Fig 2: Inhibition of intestinal lipid absorption by FACI. (A–D) Transcriptomic profiles of intestinal epithelium from jejunum between WT and Faci-/- mice (male, n = 3) by RNA-seq analysis. (A) Heatmap depicting the correlation of 6 RNA-seq samples, that is, intestinal epithelium from 3 WT (WT1, WT2, and WT3) and 3 Faci-/- mice (KO1, KO2, and KO3) by Pearson correlation coefficient analyses. (B) The volcano plot illustrates DEGs. DEGs were selected with the criteria of FDR (false discovery rate) less than 0.01 and log2 (fold change) (FC) of 2 or greater. Up-regulated and down-regulated DEGs are shown in red and green, respectively. (C) The bubble plot depicts gene ontology of up-regulated DEGs. The Y-axis represents GO terms. The X-axis indicates the gene ratio. Bubble colors represent log10 (FDR) and bubble sizes indicate gene counts. (D) Heatmap illustrating the fold changes of lipid absorption–related DEGs in WT (WT1, WT2, and WT3) and Faci-/- mice (KO1, KO2, and KO3). Scaled FPKM values were used for heatmap generation (Supplementary Table 2). Up-regulation and down-regulation are highlighted in yellow and blue, respectively. (E) RT-qPCR analysis. Total mRNA of intestinal epithelium from jejunum of WT and Faci-/- mice (male, n = 4) was extracted. The mRNA levels of the indicated genes were analyzed by RT-qPCR. Results were statistically assessed by unpaired 2-tailed Student t test. (F) Immunoblotting. Total proteins of intestinal epithelium from jejunum of WT and Faci-/- mice (male, n = 3) were extracted. Expression of the indicated proteins was analyzed. β-actin was detected as the internal control. (G) Small intestine length of male WT and Faci-/- mice at 3 months (n = 18–20 mice per group). (H) Duodenum villus length of male WT and Faci-/- mice at 3 months (n = 4 per group). (I) Plasma TG measurement. Male mice (n = 4 per group) were HFD-fed for 5 days, fasted (6 hours), and injected intraperitoneally with 30% wt/wt lipoprotein lipase inhibitor poloxamer 407 (1.5 g/kg body weight). After 30 minutes, mice were orally gavaged with olive oil (10 mL/g body weight). Plasma TGs at the indicated time points were measured. Statistical analysis was based on 2-way ANOVA with repeated measures, followed by the Sidak test. (J) Very-low-density lipoprotein secretion assay. Female mice (n = 3 per group) were fed with HFD for 5 days, fasted overnight, and injected intraperitoneally with Poloxamer 407 (1.5 g/kg). Plasma TG was measured at the indicated time points. Data are shown as means ± SD. (K) Measurement of TG in small intestines. Male mice (n = 3 per group) were HFD-fed for 5 days, fasted overnight, followed by high-fat refeeding. Small intestines of mice were removed, washed, and homogenized. Lipids were extracted following the Bligh and Dyer method.69 Statistical analysis was performed with an unpaired 2-tailed Student t test. (L) Oil red O staining of proximal jejunum. Male mice were HFD-fed for 5 days, fasted overnight, followed by high-fat refeeding. Intestinal neutral lipids were visualized with Oil red O staining. (M and N) Fatty acid uptake assay. BODIPY-C12 fatty acid uptake was measured using the QBT fatty acid uptake kit (Molecular Devices). (M) Intracellular fluorescence signals were detected every 20 seconds for up to 80 minutes. Fatty acid uptake was compared between FACI-expressing (Dox group, n = 6 wells) and mock-treated (no Dox group, n = 3 wells) Caco2 cells. (N) Areas under the curve (AUC) for kinetic FA uptake were calculated. An unpaired 2-tailed Student t test was performed to assess statistical significance. ∗P < .05, ∗∗P < .01. adj, adjusted; BP, biological process; CC, cellular component; MF, molecular function; RFU, Relative fluorescence unit.