.

AMPKα2 T172A knock-in mice demonstrated impaired endurance exercise capacity.

• Phenotypic changes were observed specifically in Ampkα2 KI mice but not in Ampkα1 KI mice.
• Impaired endurance exercise capacity was one of three key phenotypic changes identified in AMPKα2 KI mice.
• The finding establishes AMPKα2 T172 activation as critical for exercise performance in skeletal muscle.
• Both Ampkα1 and Ampkα2 KI lines were generated using the same CRISPR-Cas9 T172A mutation strategy, enabling isoform-specific comparison.

AMPKα2 T172A knock-in mice exhibited diminished mitochondrial maximal respiration and conductance in skeletal muscle.

• Mitochondrial maximal respiration was reduced in skeletal muscle of Ampkα2 KI mice.
• Mitochondrial conductance was also diminished in these mice.
• This phenotype was specific to Ampkα2 KI mice and not observed in Ampkα1 KI mice.
• These functional mitochondrial deficits were among the key phenotypic changes identified in Ampkα2 T172A KI mice.

Integrated temporal multiomics analysis revealed a pleiotropic yet imperative role of AMPKα2 T172 activation in glycolytic and oxidative metabolism, mitochondrial respiration, and contractile function in skeletal muscle.

• The multiomics approach combined proteomics, phosphoproteomics, and metabolomics.
• Samples were collected at rest and during exercise to capture temporal changes.
• Analysis was performed in skeletal muscle tissue.
• The integrated analysis identified roles spanning glycolytic metabolism, oxidative metabolism, mitochondrial respiration, and contractile function.

There is substantial overlap between skeletal muscle proteomic changes in AMPKα2 T172A knock-in mice and proteomic changes observed in patients with type 2 diabetes.

• The proteomic comparison was made between AMPKα2 T172A KI mouse skeletal muscle and human skeletal muscle from type 2 diabetes patients.
• This overlap suggests a potential mechanistic link between impaired AMPKα2 T172 activation and type 2 diabetes pathology.
• The authors suggest AMPKα2 T172 activation may serve as a therapeutic target for type 2 diabetes based on this finding.
• The finding emerged from the broader temporal proteomics dataset collected at rest and during exercise.

The T172A knock-in approach using CRISPR-Cas9 was specifically designed to overcome limitations of previous AMPK genetic models that disrupt protein stoichiometry.

• Previous genetic interventions targeting AMPK were limited by disruption of protein stoichiometry.
• The T172A point mutation renders the kinase nonactivatable without removing the protein.
• Separate KI lines were generated for both Ampkα1 and Ampkα2 isoforms.
• The approach allowed isoform-specific functional dissection of AMPKα1 versus AMPKα2 T172 phosphorylation in vivo.