Andrographolide ameliorates hepatic steatosis by suppressing FATP2-mediated fatty acid uptake in mice with nonalcoholic fatty liver disease

Fig. 1. Andro treatment improved metabolic syndrome in HFD-fed mice. a The chemical structure of Andro. b. Workflow of the animal experiment. c–e At the end of experiment (age of week 24), body weight (c), fasting blood glucose (FBG, d), HbA1c (e), insulin resistance (IPITT, f and g), glucose tolerance (OGTT, h and i), serum insulin (j), C-peptide (k), total triglyceride (TG, l), and total cholesterol (TC, m) were determined. g, i are quantitation of area under curve (AUC) of (f, h), respectively. Each dot represents data from one animal. **p < 0.01 and ***p < 0.001 versus vehicle. #p < 0.05, ##p < 0.01, and ###p < 0.001 versus HFD group

Fig. 2. Andro treatment attenuated steatosis in HFD-fed mice. a Representative images of liver sections stained with HE. b Representative images of liver sections with Oil Red O staining to show the lipid droplets. c Quantitation of triglyceride in liver tissue. TG, triglyceride. Scale bar in (a, b) is 50 μm

Fig. 3. Andro suppressed the oleic acid-induced lipid accumulation in LO2 cells. a LO2 cells were treated with indicated concentrations of Andro for 24 h followed by the determination of viability with CCK8 reagent. ***p < 0.001 versus blank control (0). b LO2 cells were treated with oleic acid alone or in combination with Andro of indicated concentrations for 48 h. Intracellular lipid droplets were revealed by Oil Red O staining. Scale bar, 100 μm. c The Oil Red O staining was resolved by isopropanol followed by colorimetric quantitation. ***p < 0.001. OA, oleic acid

Fig. 4. Andro differentially regulated the expression of lipid metabolism-related genes. a, b LO2 cells were treated with oleic acid or its vehicle (BSA) in combination with or without 20 μM Andro for 24 h. RT-PCR was performed to quantitate the relative expression of indicated genes associated with lipid anabolism (a) and catabolism (b), respectively. Each treatment was performed in triplicates. *p < 0.05, **p < 0.01, and ***p < 0.001 versus BSA. #p < 0.05 and ##p < 0.01 versus OA. c–g RT-PCR to quantitate the relative expression of FATP1 to 5 in liver tissues of mice with indicated treatments. Each dot represents one animal. ***p < 0.001 versus vehicle. ###p < 0.001 versus HFD. OA, oleic acid

Fig. 5. FATP2 was suppressed by Andro treatment in vivo and in vitro. a LO2 cells were treated with 0.5 mM BSA-conjugated OA or vehicle (BSA) in combination with or without 20 μM Andro for 48 h. The FATP2 protein level was determined by western blot. b Quantitation of (a). n = 3 for each treatment. ***p < 0.001. c–f The protein level of FATP2 in liver tissues of indicated mouse groups was checked by western blot (c, d) and immunohistochemistry (e, f). d, f Quantitation of (c, e), respectively. Each dot in (d, f) represents one animal. *p < 0.05 and ***p < 0.001 versus vehicle. #p < 0.05 and ###p < 0.001 versus HFD. Scale bar in e is 50 μm. OA, oleic acid

Fig. 6. Overexpression of FATP2 abolished the anti-steatosis effect by Andro. a LO2 cells were transfected with pLVX-FATP2 for 48 h followed by the determination of FATP2 expression by western blot. b LO2 cells were transfected with or without pLVX-FATP2 followed by treatment with the indicated reagents for 48 h. Oil Red O staining was performed to show intracellular lipid droplets. Scale bar, 50 μm. OA, oleic acid

Fig. 7. Andro suppressed oleic acid-induced fatty acid uptake which was abolished by FATP2 overexpression. a The LO2 cells were transfected with or without pLVX-FATP2 plasmid followed by treatment with indicated reagents. Fatty acid uptake was monitored by incubating the cells with fluorescent dye-labeled fatty acid and checked with a fluorescent microscope. Scale bar is 50 μm. b Flow cytometry analysis to quantitate the incorporated fluorescent dye-labeled fatty acid. Each treatment was performed in triplicates. ***p < 0.001 versus BSA group. #p < 0.05 versus OA group. $$p < 0.01 versus OA + Andro group. OA, oleic acid

Fig. 8. Schematic graph to summarize the anti-steatosis mechanism of Andro in hepatic cells. Andro executes its anti-steatosis effect by not only suppressing de novo fatty acid and triglyceride synthesis as previously reported (indicated by the black crosses) but also attenuating fatty acid uptake by targeting FATPs as demonstrated in this study (indicated by the green cross)