AKT2 is the Predominant Akt Isoform Expressed in Human Skeletal Muscle

Abstract

AKT is a serine/threonine kinase that mediates a number of intracellular processes including proliferation, survival, and metabolism. Three Akt isoforms have been identified in mammals (AKT1, AKT2, and AKT3), and there exist distinct and redundant functions for each. While a great deal of our knowledge regarding the functions of the three AKT isoforms in skeletal muscle has been derived from the use of cell and animal models, there is limited information regarding the expression and actions of AKT isoforms in human skeletal muscle. Given this, we performed a series of experiments to determine the abundances of each AKT isoform in human skeletal muscle, and to identify the principal AKT isoform involved in human myogenesis. We first examined the RNA transcript expression of each AKT isoform from biopsies obtained from human quadriceps. AKT2 was the most highly expressed AKT transcript, exhibiting a15.5‐fold increase over AKT1 and AKT3 transcripts (P<0.001). No significant difference between expression of AKT1 and AKT3 was observed. We next determined the abundance of AKT protein isoforms by using antibody immunoprecipitation followed by Liquid Chromatography‐Parallel Reaction Monitoring/Mass Spectrometry (LC‐PRM/MS). Immunoprecipitation was performed using both mouse and rabbit pan AKT antibodies that were immunoreactive with purified peptides of all three AKT isoforms. Analysis of immunoprecipitates by LC‐PRM/MS revealed that AKT2 was the most highly expressed AKT in quadriceps muscle (4.2‐fold greater than AKT1 using the rabbit antibody (P<0.001) and 1.6‐fold greater than AKT1 using the mouse antibody(P<0.05)). Expression of AKT3 protein was virtually undetectable in quadriceps, suggesting that AKT3 plays little or no role in skeletal muscle physiology. Having found that AKT2 is the most highly expressed AKT isoform in human quadriceps, we next asked whether AKT2 was the principal isoform regulating human myotube formation. To test this, cultured primary human myoblasts were virally‐transduced with cDNAs encoding either wild‐type (WT) or kinase‐dead (KD) AKT1 or AKT2 and allowed to terminally differentiate. Myotubes expressing either WT‐AKT1 or WT‐AKT2 showed enhanced fusion compared to control myotubes(P<0.05 for each), while myotubes expressing KD‐AKT1 showed a 10% reduced fusion index (P<0.05). However, compared to control myotubes, myotubes expressing KD‐AKT2 showed a 63% decrease in fusion index (P<0.001). In conclusion, our findings identify AKT2 as the predominant AKT isoform expressed in human quadriceps muscle and as the principal AKT isoform that regulates differentiation of cultured human skeletal myoblasts.Support or Funding InformationThe opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Army or the Department of Defense.