Figure 5

Cholesterol treatment of zebrafish larvae increased the extrahepatic ATP and Ado levels. (a) Oil Red O staining of the liver (black line) of 8 days postfertilization (dpf) wild-type larvae fed a normal (control) or 5% cholesterol (HCD) diet. Lower magnification sagittal images (top panel) and higher magnification images (bottom panel) are shown. Scale bar 200 µm. (b) Representative hematoxylin and eosin (HE)-stained images of the liver sections (red dotted line) from zebrafish larvae. Lipid droplets are seen in larvae fed a HCD (white arrowheads). Scale bar 20 µm. (c) Representative electron micrographs of the control and HCD groups. Lipid droplets (asterisks), glycogen accumulation (red painted area) and lysosomal phagocytosis (black arrows) are visible in the HCD group. The vacuoles often found in control samples were mitochondria. Scale bar 5.0 μm for (i,iii); 2.0 μm for (ii); 500 nm for (iv). (d) Hepatic mRNA levels of inflammatory genes tnfa and il1b, inflammasome-related gene nlrp3, and fibrosis marker mmp9 were determined in the control and HCD groups of zebrafish larvae using qRT-PCR. (e,f) Representative images of the liver of GRAB_ATP zebrafish at 8 dpf. The GFP fluorescence intensity in the liver was increased in response to HCD treatment (bottom panel). Liver: white dotted line. Scale bar 100 μm (e). Quantification of the change in the GFP fluorescence intensity in GRAB_ATP zebrafish larvae with or without cholesterol treatment (n = 10, each group) (f). (g,h) Representative images of the liver in GRAB_Ado zebrafish larvae at 8 dpf. The GFP fluorescence intensity in the liver was increased in response to HCD treatment (bottom panel). Liver: white dotted line. Scale bar 100 μm (g). Quantification of the change in the GFP fluorescence intensity in GRAB_Ado zebrafish larvae with or without HCD treatment (n = 9, 15, each group) (h). Data are mean ± SEM. *P < 0.05; **P < 0.01 vs. control. N.D. not detected, N nucleus, n.s. non-significant.