MFGE8 links absorption of dietary fatty acids with catabolism of enterocyte lipid stores through HNF4γ-dependent transcription of CES enzymes

**Background:** Enterocytes modulate postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs) and mobilizing them for chylomicron production. The integrin ligand MFGE8 links dietary fat absorption with activation of triglyceride (TG) hydrolases that catabolize cLDs, but the downstream molecular pathway was unknown. While ATGL/CGI-58 regulates cLDs derived from the basolateral circulation, the hydrolase(s) regulating diet-derived cLDs had not been identified.

**Methods:** The authors used 3′ tag RNA-seq of proximal small intestinal enterocytes from WT and Mfge8 KO mice to identify differentially expressed genes. Activity-based protein profiling (ABPP) with a serine hydrolase-specific fluorophosphonate biotin probe was performed to assess hydrolase activity. TG hydrolase activity was measured using radiolabeled triolein substrate. Protein levels were assessed by western blot. Acute fat challenges used olive oil gavage with or without ³H-oleic acid, with tyloxapol to inhibit lipoprotein lipase. Chronic high-fat diet feeding was performed for 3 weeks. ChIP-seq data were analyzed for HNF4γ binding sites. Luciferase reporter assays and cycloheximide pulse-chase experiments assessed transcriptional regulation and protein stability. Multiple KO mouse strains were used: Mfge8 KO, Ces1d global KO, Ces1d intestine-specific KO (villin-Cre), Hnf4γ KO, Cd36 KO, and double KOs.

**Key Results:** RNA-seq of Mfge8 KO enterocytes identified 530 differentially regulated genes with enrichment for triacylglycerol degradation pathways. Multiple CES1 family hydrolases (Ces1d, Ces1e, Ces1f) were downregulated. ABPP confirmed decreased activity of CES1 enzymes in Mfge8 KO enterocytes. HNF4γ protein (but not transcript) was markedly reduced in Mfge8 KO enterocytes. Hnf4γ KO mice showed reduced CES1D protein, reduced enterocyte TG hydrolase activity at baseline and 2 h after olive oil gavage (p<0.05), increased proximal small intestinal tissue TG content, and lower serum TG levels. After ³H-oleic acid gavage, Hnf4γ KO mice had greater intestinal radioisotope accumulation and reduced serum radiolabel. After 3 weeks of HFD, Hnf4γ KO mice had greater intestinal TG and lower serum TG vs. WT. ChIP-seq data showed HNF4γ binding sites in Ces1d enhancer regions. Forced expression of HNF4γ in Hnf4γ KO enterocytes rescued CES1D protein. Ces1d global KO and intestine-specific KO mice both showed reduced enterocyte TG hydrolase activity, increased intestinal TG content, and reduced serum TG after olive oil gavage. ³H-oleic acid accumulated in the cytosolic (cLD) fraction of Ces1d int-KO enterocytes with no difference in the microsomal fraction. rMFGE8 increased TG hydrolase activity in WT and Mfge8 KO enterocytes but had no effect on Ces1d KO enterocytes. Ces1d/Mfge8 double KO mice showed no additive effect vs. single KOs. Cd36 KO enterocytes showed reduced HNF4γ and CES1D protein, reduced TG hydrolase activity, and rMFGE8 failed to increase activity in Cd36 KO cells. In HEK293 cells, oleic acid further induced HNF4γ-dependent Ces1d luciferase activity and prevented HNF4γ protein decay in cycloheximide pulse-chase experiments. CES1D protein increased 2 h after olive oil gavage in WT but not Hnf4γ KO mice. Fasting for 4 h reduced HNF4γ and CES1D protein. β5 blocking antibody reduced enterocyte TG hydrolase activity, increased intestinal ³H retention, and decreased serum ³H after gavage.

**Clinical Implications:** This study identifies a complete regulatory network—MFGE8 → CD36-mediated fatty acid uptake → HNF4γ protein stabilization → CES1D transcription → cLD TG hydrolysis—that controls postprandial lipemia. The pathway is specific to diet-derived cLDs, as MFGE8 did not affect basolateral-derived cLDs. The authors note that MFGE8/αvβ5 blockade could be therapeutically targeted to reduce postprandial lipemia in obese, insulin-resistant patients while also reducing fat absorption and enhancing peripheral insulin sensitivity, though risks remain to be determined. Limitations include potential post-translational regulation of other CES enzymes, possible effects of HNF4γ on lipid droplet synthesis or chylomicron secretion not addressed, and the unknown mechanism of fatty-acid-dependent HNF4γ protein stabilization.