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

Abstract

Background/Aims: Carnivores exhibit poor utilization of dietary carbohydrates and glucose intolerant phenotypes, yet it remains unclear what are the causal factors and underlying mechanisms. We aimed to evaluate excessive amino acids (AAs)-induced effects on insulin signaling, fatty acid biosynthesis and glucose metabolism in rainbow trout and determine the potential involvement of mTORC1 and p38 MAPK pathway. Methods: We stimulated trout primary hepatocytes with different AA levels and employed acute administration of rapamycin to inhibit mTORC1 activation. Results: Increased AA levels enhanced the phosphorylation of ribosomal protein S6 kinase (S6K1), S6, and insulin receptor substrate 1 (IRS-1) on Ser³⁰² but suppressed Akt and p38 phosphorylation; up-regulated the expression of genes related to gluconeogenesis and fatty acid biosynthesis. mTORC1 inhibition not only inhibited the phosphorylation of mTORC1 downstream targets, but also blunted IRS-1 Ser³⁰² phosphorylation and restored excessive AAs-suppressed Akt phosphorylation. Rapamycin also inhibited fatty acid biosynthetic and gluconeogenic gene expression. Conclusion: High levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser³⁰² phosphorylation, which in turn impairs Akt activation and thereby weakening insulin action. We propose that p38 MAPK probably also involves in these AAs-induced metabolic changes.

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

“…In vitro data showed that elevated fatty acid biosynthetic gene expression by a high level of AAs was associated with the enhanced phosphorylation of S6K1 and S6, which is totally consistent with our previous investigation (15). Furthermore, we also reported that elevated fatty acid biosynthetic gene expression was markedly suppressed by mTORC1 inhibition (15).…”

“…However, our latest data have also showed that high levels of AAs promote hepatic fatty acid biosynthesis through upregulating the expression of genes related to DNL, NADPH production, and fatty acid bioconversion, and by elevating the amino acid catabolic pathway in trout hepatocytes (15). In this study, we further evaluated and compared the capacity of dietary protein intake/AAs and dietary carbohy- Fig.…”

“…Protein metabolism in rainbow trout is intimately linked to hepatic glucose metabolism since protein and amino acid catabolism provides substrates that channel into gluconeogenic pathways, and excessive AAs attenuate insulin action on hepatic gluconeogenesis via an mTORC1/S6K1-induced feedback loop (15,42). However, our latest data have also showed that high levels of AAs promote hepatic fatty acid biosynthesis through upregulating the expression of genes related to DNL, NADPH production, and fatty acid bioconversion, and by elevating the amino acid catabolic pathway in trout hepatocytes (15).…”

“…Ins*AA denotes interaction between Ins and AA. (15) and in vivo observations reporting that high-proteinlow-carbohydrate diets stimulated lipogenic gene expression (FAS and SREBP1) and enzyme activity (FAS, G6PDH, and ME) compared with low-protein-high-carbohydrate diets in rainbow trout and blackspot seabream, respectively (23,75). However, these results are different from the demonstrations in rodents, chickens, or humans reporting that high-protein (HP) diets induce downregulations of fatty acid biosynthesis in liver at both gene expression and enzymatic levels (1,2,7,56,64,67,87,90).…”

“…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).…”