Postprandial regulation of hepatic glucokinase and lipogenesis requires the activation of TORC1 signaling in rainbow trout (Oncorhynchus mykiss)

Abstract

SUMMARYTo assess the potential involvement of TORC1 (target of rapamycin complex 1) signalling in the regulation of post-prandial hepatic lipid and glucose metabolism-related gene expression in trout, we employed intraperitoneal administration of rapamycin to achieve an acute inhibition of the TOR pathway. Our results reveal that rapamycin inhibits the phosphorylation of TORC1 and its downstream effectors (S6K1, S6 and 4E-BP1), without affecting Akt and the Akt substrates Forkhead-box Class O1 (FoxO1) and glycogen synthase kinase 3α/β (GSK 3α/β). These results indicate that acute administration of rapamycin in trout leads to the inhibition of TORC1 activation. No effect is observed on the expression of genes involved in gluconeogenesis, glycolysis and fatty acid oxidation, but hepatic TORC1 inhibition results in decreased sterol regulatory element binding protein 1c (SREBP1c) gene expression and suppressed fatty acid synthase (FAS) and glucokinase (GK) at gene expression and activity levels, indicating that FAS and GK activity is controlled at a transcriptional level in a TORC1-dependent manner. This study demonstrates for the first time in fish that post-prandial regulation of hepatic lipogenesis and glucokinase in rainbow trout requires the activation of TORC1 signalling.

Amino Acids Attenuate Insulin Action on Gluconeogenesis and Promote Fatty Acid Biosynthesis via mTORC1 Signaling Pathway in trout Hepatocytes

“…As expected, rapamycin partially inhibited the phosphorylation of TOR and 4E-BP1 and totally abolished the phosphorylation of S6K1 and S6 as previously observed [27,32]. More importantly, we observed that rapamycin significantly inhibited the phosphorylation of IRS-1 on Ser 302 and strongly increased the Akt phosphorylation (on Thr 308 and Ser 473 ) (Fig.…”

“…As for the regulation of lipogenesis, previous in vivo studies tend to indicate that dietary protein play a potent role in regulating mTOR signaling pathway [28], and lipogenic and glycolytic pathways in fish [28][29][30]. Moreover, our in vitro studies demonstrated that AAs (particularly leucine) together with insulin successfully stimulated the activation of mTOR signaling pathway [27,31], which was proven as an upstream activator for hepatic lipogenesis in trout liver and hepatocytes [27,32]. However, whether different AA levels affect mTORC1 activation and thereby regulating hepatic lipogenesis in fish is still unknown.…”

“…Lysates (2 µg of total protein per lane for Akt/S6/4EBP1, 7 µg for TOR/ S6K1/p38 MAPK and 17 µg for IRS-1, respectively) were subjected to SDS-PAGE and Western blotting using the appropriate antibodies. Anti-phospho-Akt (Ser ), anti-phospho-p38 MAPK and anti-p38 MAPK) were successfully crossreacted with rainbow trout [32]. For anti-phospho-IRS-1 (Ser 302 ), anti-phospho-p38 MAPK and anti-p38 MAPK antibody, the molecular weight and amino acid sequences were monitored in the SIGENAE database [37] to check for a good conservation of the antigen sequence.…”

Hepatic fatty acid biosynthesis is more responsive to protein than carbohydrate in rainbow trout during acute stimulations

“…2146) were purchased from Cell Signaling Technologies (Ozyme). We have confirmed that all of these antibodies successfully cross-reacted with rainbow trout (14). After washing, membranes were incubated with an IRDye infrared secondary antibody (LI-COR Biosciences, Lincoln, NE).…”

“…To prevent the other nutrient intake changes (except dietary carbohydrate or protein intake), we employed a controlled-feeding method for in vivo studies. As our recent work demonstrated that hepatic lipogenesis requires the activation of mechanistic target of rapamycin complex 1 (mTORC1) in rainbow trout (14,15,42) as in mammals (9,41,63), and elevated amino acid levels enhanced hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner (15), we also investigated the potential involvement of mTORC1 signaling pathway in the present study. Acute administration of rapamycin, a pharmacological inhibitor of mTOR, was employed to achieve an inhibition of mTORC1 and its downstream effectors, including p70 ribosomal S6 kinase 1 (S6K1), S6, and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (14 -16, 42, 74).…”