Characterization of AKT Isoform Responses to Insulin and IGF‐I in Primary Human Skeletal Myotubes

Abstract

Insulin and Insulin‐like GrowthFactor‐I (IGF‐I) are peptide hormones that regulate numerous cell and tissueprocesses including metabolism, growth, and survival. One principal mediator of insulin and IGF‐Isignal transduction is the serine/threonine kinase AKT. Three AKT isoforms have been identified inmammals, and they have been found to possess both distinct and redundantfunctions. However, little is knownregarding the effects of insulin and IGF‐I on AKT isoform activation in humanskeletal muscle. Thus, we sought todetermine the abundances of each AKT isoform in primary human skeletal myotubesand their responses to insulin or IGFI. Analysis of protein lysates by Liquid Chromatography‐Parallel ReactionMonitoring/Mass Spectrometry revealed that AKT1 was the mosthighly‐expressed AKT isoform, expressed ~3‐ fold greater than AKT2 (P<0.001). AKT3 was the least‐abundant AKT isoform, expressed at a level ~2% that of AKT1 (P<0.001). Next, primary human myotubes were treatedwith either 100nM insulin or 10nM IGF‐I for 5, 20, 45, or 60 minutes andanalyzed for AKT phosphorylation by Western immunoblotting. AKT phosphorylation was determined using anAKT antibody that detects phosphorylation of all AKT isoforms in thehydrophobic motif (HM)(Ser 473 in AKT1, Ser 474 in AKT2, and Ser 472 inAKT3). Isoform‐specific AKT1 and AKT2phosphorylation was determined using antibodies specific for AKT1 S473 or AKT2S474, while phosphorylation of AKT3 was determined by immunoprecipitatinglysates using an AKT3‐specific antibody followed by Western blotting using the AKTHM antibody. Two‐way ANOVA was performedto compare treatment (insulin or IGF‐I) and time. In response to insulin, there was asignificant treatment effect on phosphorylation of AKT at the HM, (~2.5 – 4.3‐foldincrease at all time points compared to vehicle‐treated control myotubes (F=88.1;P<0.001)). There was also asignificant effect of insulin treatment on phosphorylation of AKT1 (elevated ~1.5‐ 2‐fold at all time points (F=29.9; P<0.001)), and AKT2 (~2.4 – 3.4‐foldgreater than control (F=45.8; P<0.001). Insulin‐stimulated AKT3 phosphorylation was observed at all time points, whereas AKT3 phosphorylation was minimal in unstimulated control myotubes. In response to IGF‐I, there was a significanttreatment effect on phosphorylation of AKT at the HM (~4 – 5‐fold increase atall time points compared to controls (F=260; P< 0.001)). There was a significant treatment effect ofIGF‐I on phosphorylation of AKT1 (elevated ~2.5 – 3.4‐fold at all time points (F=86.6;P<0.001), and AKT2 (~2.9–6‐fold greater than control (F=162.3; P<0.001)). AKT3 phosphorylation was detected at all timepoints in response to IGF‐I; in contrast, AKT3 phosphorylation was barely detectedin control myotubes. Together, thesedata demonstrate that AKT1 is the most abundant AKT isoform in primary humanmyotubes, and that activation of AKT isoforms is differentially regulated byinsulin and IGF‐I.The views expressed in thisabstract are those of the authors and do not reflect the official policy of theDepartment of Army, Department of Defense, or the U.S. Government. Thisabstract has been approved for public release with unlimited distributionThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.