US Army Research of Environmental Medicine, Natick, Massachusetts, USA.
Military Operational Medicine Research Program, Ft. Detrick, Maryland, USA.
Physiol Rep. 2023 Oct;11(20):e15805. doi: 10.14814/phy2.15805.
AKT signaling plays a crucial role in muscle physiology, and is activated by stimuli, including insulin, growth factors, and exercise. Three AKT isoforms have been identified in mammals, and they possess both distinct and redundant functions. However, it is currently unknown what the predominant AKT isoform is in primary human skeletal myotubes, and very little is known regarding the effects of insulin and insulin-like growth factor-I (IGF-I) on AKT isoforms activation in human myotubes. Thus, we sought to determine the abundances of each AKT isoform in primary human skeletal myotubes and their responses to insulin or IGF-I. Analysis of protein lysates by liquid chromatography-parallel reaction monitoring/mass spectrometry revealed that AKT1 was the most abundant AKT isoform and AKT3 was the least-abundant isoform. Next, myotubes were treated with either 100 nM insulin or 10 nM IGF-I for 5, 20, 45, or 60 min. In response to insulin, there was a significant treatment effect on phosphorylation of AKT1 and AKT2, but not AKT3 (p < 0.01). In response to IGF-I, there was a significant treatment effect on phosphorylation of pan-AKT at all timepoints compared to control (p < 0.01). Next, we determined how much of the total AKT isoform pool was phosphorylated. For insulin stimulation, AKT1 was significantly higher than AKT2 at 5 min and 60 min posttreatment (p < 0.05 both) and significantly different than AKT3 at all timepoints (p < 0.05). For IGF-I stimulation, AKT1 was significantly higher than AKT2 at 45 and 60 min posttreatment (p < 0.05 both) and significantly higher than AKT3 at all timepoints (p < 0.05). Our findings reveal the differential phosphorylation patterns among the AKT isoforms and suggest a potential explanation for the regulatory role of AKT1 in skeletal muscle.
AKT 信号转导在肌肉生理学中起着至关重要的作用，并且可以被包括胰岛素、生长因子和运动在内的刺激激活。哺乳动物中已经鉴定出三种 AKT 同工型，它们具有独特且冗余的功能。然而，目前尚不清楚主要的 AKT 同工型在原代人骨骼肌肌管中是什么，并且对于胰岛素和胰岛素样生长因子-I（IGF-I）对人肌管中 AKT 同工型激活的影响知之甚少。因此，我们试图确定原代人骨骼肌肌管中每种 AKT 同工型的丰度及其对胰岛素或 IGF-I 的反应。通过液相色谱-平行反应监测/质谱分析蛋白质裂解物表明，AKT1 是最丰富的 AKT 同工型，AKT3 是最少的同工型。接下来，用 100 nM 胰岛素或 10 nM IGF-I 处理肌管 5、20、45 或 60 min。对胰岛素的反应，AKT1 和 AKT2 的磷酸化有显著的处理效应，但 AKT3 没有（p<0.01）。对 IGF-I 的反应，与对照相比，所有时间点的 pan-AKT 磷酸化均有显著的处理效应（p<0.01）。接下来，我们确定了总 AKT 同工型库中有多少被磷酸化。对于胰岛素刺激，AKT1 在处理后 5 和 60 min 时明显高于 AKT2（p<0.05），并且在所有时间点都明显不同于 AKT3（p<0.05）。对于 IGF-I 刺激，AKT1 在处理后 45 和 60 min 时明显高于 AKT2（p<0.05），并且在所有时间点都明显高于 AKT3（p<0.05）。我们的发现揭示了 AKT 同工型之间的差异磷酸化模式，并为 AKT1 在骨骼肌中的调节作用提供了潜在的解释。
