Yasuhiko Okimura

Sestrin 2 (SESN2) is a conserved protein whose expression is upregulated under various cellular stresses including hepatic injury. In the injured liver, hepatic stellate cells (HSCs) become activated and produce collagen, contributing to fibrosis; however, SESN2 overexpression has been shown to suppress collagen synthesis. Amino acids are known to influence SESN2 expression; however, their specific effects remain unclear. In the present study, it was investigated whether specific amino acids regulate SESN2 expression and the function of quiescent RI‑T HSCs, which are responsible for collagen production, using reverse transcription‑quantitative PCR, western blotting, and cell proliferation assay. It was found that supplementation with asparagine (Asn) and proline (Pro) (AP), both non‑essential amino acids, led to a complete reduction in SESN2 and activating transcription factor 4 (ATF4) mRNA levels after 5 h of incubation. Additionally, AP partially reduced collagen type I α1 (COL1A1) mRNA levels. However, knockdown of SESN2 or ATF4 resulted in a more substantial reduction in COL1A1 mRNA levels than the supplementation with AP. These results suggest that SESN2, which is induced by amino acid insufficiency, contributes to the upregulation of COL1A1 mRNA levels and that AP may increase COL1A1 mRNA levels through pathways independent of SESN2. The COL1A1‑inducing effect of SESN2 contrasted with the inhibitory effect of SESN2 on activated HSCs. In long‑term cultures, AP supplementation increased COL1A1 mRNA and protein levels, as well as RI‑T cell proliferation, while SESN2 and ATF4 mRNA levels remained suppressed. These findings suggested that the absence of AP induces relative amino acid starvation, leading to increased ATF4/SESN2 expression. By contrast, long‑term AP supplementation alleviated this stress, promoting cell proliferation and COL1A1 synthesis. The present results indicate that SESN2 function in quiescent HSCs may differ from its role in activated cells, providing new insights into its regulatory mechanisms in collagen production.

Branched-chain amino acids (BCAAs) and IGF-I, the secretion of which is stimulated by growth hormone (GH), prevent muscle atrophy. mTOR plays a pivotal role in the protective actions of BCAA and IGF-1. The pathway by which BCAA activates mTOR is different from that of IGF-1, which suggests that BCAA and GH work independently. We tried to examine whether BCAA exerts a protective effect against dexamethasone (Dex)-induced muscle atrophy independently of GH using GH-deficient spontaneous dwarf rats (SDRs). Unexpectedly, Dex did not induce muscle atrophy assessed by the measurement of cross-sectional area (CSA) of the muscle fibers and did not increase atrogin-1, MuRF1 and REDD1 expressions, which are activated during protein degradation. Glucocorticoid (GR) mRNA levels were higher in SDRs compared to GH-treated SDRs, indicating that the low expression of GR is not the reason of the defect of Dex’s action in SDRs. BCAA did not stimulate the phosphorylation of p70S6K or 4E-BP1, which stimulate protein synthesis. BCAA did not decrease the mRNA level of atrogin-1 or MuRF1. These findings suggested that Dex failed to modulate muscle mass and that BCAA was unable to activate mTOR in SDRs because these phosphorylations of p70S6K and 4E-BP1 and the reductions of these mRNAs are regulated by mTOR. In contrast, after GH supplementation, these responses to Dex were normalized and muscle fiber CSA was decreased by Dex. BCAA prevented the Dex-induced decrease in CSA. BCAA increased the phosphorylation of p70S6K and decreased the Dex-induced elevations of atrogin-1 and Bnip3 mRNAs. However, the amount of mTORC1 components including mTOR was not decreased in the SDRs compared to the normal rats. These findings suggest that GH increases mTORC1 activity but not its content to recover the action of BCAA in SDRs and that GH is required for actions of Dex and BCAA in muscles.