Alcohol Acutely Antagonizes Refeeding-Induced Alterations in the Rag GTPase-Ragulator Complex in Skeletal Muscle

• Published in: Nutrients Vol. 13; no. 4; p. 1236
• Main Authors: Laufenberg, Lacee J, Crowell, Kristen T, Lang, Charles H
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 09.04.2021; MDPI
• Subjects: Adenosine triphosphatase; Alcohol; Alcohols; Amino Acid Transport Systems - genetics; Amino Acid Transport Systems - metabolism; Amino acids; anabolic resistance; Animals; Antibodies; Calories; Complex formation; Dietary restrictions; Ethanol; Ethanol - toxicity; Feeding Behavior; Gene expression; Glutamine - blood; H+-transporting ATPase; Insulin; Insulin-like growth factors; Kinases; Laboratories; Leucine - blood; leucine: LAMPTOR1: V-ATPase; Male; Membrane proteins; Mice; Mice, Inbred C57BL; Monomeric GTP-Binding Proteins - metabolism; mTORC1; Multiprotein Complexes - metabolism; Muscle Proteins - metabolism; Muscle, Skeletal - drug effects; Muscle, Skeletal - metabolism; Muscles; Musculoskeletal system; Peptides; Phosphorylation; Protein biosynthesis; Protein Biosynthesis - drug effects; Protein synthesis; Proteins; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sarcoplasmic Reticulum - drug effects; Sarcoplasmic Reticulum - metabolism; Signal transduction; Skeletal muscle; TOR protein; United States > US; anabolic resistance; protein synthesis; leucine: LAMPTOR1: V-ATPase; mTORC1
• ISSN: 2072-6643; 2072-6643
• DOI: 10.3390/nu13041236

The Ragulator protein complex is critical for directing the Rag GTPase proteins and mTORC1 to the lysosome membrane mediating amino acid-stimulated protein synthesis. As there is a lack of evidence on alcohol's effect on the Rag-Ragulator complex as a possible mechanism for the development of alcoholic skeletal muscle wasting, the aim of our study was to examine alterations in various protein-protein complexes in the Rag-Ragulator pathway produced acutely by feeding and how these are altered by alcohol under in vivo conditions. Mice (C57Bl/6; adult males) were fasted, and then provided rodent chow for 30 min ("refed") or remained food-deprived ("fasted"). Mice subsequently received ethanol (3 g/kg ethanol) or saline intraperitoneally, and hindlimb muscles were collected 1 h thereafter for analysis. Refeeding-induced increases in myofibrillar and sarcoplasmic protein synthesis, and mTOR and S6K1 phosphorylation, were prevented by alcohol. This inhibition was not associated with a differential rise in the intracellular leucine concentration or plasma leucine or insulin levels. Alcohol increased the amount of the Sestrin1•GATOR2 complex in the fasted state and prevented the refeeding-induced decrease in Sestrin1•GATOR2 seen in control mice. Alcohol antagonized the increase in the RagA/C•Raptor complex formation seen in the refed state. Alcohol antagonized the increase in Raptor with immunoprecipitated LAMPTOR1 (part of the Ragulator complex) after refeeding and decreased the association of RagC with LAMPTOR1. Finally, alcohol increased the association of the V domain of v-ATPase with LAMPTOR1 and prevented the refeeding-induced decrease in v-ATPase V1 with LAMPTOR1. Overall, these data demonstrate that acute alcohol intake disrupts multiple protein-protein complexes within the Rag-Ragulator complex, which are associated with and consistent with the concomitant decline in nutrient-stimulated muscle protein synthesis under in vivo conditions.