Fig 2: Endocytosis of apoA5 in hypertrophied adipocytes (21 days of differentiation) is reduced compared with the non-hypertrophied control (8 days of differentiation). (A) Western blot analysis of apoA5 expression in hypertrophied adipocytes. Differentiated 3T3-L1 adipocytes at day 8 and day 21 were starved in serum-free medium for 12 h. Cells were then incubated in serum-free medium in the presence of 600 ng/ml recombinant human apoA5 for 4 h. β-actin served as a loading control. #P<0.05 vs. control. (B) Differentiated 3T3-L1 adipocytes at day 8 and 21 were pulsed with 125I-apoA5 for 2 h in serum-free medium and chased for various time-points up to 24 h. Radioactivity (cpm) was normalized to cell protein, and data are expressed as total cpm/mg protein at each time-point. Data are expressed as the mean ± standard error of 3 independent experiments. ApoA5, apolipoprotein A5.

Fig 3: ApoA5 protein levels in obese and non-obese human adipose tissue. Western blot analysis of apoA5 in subcutaneous abdominal adipose tissue from obese (BMI ≥30) (n=3) and non-obese (BMI <25) humans (n=3). The experiment was performed three times. *P<0.05 vs. control. ApoA5, apolipoprotein A5.

Fig 4: ApoA5 treatment reduces the TG concentration but did not affect lipid droplet formation in human adipocytes. Differentiated adipocytes were starved in serum-free medium for 12 h. Cells were then incubated in serum-free medium in the presence or absence of 200 ng/ml apoA5 for 48 h. (A) TG concentration in adipocytes was determined. ***P<0.001 vs. control. (B) Cells were stained with Oil Red O and 4′,6-diamidino-2-phenylindole. Representative images from confocal microscopy analysis are presented. Scale bar, 30 µm. (C) Quantification of droplet number per cell (left graph) and mean droplet size (right graph) after treatment with or without apoA5. Data are expressed as the mean ± standard error of at 5 independent experiments. ApoA5, apolipoprotein A5; TG, triglyceride.

Fig 5: ApoA5 reduces the expression levels of the genes encoding cidec and perilipin in human adipocytes. Differentiated adipocytes were starved in serum-free medium for 12 h. Cells were then incubated in serum-free medium in the presence or absence of 200 ng/ml apoA5 for various time-points up to 48 h. mRNA expression levels of (A) CIDEC and (B) PLIN1. The mRNA expression levels of each gene normalized relative to 18S rRNA expression, and presented as mRNA levels relative to the 0 h control (without apoA5). ***P<0.001 vs. control. (C) Western blot and densitometric analysis of cidec, perilipin and β-actin (control) expression levels in adipocytes 24 h after 200 ng/ml apoA5 treatment. Data are expressed as the mean ± standard error of 3 independent experiments. *P<0.05 and **P<0.01 vs. control. ApoA5, apolipoprotein A5; CIDEC, cidec gene; PLIN1, perilipin gene.