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Amino acid effects on translational repressor 4E-BP1 are mediated primarily by L-leucine in isolated adipocytes
H L Fox1, P T Pham, S R Kimball
1Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Abstract:
Previous studies indicated that amino acids may activate the protein kinase activity of the target of rapamycin (TOR) and thereby augment and/or mimic the effects of insulin on protein synthesis, p70(S6k) phosphorylation, and multicellular clustering in adipocytes. To identify the individual amino acids responsible for these effects, the present study focused on the TOR substrate and translational repressor 4E-BP1. A complete mixture of amino acids stimulated the phosphorylation of 4E-BP1, decreasing its association with eukaryotic initiation factor eIF-4E. Studies on subsets of amino acids and individual amino acids showed that L-leucine was the amino acid responsible for most of the effects on 4E-BP1 phosphorylation; however, the presence of other amino acids was required to observe a maximal effect. The stimulatory effect of leucine was stereospecific and not mimicked by other branched chain amino acids but was mimicked by the leucine metabolite alpha-ketoisocaproate (alpha-KIC). The effect of alpha-KIC, but not leucine, was attenuated by the transaminase inhibitor (aminooxy)acetate. The latter result indicates that the effects of alpha-KIC required its conversion to leucine. Half-maximal stimulation of 4E-BP1 phosphorylation occurred at approximately 430 microM; therefore, the response was linear within the range of circulating concentrations of leucine found in various nutritional states.
Insights
L-leucine activates the target of rapamycin (TOR) pathway by promoting 4E-BP1 phosphorylation, crucial for protein synthesis. Maximal effects require other amino acids, with leucine
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Amino acids may activate the target of rapamycin (TOR) kinase activity.
- TOR activation influences insulin signaling, protein synthesis, and adipocyte function.
- Previous studies suggested amino acids mimic insulin's effects on cellular processes.
Purpose of the Study:
- To identify specific amino acids responsible for TOR pathway activation.
- To investigate the role of individual amino acids in regulating 4E-BP1 phosphorylation.
- To elucidate the mechanism by which amino acids modulate protein synthesis initiation.
Main Methods:
- Treatment of adipocytes with amino acid mixtures, subsets, and individual amino acids.
- Assessment of 4E--BP1 phosphorylation and its association with eukaryotic initiation factor eIF-4E.
- Stereospecificity studies and investigation of leucine metabolites like alpha-ketoisocaproate (alpha-KIC).
- Use of transaminase inhibitor (aminooxy)acetate to study alpha-KIC metabolism.
Main Results:
- A complete amino acid mixture stimulated 4E-BP1 phosphorylation, reducing eIF-4E binding.
- L-leucine was the primary driver of 4E-BP1 phosphorylation, though other amino acids enhanced the effect.
- Leucine's effect was stereospecific and mimicked by alpha-KIC, indicating metabolic conversion to leucine.
Conclusions:
- L-leucine is a key amino acid in activating the TOR pathway via 4E-BP1 phosphorylation.
- Optimal TOR activation requires L-leucine in conjunction with other amino acids.
- The observed effects are physiologically relevant, occurring within physiological leucine concentrations.