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Smith CD, Compton RA, Bowler JS, Kemp JT, Sudweeks SN, Thomson DM, Winder WW. Characterization of the liver kinase B1-mouse protein-25 -Ste-20-related adaptor protein complex in adult mouse skeletal muscle. J Appl Physiol 111: 1622-1628, 2011. First published September 8, 2011 doi:10.1152/japplphysiol.00160.2011In liver, the AMP-activated protein kinase kinase (AMPKK) complex was identified as the association of liver kinase B1 (LKB1), mouse protein 25 (MO25␣/␤), and Ste-20-related adaptor protein (STRAD␣/ ␤); however, this complex has yet to be characterized in skeletal muscle. We demonstrate the expression of the LKB1-MO25-STRAD complex in skeletal muscle, confirm the absence of mRNA splice variants, and report the relative mRNA expression levels of these proteins in control and muscle-specific LKB1 knockout (LKB1 Ϫ/Ϫ ) mouse muscle. LKB1 detection in untreated control and LKB1 Ϫ/Ϫ muscle lysates revealed two protein bands (50 and 60 kDa), although only the heavier band was diminished in LKB1 Ϫ/Ϫ samples [55 Ϯ 2.5 and 13 Ϯ 1.5 arbitrary units (AU) in control and LKB1 Ϫ/Ϫ , respectively, P Ͻ 0.01], suggesting that LKB1 is not represented at 50 kDa, as previously cited. The 60-kDa LKB1 band was further confirmed following purification using polyethylene glycol (43 Ϯ 5 and 8.4 Ϯ 4 AU in control and LKB1 Ϫ/Ϫ , respectively, P Ͻ 0.01) and ionexchange fast protein liquid chromatography. Mass spectrometry confirmed LKB1 protein detection in the 60-kDa protein band, while none was detected in the 50-kDa band. Coimmunoprecipitation assays demonstrated LKB1-MO25-STRAD complex formation. Quantitative PCR revealed significantly reduced LKB1, MO25␣, and STRAD␤ mRNA in LKB1 Ϫ/Ϫ muscle. These findings demonstrate that the LKB1-MO25-STRAD complex is the principal AMPKK in skeletal muscle.AMP-activated protein kinase; AMP-activated protein kinase kinase; diabetes; metabolism AMP-ACTIVATED PROTEIN KINASE (AMPK) was first reported to be activated by muscle contraction nearly 15 years ago (14,29,32). Since that time, this signaling system has been the focus of a very large number of studies demonstrating its important roles in regulation of muscle metabolism, including stimulation of glucose transport, fatty acid oxidation, and mitochondrial biogenesis (6,7,16,30,31). Although it became clear that phosphorylation of the ␣-subunit of AMPK on Thr 172 is essential for activity, identification of the upstream kinase was elusive. Studies in liver by Hawley and co-workers (10) demonstrated purified protein fractions with AMPK kinase (AMPKK) activity, but the specific proteins contributing to this activity were not identified until 2003, when the protein complex consisting of liver kinase B-1 (LKB1), mouse protein-25 (MO25), and Ste-20-related adaptor protein (STRAD) was identified as AMPKK by three different research groups (9, 21, 34). The work identifying this AMPKK was done on liver and cells in culture, and not skeletal muscle. LKB1, MO25 protein, and STRAD mRNA were subsequently shown to be expressed in skeletal muscle (13,24). T...

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Many cell processes that are disrupted in aging skeletal muscle are also regulated by AMP‐activated protein kinase (AMPK). However, the effect of aging on skeletal muscle AMPK activation is controversial and unclear. Our purpose was to determine the effect of old‐age on endurance‐type contraction‐induced AMPK activation in skeletal muscle. Gastrocnemius muscles from YA (8 mo old) and O (30 mo old) male Fischer344 x Brown Norway F1 hybrid rats were removed after 10 minutes of electrically stimulated in situ contractions (STIM). Muscles from the resting legs served as controls (REST). AMPK phosphorylation and AMPKα2 activity were 63% and 19% lower, respectively, in O vs. YA muscles after STIM. AMPKα1 activity was unaffected by STIM in YA, but increased by 30% in O muscles. AMPK α1 protein concentration was 45% greater, while α2 content was 18% lower in O vs. YA muscles. AMPK β1, β2, and γ1 proteins were unaffected by age, but AMPK γ2 and γ3 concentrations were 75% and 85% lower in O vs. YA muscles, respectively. In conclusion, AMPKα2 activation is impaired while AMPKα1 activation is enhanced after endurance‐type in situ contractions in aged skeletal muscle. This work was funded by NIAMSD Grant AR‐51928.