Fig. 4: GRASP55 is required for intestinal fat absorption and chylomicron secretion.

a An oral fat tolerance test was performed with 12-week-old male mice after oral gavage of olive oil (10 μl g⁻¹ of body weight). Tyloxapol (Triton WR-1339) was administered 20 min after the olive oil gavage (arrow, 0.5 g kg⁻¹ intravenous injection) to inhibit lipoprotein catabolism. Plasma triglyceride (TG) concentrations were measured at the indicated time points (n = 6). b Photograph of blood plasma taken from WT and Grasp55^−/− male mice 2 h after application of olive oil. c, d Fractionation analysis of plasma lipids from mice using fast performance liquid chromatography. Blood samples were taken 2 h after oral garage of olive oil. TG concentrations were determined in each fraction. The area under curve (AUC) of plasma lipoprotein fractions were measured using ImageJ software and summarized in (d, n = 3 independent experiments from pooled blood plasma samples of 3–4 mice). e Schematic diagram of the in vitro two-chamber system with differentiated Caco-2 cells. f Immunoblot results showed that the short-hairpin RNA against GRASP55 (shGRASP55) specifically depleted endogenous GRASP55 protein expression and GRASP55 with a COOH-terminal Myc-tag (GRASP55-Myc) induced exogenous GRASP55 protein. The filled arrowhead and the open arrowhead indicate levels of exogenous GRASP55 and endogenous GRASP55, respectively. Aldolase A was monitored as a cytosolic protein loading control. g Transepithelial lipid transport of Caco-2 monolayers were determined using ¹⁴C-labeled oleic acid. The majority of the exogenous oleic acids were incorporated into TGs and secreted as TGs in Caco-2 cells⁴⁷. Basolateral (B) to apical (A) transport of lipids (left, B to A) was much smaller than that of apical to basolateral transport (middle, A to B), and was not affected by GRASP55 depletion or overexpression. Specific transepithelial lipid absorption was determined by subtracting values of (B to A) from those of (A to B) in each paired experiment (right, n = 5). GRASP55 depletion reduced lipid absorption by 49.2 ± 14.5%. GRASP55 overexpression rescued the lipid absorption defect in GRASP55-depleted Caco-2 cells. CPM, counts per min. CM, chylomicron, VLDL, very-low-density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein. Unprocessed blots can be found in Supplementary Fig. 22. Data are shown as mean ± SEM. n.s.: not significant, *p < 0.05, **p < 0.01. P values were calculated by unpaired (a, c), paired (d) two-tailed Student’s t tests or ANOVA followed by Tukey’s multiple comparison tests (g). Source data are provided as a Source Data file.