BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival in septic mice

Abstract

Endotoxin (LPS) and sepsis decrease mammalian target of rapamycin (mTOR) activity in skeletal muscle, thereby reducing protein synthesis. Our study tests the hypothesis that inhibition of branched-chain amino acid (BCAA) catabolism, which elevates circulating BCAA and stimulates mTOR, will blunt the LPS-induced decrease in muscle protein synthesis. Wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout mice were studied 4 h after Escherichia coli LPS or saline. Basal skeletal muscle protein synthesis was increased in knockout mice compared with WT, and this change was associated with increased eukaryotic initiation factor (eIF)-4E binding protein-1 (4E-BP1) phosphorylation, eIF4E.eIF4G binding, 4E-BP1.raptor binding, and eIF3.raptor binding without a change in the mTOR.raptor complex in muscle. LPS decreased muscle protein synthesis in WT mice, a change associated with decreased 4E-BP1 phosphorylation as well as decreased formation of eIF4E.eIF4G, 4E-BP1.raptor, and eIF3.raptor complexes. In BCATm knockout mice given LPS, muscle protein synthesis only decreased to values found in vehicle-treated WT control mice, and this ameliorated LPS effect was associated with a coordinate increase in 4E-BP1.raptor, eIF3.raptor, and 4E-BP1 phosphorylation. Additionally, the LPS-induced increase in muscle cytokines was blunted in BCATm knockout mice, compared with WT animals. In a separate study, 7-day survival and muscle mass were increased in BCATm knockout vs. WT mice after polymicrobial peritonitis. These data suggest that elevating blood BCAA is sufficient to ameliorate the catabolic effect of LPS on skeletal muscle protein synthesis via alterations in protein-protein interactions within mTOR complex-1, and this may provide a survival advantage in response to bacterial infection.

Fig. 1.

Effect of LPS on in vivo protein synthesis in skeletal muscle (top) and heart (bottom) from wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout (KO) mice. Phe, [³H]-L-phenylalanine. Values in bar graph are means ± SE; n = 10 mice per group. Values with different letters are significantly different from each other (P < 0.05).

Fig. 2.

Effect of LPS on 4E binding protein-1 (4E-BP1) phosphorylation and the distribution of eukaryotic initiation factor (eIF)-4E (eIF4E) in skeletal muscle of WT and BCATm KO mice. A: bar graph quantitating the densitometric analysis of Western blot analysis for phosphorylated 4E-BP1 in total muscle homogenate. In the total homogenate, there was no effect of gene deletion (e.g., KO) or LPS on the total amount eIF4E, eIF4G, or 4E-BP1 (data not shown). Additionally, eIF4E was immunoprecipitated from the homogenate and then immunoblotted for 4E-BP1 (B), eIF4G (C), and eIF4E [data not shown; P = not significant (NS)]. For all bar graphs, the value from the control WT group was set at 1.0 arbitrary unit (AU). Values in bar graph are means ± SE; n = 9–10 mice per group. Values with different letters are significantly different from each other (P < 0.05).

Fig. 3.

Effect of LPS on proline-rich Akt substrate 40 kDa (PRAS40) and raptor phosphorylation in skeletal muscle of WT and BCATm KO mice. Bar graphs represent, quantitation of the densitometric analysis of all immunoblots for either phosphorylated PRAS40 (top) or raptor (bottom) in which the value from the control WT group was set at 1.0 AU. No difference in total PRAS40 or raptor among groups (data not shown). Values in bar graph are means ± SE; n = 8–10 mice per group. Values with different letters are significantly different from each other (P < 0.05).

Fig. 4.

Effect LPS on mamalian target of rapamycin (mTOR), 4E-BP1, and PRAS40 binding with raptor in skeletal muscle of WT and BCATm KO mice. Raptor was immunoprecipitated, and the amount of mTOR (A), 4E-BP1 (B) and PRAS40 (C) bound to the raptor immunoprecipitate was determined. The amount of raptor in the immunoprecipitate did not differ among the 4 groups (data not shown). D: eIF3f was immunoprecipitated and the amount of raptor bound was determined by immunoblotting. The amount of eIF3 in the immunoprecipitate did not differ between groups (data not shown). Bar graphs, represent quantitation of the densitometric analysis of all immunoblots where the value from the control WT group was set at 1.0 AU per either total raptor or eIF3 in immunoprecipitate. Values in bar graph are means ± SE; n = 8–10 mice per group. Values with different letters are significantly different from each other (P < 0.05).

Fig. 5.

Effect of LPS on muscle cytokine mRNA content in WT and BCATm KO mice. The mRNA content for TNF-α (A), IL-6 (B), nitric oxide synthase-2 (NOS2; C), and IκBζ (D) was determined by ribonuclease protection assay. Bar graphs, values are means ± SE; n = 8–10 mice per group. Values with different letters are significantly different from each other (P < 0.05).

Fig. 6.

Survival and food consumption of WT and BCATm KO mice to peritonitis produced by cecal ligation and puncture (CLP). A: survival was determined in all 4 experimental groups over a 7-day period. *P < 0.05, compared with BCATm KO + sepsis group. B: food consumption was determined daily for all 4 groups. *P < 0.05, septic groups, compared with nonseptic control values. There was no significant difference in food consumption between WT-septic and BCATm KO-septic mice at any time point.

Fig. 7.

Body weight, lean body mass, and gastrocnemius weight in WT and BCATm KO mice 7 days after sepsis produced by CLP. Bar graphs, values are means ± SE; n = 7–8 mice per group. Values with different letters are significantly different from each other (P < 0.05).