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Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. Methods: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S]methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. Results: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. Conclusions: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.

Two dogs with simultaneous congenital and acquired portosystemic shunts are reported. The first dog was an eight–month-old, male Golden Retriever with a history of peritoneal effusion, polyuria/ polydipsia, and stunted growth. The dog had a microcytic, hypochromic anemia, a mildly elevated AST, and a moderate to severely elevated preprandial and postprandial serum bile acids. Transcolonic portal scintigraphy confirmed the presence of a portosystemic shunt. An intraoperative mesenteric portogram was performed. Two conjoined congenital extrahepatic portosystemic shunts and multiple acquired extrahepatic portosystemic shunts were identified. The second dog was a five-month-old, mixed breed with two week history of peritoneal effusion. Abdominal ultrasound and transcolonic scintigraphy were used to diagnose a portosystemic shunt. A single extrahepatic portosystemic shunt, portal hypertension, and multiple acquired collateral shunts were identified at surgery. The histologic alterations observed in these dogs were consistent with a portosystemic shunt. In these dogs, the presence of congenital and acquired portosystemic shunts and histopathologic findings are considered to represent a combination of congenital portosystemic shunts and noncirrhotic portal hypertension or portal vein hypoplasia.

Background: The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1. Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein–protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by 35S-methionine/cysteine incorporation into cellular protein. Results: EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts. Conclusions: EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis.

The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GbetaL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS-1. EtOH also caused changes in mTORC1 protein-protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14-3-3 to raptor, while the PRAS40 and 14-3-3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14-3-3, whereas decreased GbetaL-mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GbetaL with mTOR, while likewise increasing the interaction of raptor with 14-3-3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes.