Fig 2: AMPK is required for Beclin1 phosphorylation in phosphorus-induced lipophagy in tilapia hepatocytes. Tilapia hepatocytes were incubated with Pi and AMPKa1-knockdown experiment for 48 h in L-15 medium. (A) The presence of Lyso-Tracker Red-stained autolysosomes was demonstrated by the flow cytometry, showing the mean fluorescence intensity. (B) The Lyso-Tracker Red (50 nM)-stained autolysosome was calculated by the flow cytometric analysis of the mean fluorescence intensity. (C) The presence of AO-stained autophagic vacuole was demonstrated by the flow cytometry analysis of the mean fluorescence ratio. (D) The AO (1 µM)-stained autophagic vacuole was calculated by the flow cytometric analysis of the mean fluorescence ratio. (E) Representative confocal microscopy image of hepatocytes stained with acridine orange. (F, G) Western blot analysis and quantification analysis of the Beclin1, p-Beclin1, p62 and LC3B protein levels. (H) TG content. (I) The presence of LDs with Bodipy 493/503 staining was demonstrated by the flow cytometry of tilapia hepatocytes. (J) Bodipy 493/503-stained LDs was quantified by the flow cytometric analysis of FL1 (green) mean fluorescence intensity of tilapia hepatocytes. (K) Representative confocal microscopy image of tilapia hepatocytes with Bodipy 493/503 staining. All data are mean ± SEM (n = 3), *P = 0.05, **P = 0.01, ***P = 0.001, the P Value was calculated by the one-way ANOVA with the Bonferroni post-hoc test.

Fig 3: The mechanisms of dietary phosphorus inducing lipid degradation and lipophagy through AMPK/Beclin1 pathways. Dietary phosphorus activated AMPKa1 phosphorylation, triggered lipophagy by phosphorylating Beclin1, and induced lipid degradation of the liver tissues of tilapia.

Fig 4: Phosphorus incubation activated autophagy in tilapia hepatocytes. (A,B) Western blot analysis and quantification analysis of Beclin1, p-Beclin1, p62 and LC3B protein levels. (C) The Lyso-Tracker Red (50 nM)-stained autolysosome was calculated by the flow cytometric analysis of the mean fluorescence intensity. (D) The presence of Lyso-Tracker Red-stained autolysosome was demonstrated by the flow cytometry analysis of the mean fluorescence intensity. (E) The presence of AO-stained autophagic vacuole was demonstrated by the flow cytometry analysis of the mean fluorescence ratio. (F) The AO (1 µM)-stained autophagic vacuoles were calculated by the flow cytometric analysis of red/green (FL2/FL1) fluorescence ratio. (G) Lyso-Tracker Red (50 nM)-stained. Scale bar, 5 µm. (H) Representative confocal microscopic images of hepatocytes stained with acridine orange. Scale bar, 25 µm. All data are expressed as mean ± S.E.M (n = 3). P value was calculated by the Student's t tests. *P = 0.05, **P = 0.01, compared with control.

Fig 5: Dietary phosphorus addition increased autophagosome formation in tilapia liver. (A) Representative image of liver ultrastructure (TEM). N, hepatocyte nucleus, Ld, lipid droplet. Arrows indicate the autophagosomes and autolysosomes. Arrowheads indicate the lipophagy. (B) Relative mRNA levels of genes related to autophagy. (C,D) Western blot analysis of Beclin1, p-Beclin1 and LC3B protein levels. (E) mRNA levels of the FA ß-oxidation-related genes. (F) NEFA content. Data are mean ± SEM (n = 3). “a-c” denote significance at P = 0.05. The P Value was calculated by the one-way ANOVA and further the post-hoc Duncan's multiple range test.