Abstract
ATF4 is a transcription factor that induces a genetic program for amino acid synthesis and amino acid uptake. Previous work demonstrated that ATF4 expression is increased either by insulin or by the general amino acid control (GAAC) response, an evolutionarily ancient pathway that is activated when eukaryotic cells are deprived of amino acids. It is not known whether insulin and the GAAC pathway increase ATF4 expression by the same or different mechanisms. In these studies, we demonstrate that insulin-mediated ATF4 expression occurs as part of a coordinated anabolic program that does not require an essential component of the GAAC pathway, the protein kinase GCN2. Moreover, insulin and the GAAC pathway have an additive effect on expression of ATF4 and downstream mRNAs for amino acid synthesis and uptake. These data suggest that the GAAC pathway may facilitate insulin-mediated anabolism when exogenous amino acids are limiting. We conclude that insulin signaling and the GAAC response comprise two distinct yet complimentary pathways to ATF4 expression, allowing anabolism to be finely tuned to amino acid availability.
Highlights
In mammals, changes in nutrient availability induce changes in levels of metabolic hormones, which in turn orchestrate the metabolism of trillions of cells, allowing them to cooperate and function as a single organism [1, 2]
We demonstrate that insulin-mediated ATF4 expression occurs as part of a coordinated anabolic program that does not require an essential component of the general amino acid control (GAAC) pathway, the protein kinase GCN2
GCN2 phosphorylates the eukaryotic translation initiation factor 2␣, thereby inhibiting global protein synthesis but increasing synthesis of a few specific proteins. One such protein that is increased under these conditions is a basic leucine zipper transcription factor that activates a large panel of genes encoding amino acid transporters and amino acid biosynthetic enzymes
Summary
ATF4 modified Eagle’s medium containing 1 g/liter of glucose (Mediatech catalog number 10-014) containing antibiotics (100 units/ml penicillin, 100 g/ml streptomycin sulfate) and 10% (v/v) fetal bovine serum;. SYNTHESIS CATABOLISM ments in medium A on day 0 at a density of 7 ϫ 105 cells/100-mm dish (for use on day 2) or 3.5 ϫ 105. Cells/100-mm dish (for use on day 3). RNA Interference—Mouse L cells were set up at a density of 2.5 ϫ 105 cells/60-mm dish on day 0. On day 1, cells were washed with PBS, refed with 3 ml of medium B, followed by the addition of a mixture containing 1 ml of OptiMEM (Invitrogen), 10 l of Lipofectamine 2000 reagent (Invitrogen) and 200 nM siRNA duplex (to give a final siRNA concentration of 50 nM).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
