Preservation of Liver Protein Synthesis during Dietary Leucine Deprivation Occurs at the Expense of Skeletal Muscle Mass in Mice Deleted for eIF2 Kinase GCN2

Tracy G Anthony,Brent J Mcdaniel,Rachel L Byerley,Barbara C Mcgrath,Douglas R Cavener,Margaret A Mcnurlan,Ronald C Wek

doi:10.1074/jbc.m404559200

Abstract

In eukaryotic cells, amino acid depletion reduces translation by a mechanism involving phosphorylation of eukaryotic initiation factor-2 (eIF2). Herein we describe that mice lacking the eIF2 kinase, general control nonderepressible 2 (GCN2) fail to alter the phosphorylation of this initiation factor in liver, and are moribund in response to dietary leucine restriction. Wild-type (GCN2(+/+)) and two strains of GCN2 null (GCN2(-/-)) mice were provided a nutritionally complete diet or a diet devoid of leucine or glycine for 1 h or 6 days. In wild-type mice, dietary leucine restriction resulted in loss of body weight and liver mass, yet mice remained healthy. In contrast, a significant proportion of GCN2(-/-) mice died within 6 days of the leucine-deficient diet. Protein synthesis in wild-type livers was decreased concomitant with increased phosphorylation of eIF2 and decreased phosphorylation of 4E-BP1 and S6K1, translation regulators controlled nutritionally by mammalian target of rapamycin. Whereas translation in the liver was decreased independent of GCN2 activity in mice fed a leucine-free diet for 1 h, protein synthesis in GCN2(-/-) mice at day 6 was enhanced to levels measured in mice fed the complete diet. Interestingly, in addition to a block in eIF2 phosphorylation, phosphorylation of 4E-BP1 and S6K1 was not decreased in GCN2(-/-) mice deprived of leucine for 6 days. This suggests that GCN2 activity can also contribute to nutritional regulation of the mammalian target of rapamycin pathway. As a result of the absence of these translation inhibitory signals, liver weights were preserved and instead, skeletal muscle mass was reduced in GCN2(-/-) mice fed a leucine-free diet. This study indicates that loss of GCN2 eIF2 kinase activity shifts the normal maintenance of protein mass away from skeletal muscle to provide substrate for continued hepatic translation.

Highlights

Mammals require an adequate supply of dietary essential amino acids to grow and thrive
We report the following novel observations: 1) general control nonderepressible 2 (GCN2) is the dominant kinase involved in the phosphorylation of eukaryotic initiation factor-2 (eIF2)␣ in the liver in response to amino acid deprivation, because GCN2Ϫ/Ϫ mice do not display increased eIF2␣ phosphorylation in the liver following 1 h and 6 days of feeding a leucine-devoid diet; 2) GCN2 is important for viability under nutritional stress
GCN2Ϫ/Ϫ mice that consumed a leucinedevoid diet became scruffy in appearance and either lost significantly more weight than GCN2ϩ/ϩ mice, or died before the end of the study period; 3) mice lacking functional GCN2 had reduced abilities to chronically down-regulate hepatic protein synthesis in response to leucine starvation over time, resulting in preservation of liver mass relative to body size; 4) the inability to adequately restrain protein synthesis in the liver results in increased loss of skeletal muscle to supply needed substrate; and 5) loss of GCN2 reduces dephosphorylation of 4E-BP1 and S6K1 in response to leucine deprivation, suggesting a role for this eIF2␣ kinase in the regulation of the mTORdirected pathway

Summary

EXPERIMENTAL PROCEDURES

Animals and Diets—The following study protocol was approved by the Institutional Care and Use Committee at the Indiana University School of Medicine, Evansville Center for Medical Education. Each tissue sample was processed to determine the enrichment of labeled phenylalanine in liver protein as previously described [28]. Time course studies performed using the intraperitonal route of injection demonstrated constant enrichment of the free phenylalanine within the liver and linear incorporation of injected tracer into tissue protein for at least 20 min (data not shown). Phosphorylation of 4E-BP1—Phosphorylation of 4E-BP1 was measured as a change in migration during SDS-polyacrylamide gel electrophoresis as detected by immunoblot analysis as described previously [4]. The resultant supernatant was added to 1 volume of SDS sample buffer and subjected to protein immunoblot analysis using a polyclonal 4E-BP1 antibody (Bethyl Laboratories, Montgomery, TX). Phosphorylation of S6K1—Phosphorylation of S6K1 was measured as a decrease in mobility during SDS-polyacrylamide gel electrophoresis as described previously [4].

RESULTS

Mortality rate

DISCUSSION

Whereas genetic diversity impacted the gross phenotype in