Mfge8 regulates enterocyte lipid storage by promoting enterocyte triglyceride hydrolase activity

Abstract

The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the αvβ3 and αvβ5 integrins. Mice deficient in Mfge8 or the αvβ3 and αvβ5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2-dependent signaling pathway. These data identify a key role for Mfge8 and the αvβ3 and αvβ5 integrins in regulating enterocyte lipid processing.

Figure 1. Accumulation of intracellular triglyceride (TG) in Mfge8^–/– and αvβ3/αvβ5^–/– mice after a dietary fat challenge.

(A) TG content in primary enterocytes isolated from the proximal jejunum 2 hours after olive oil gavage. n = 5. (B and C) Serum TG concentrations over time after olive oil gavage in the presence (B) or absence (C) of i.p. administration of the lipoprotein lipase inhibitor Triton WR-1339. n = 5. (D and E) Baseline fecal TGs (D) and fecal TGs 2 hours after olive oil gavage (E). n = 5–6. (F and G) Radiolabel signal in proximal jejunal segments (F) or in the serum (G) 2 hours after gavage with ¹⁴C-triolein. n = 4–5. (H) Oil Red O staining of proximal jejunal segments 2 hours after olive oil gavage. (I) Jejunal sections obtained 30 minutes after olive oil gavage were stained with anti–perilipin 3 antibody. Female mice were used in all panels. *P < 0.05, **P < 0.01, ***P < 0.001. Paired data were analyzed by Student’s t test and group data were analyzed by 1-way ANOVA followed by a post-hoc Bonferroni test for multiple comparisons and expressed as the mean ± SEM.

Figure 2. Accumulation of intracellular triglyceride in Mfge8^–/– and αvβ3/αvβ5^–/– mice after a high-fat diet (HFD).

(A) Oil Red O staining of jejunal segments after a 12-hour fast of mice that had been on an HFD for 3 weeks. (B and C) Triglyceride content of the proximal jejunal segments after a 12-hour fast of mice that had been on an HFD for 3 weeks. n = 4–7. Female mice were used for all panels. *P < 0.05, ***P < 0.001. Data were analyzed by Student’s t test and expressed as the mean ± SEM.

Figure 3. Accumulation of cytoplasmic lipid droplets in Mfge8^–/– and αvβ3/αvβ5^–/– mice after a dietary fat challenge.

(A and B) ¹⁴C signal in the cytoplasmic versus microsomal compartments 2 hours after gavage with ¹⁴C–oleic acid expressed as absolute radioactive counts (A) or as ratio of cytosolic to microsomal radioactivity (B). n = 6. (C) Incorporation of radiolabel into triglyceride (TG), diacylglycerol (DAG), and fatty acids (FA) in primary enterocytes isolated from the proximal jejunum 2 hours after gavage with ¹⁴C-triolein. Lipids were resolved by TLC. The distribution of ¹⁴C in the different lipid species is expressed as a percentage of total ¹⁴C counts, with levels in WT cells set at 100%. n = 6. (D) Incorporation of ¹⁴C into TG, DAG, and FA in primary enterocytes isolated from the proximal jejunum incubated for 30 minutes with bile salt micelles containing ¹⁴C–oleic acid ([¹⁴C]OA). Lipids were isolated by TLC 1.5 hours after removal of bile salt micelles. n = 6. (E) Incorporation of radiolabel in TG secreted into the media of primary enterocytes after 30 minutes of incubation with bile salt micelles containing [¹⁴C]OA, followed by a 1.5-hour incubation with fresh media. n = 5. (F) Incorporation of ¹⁴C into TG in primary enterocytes incubated with [¹⁴C]OA-containing micelles followed by washing off micelles and a 2-hour incubation with fresh media. Lipids were resolved by TLC and the TG level is shown at time –30 (30 minutes before washing), –15, immediately before washing, and 0 (immediately after washing), 30, 60, and 120 minutes after wash. n = 3. (G) Total radioactivity in the media of primary enterocytes incubated with bile salt micelles containing [¹⁴C]OA 30, 15, and 0 minutes before washing and replacing with fresh media. (H) Incorporation of [¹⁴C]OA in TG secreted into the media of primary enterocytes after 30 minutes of incubation with bile salt micelles containing [¹⁴C]OA. Lipids were resolved by TLC and the TG level is shown at time 0, 30, 60, and 120 minutes after replacing media. Both female and male mice were used in A and B, and female mice were used for the remaining panels. *P < 0.05, **P < 0.01, ***P < 0.001. Paired data were analyzed by Student’s t test and group data were analyzed by 1-way ANOVA followed by a post-hoc Bonferroni test for multiple comparisons and expressed as the mean ± SEM.

Figure 4. Impaired triglyceride (TG) hydrolase activity in enterocytes from Mfge8^–/– and αvβ3/αvβ5^–/– mice.

(A) TG hydrolase activity in the proximal jejunal segments of mice measured 2 hours after olive oil gavage. n = 5–6. (B) Effect of rMfge8 (10 μg/ml), RGE (an rMfge8 mutant that does not bind integrin, 10 μg/ml), and integrin-blocking (αv, β3, β5, and β1; 5 μg/ml) antibodies on TG hydrolase activity in the Caco-2 cell line. n = 4–6. (C) Effect of rMfge8 (10 μg/ml) in the absence and presence of integrin-blocking (αv, β3, β5, and β1; 5 μg/ml) antibodies on TG hydrolase activity in the proximal jejunal enterocytes of mice. n = 5–7. (D) Effect of rMfge8 (10 μg/ml) on TG hydrolase activity in the primary enterocytes from αvβ3/αvβ5^–/– mice. n = 3. (E) TG hydrolase activity in Caco-2 cells treated with wortmannin (100 ng/ml) and rMfge8. n = 4–6. (F) Western blot of differentiated Caco-2 cells after incubation with siRNA targeting RICTOR or control siRNA. (G) TG hydrolase activity in differentiated Caco-2 cells treated with siRNA targeting RICTOR or control siRNA and rMfge8. n = 4–6. Female mice were used for panel A. Both female and male mice were used in panel C. **P < 0.01, ***P < 0.001. Paired data were analyzed by Student’s t test and group data were analyzed by 1-way ANOVA followed by a post-hoc Bonferroni test for multiple comparisons and expressed as the mean ± SEM. Ctrl., control; No Tx, no treatment.

Figure 5. Model to describe the mechanism by which Mfge8 regulates triglyceride (TG) hydrolysis.

Mfge8 binding to the αvβ3 and αvβ5 integrins on the surface of enterocytes activates a PI3 kinase/mTORC2 pathway that increases TG hydrolase activity. The increase in TG hydrolase activity leads to the release of free fatty acids from cytoplasmic lipid droplets which can then be utilized for chylomicron production. CLD, cytoplasmic lipid droplet; CM, chylomicron; FFA, free fatty acids.