Abstract
BackgroundBranched-chain amino acids, especially leucine, are known to interact with insulin signaling pathway and glucose metabolism. However, the mechanism by which this is exerted, remain to be clearly defined. In order to examine the effect of leucine on muscle insulin signaling, a set of experiments was carried out to quantitate phosphorylation events along the insulin signaling pathway in human skeletal muscle cell cultures. Cells were exposed to insulin, leucine or both, and phosphorylation events of key insulin signaling molecules were tracked over time so as to monitor time-related responses that characterize the signaling events and could be missed by a single sampling strategy limited to pre/post stimulus events.ResultsLeucine is shown to increase the magnitude of insulin-dependent phosphorylation of protein kinase B (AKT) at Ser473 and glycogen synthase kinase (GSK3β) at Ser21-9. Glycogen synthesis follows the same pattern of GSK3β, with a significant increase at 100 μM leucine plus insulin stimulus. Moreover, data do not show any statistically significant increase of pGSK3β and glycogen synthesis at higher leucine concentrations. Leucine is also shown to increase the magnitude of insulin-mediated extracellularly regulated kinase (ERK) phosphorylation; however, differently from AKT and GSK3β, ERK shows a transient behavior, with an early peak response, followed by a return to the baseline condition.ConclusionsThese experiments demonstrate a complementary effect of leucine on insulin signaling in a human skeletal muscle cell culture, promoting insulin-activated GSK3β phosphorylation and glycogen synthesis.
Highlights
Branched-chain amino acids, especially leucine, are known to interact with insulin signaling pathway and glucose metabolism
Cells were exposed to stimuli for the following durations: 0′, 2′, 5′, 10′, 30′, and 60′ and AKT, pAKT-S473, ERK1/2, ppERK1/2-T202-Y204, GSK3β, ppGSK3β-S21-S9, FOXO1a, pFOXO1-S256, mammalian target of rapamycin (mTOR), pmTOR-S2448, 4E binding protein 1 (4EBP1), pp4EBP1-T37-T46, P70S6K, and pP70S6K-T389 were measured using Western blots
In order to examine the effect of leucine, an essential branched-chain amino acid, on muscle insulin signaling, a set of experiments was carried out to quantitate phosphorylation events along the insulin signaling pathway in human skeletal muscle cell cultures
Summary
Branched-chain amino acids, especially leucine, are known to interact with insulin signaling pathway and glucose metabolism. In order to examine the effect of leucine on muscle insulin signaling, a set of experiments was carried out to quantitate phosphorylation events along the insulin signaling pathway in human skeletal muscle cell cultures. Cells were exposed to insulin, leucine or both, and phosphorylation events of key insulin signaling molecules were tracked over time so as to monitor time-related responses that characterize the signaling events and could be missed by a single sampling strategy limited to pre/post stimulus events. Insulin signaling pathway [1] is characterized by cascades of phosphorylation events, which activate and deactivate molecules critical for physiological responses such as glucose uptake, protein synthesis, and glycogen production (Figure 1). The specific function of this transcription factor has not yet been well determined; it is known to act as a regulator of cell response to oxidative stress and may play a role in myogenic growth and differentiation
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