Leucine Alleviates Endoplasmic Reticulum-Stress Mediated Lipotoxic Injury Induced by High-Fat Diet Via Sar1b/Sestrin2/Ampkα1 Pathways in Acanthopagrus Schlegelii.

BACKGROUND

With the increasing use of high-fat diet (HFD), fatty liver has become prevalent in farmed fish. Leucine regulates lipid metabolism via multiple pathways, but its role in hepatic lipotoxic injury remains unclear.

OBJECTIVE

This study aimed to determine the molecular mechanism responsible for alleviation of HFD-induced liver injury by dietary supplementation with leucine in black seabream (Acanthopagrus schlegelii).

METHODS

In vivo, juvenile black seabream (male; 1.25 ± 0.01 g) were fed 1 of 5 diets in triplicate (30 fish/replicate, n = 3) for 8-wk. The diets consisted of Control (11% lipid) and HFD (18% lipid) diets with the HFD supplemented with 0.5%, 1.0%, or 2.0% leucine. Fish were hand-fed a fixed ration twice daily. Growth, hepatic lipid deposition, and lipotoxic injury were assessed by biochemical, histological, and molecular (flow cytometry, qPCR, and Western blot) methods. In vitro, primary hepatocytes were treated with oleic acid (OA) alone or with 2 mM leucine (n = 3). To explore the mechanism, OA was combined with leucine, Ampk, Sar1b, and Sestrin2 overexpression. Oil Red O staining, immunofluorescence, qPCR, and Western blot assessed lipid accumulation, and signaling pathways. Data were means ± SEM and analyzed by t-test or 1-way analysis of variance, with P < 0.05 considered significant.

RESULTS

In vivo, leucine supplementation activated Sar1b/Sestrin2 and Ampkα1/Sirt1 pathways, reducing HFD-induced hepatic lipid deposition (36%-54%) by enhancing lipolysis (75%-309%) and suppressing lipogenesis (24%-87%). Leucine downregulated ER stress markers (p-Ire1α, 54%-67%) and inhibited p-P65 and p-Jnk (23%-33% and 23%-32%), attenuating inflammation and apoptosis. Similar patterns in vitro confirmed leucine's consistent alleviation of HFD-induced hepatic lipid deposition and lipotoxic injury across models. Overexpression of Ampkα1, Sar1b, or Sestrin2 mimicked leucine's effects, alleviating OA-induced lipid accumulation, inflammation, and apoptosis.

CONCLUSIONS

Leucine alleviated HFD-induced liver lipotoxic injury in black seabream by activating Sar1b/Sestrin2 and Ampk1/Sirt1 pathways, providing novel insights for developing nutritional strategies against diet-induced liver injury.