Figure 1
APOA4 is transcribed and translated primarily in the gut and transported to adipose tissue via circulation. (A) APOA4 gene was expressed in mouse jejunum and liver tissues, but not in gonadal adipose tissue and kidney. RT-PCR for ApoA4 and β-actin was performed on total mRNA isolated from jejunum, liver, gonadal fat, and kidney tissues from male and female 129X1/SvJ WT mice (n = 3 for each sex). ApoA4 levels were normalized with β-actin levels and data was presented as fold change over normalized ApoA4 mRNA levels in jejunum. See Supplemental Fig. S1. (B) Presence of APOA4 protein in tissues of WT but not of ApoA4−/− mice. Total protein (40 µg) from jejunum, liver, epididymal fat and kidney tissues and plasma (10 μg) were separated by SDS-PAGE and probed with anti-APOA4 antibody. The blot was stripped and probed with anti-β-actin for loading control and used for normalization. A representative western blot image shows the presence of APOA4 protein in all tissues from WT, but not ApoA4−/− mice (n = 3/group); these findings also confirmed specificity of the antibody used. (C-E) Representative immunofluorescence confocal images of APOA4 immunoreactivity (IR) in mouse gonadal fat tissues. Epididymal fat from male and periovarian fat from female mice were harvested, fixed, paraffin embedded, sectioned and stained with anti-APOA4 and anti-caveolin-1 (n = 2 technical and 3 biological replicates). Scale bar: 50 μm. (C) APOA4 immunoreactivity (APOA4-IR) was present at the cell surface of adipocytes in a discrete manner in male and female WT mice (arrows) and in the vasculature (inset: arrowhead), (D) no APOA4-IR was detected in ApoA4−/− mice, thereby also confirming specificity of the antibody used. (E) I.p. injection of r-m-APOA4 (5 μg/g body weight) in ApoA4−/− mice resulted in APOA4 uptake in gonadal fat tissues with similar pattern of localization of APOA4-IR in adipocytes as that in WT mice (arrows show discrete cell surface expression). (F) Apo-A-IV-IR co-localized with the adipocyte cell surface marker caveolin-1 (Merge, yellow arrows), but not in the vasculature (white arrowhead), confirming that exogenous APOA4 is targeted to the appropriate cell types. Scale bar: 50 μm and 10 µm.
