Lacteal junction zippering protects against diet-induced obesity

Abstract

Excess dietary lipid uptake causes obesity, a major global health problem. Enterocyte-absorbed lipids are packaged into chylomicrons, which enter the bloodstream through intestinal lymphatic vessels called lacteals. Here, we show that preventing lacteal chylomicron uptake by inducible endothelial genetic deletion of Neuropilin1 (Nrp1) and Vascular endothelial growth factor receptor 1 (Vegfr1; also known as Flt1) renders mice resistant to diet-induced obesity. Absence of NRP1 and FLT1 receptors increased VEGF-A bioavailability and signaling through VEGFR2, inducing lacteal junction zippering and chylomicron malabsorption. Restoring permeable lacteal junctions by VEGFR2 and vascular endothelial (VE)-cadherin signaling inhibition rescued chylomicron transport in the mutant mice. Zippering of lacteal junctions by disassembly of cytoskeletal VE-cadherin anchors prevented chylomicron uptake in wild-type mice. These data suggest that lacteal junctions may be targets for preventing dietary fat uptake.

Fig.1.. Nrp1;Flt1iECko mice are resistant to diet-induced obesity.

(A) Nrp1;Flt1iECko and Cre-negative littermate control (Ctrl) mice after 16wk HFD. (B-C) Growth curves (B) and weight gain (C) of Ctrl (n=8) and Nrp1;Flt1iECko (n=11) mice after 16wk HFD feeding. Data are mean±SEM. ***, p-0.001. Mann-Whitney U test. (D-F) Weight gain of normal chow (NC)- or HFD-fed mice (n=4-11/group). Data are mean±SEM. ***, p<0.001. Mann-Whitney U test. (G) Body composition of mice (n=7-8/group) after 8wk NC or HFD. Data are mean±SEM. * p<0.05, ***, p<0.001. Mann-Whitney U test. (H) Quantification of Bodipy staining intensity of liver cryosections (left) and liver triglyceride content (right) from 8wk HFD-fed mice (n=4-5/group). Data are mean±SEM. * p<0.05, ***, p<0.001. Mann-Whitney U test. (I) Calorie intake, energy expenditure and activity of Ctrl (n=7) and Nrp1;Flt1iECko (n=5) mice after 2wk on HFD. Data represent 24h, light cycle (7:00am-7:00pm) and dark cycle (7:00pm-7:00am) average, normalized to lean body mass (LBM). Error bars: SEM. (J) Intraperitoneal glucose tolerance test in HFD-fed Ctrl (n=10) and Nrp1;Flt1iECko (n=6) mice. Data are mean±SEM. *, p<0.05; **, p<0.01. Mann-Whitney U test.

Fig.2.. Endothelial Nrp1;Flt1 deletion prevents lacteal chylomicron absorption.

(A) Plasma lipid profile in 6h fasted Ctrl (n=6) and Nrp1;Flt1iECko mice (n=6) after 16wk HFD. Data are mean±SEM. **, p<0.01. Mann-Whitney U test. (B-C) Plasma triglyceride content in NC-fed adult mice following gavage with 200μ1 olive oil. All mice (n=8-10/group) received 6h fasting and mice in (C) received Triton WR1339 (0.5g/kg) i.p. 30min prior to gavage. Data are mean±SEM. **, p<0.01, ***, p<0.001. Mann-Whitney U test. (D) Plasma ³H CPM (counts per minute) in NC-fed adult mice after gavage with ³H-triolein containing lipid. Mice (n=5/group) were fasted for 6h and injected i.p. with poloxamer 407 (1g/kg) 30min prior to gavage. Data are mean±SEM. *, p<0.05; **, p<0.01, ***, p<0.001. Mann-Whitney U test. (E) Fecal bomb calorimetry analysis of 2wk HFD-fed mice (n=8/group). Data are mean±SEM. ***, p<0.001. Mann-Whitney U test. (F) Quantifications of chyle-filled lymphatics in mesenteries of P7 mice after 3X100μg TAM injection at P2-P4. Each dot represents one mouse (n=17-24/group). Error bars: SEM. ***, p<0.001. Mann-Whitney U test. (G) Transmission electron microscopy of jejunum central lacteals in P13 mice. LL: lacteal lumen; LEC: lymphatic endothelial cell; CM: chylomicron. JNC: junction. Note closed LEC junction and empty lacteal lumen in Nrp1;Flt1iECko mice.

Fig.3.. Increased VEGF-A signaling alters villus endothelial junctions.

(A) VE-Cad and LYVE-1 staining of whole-mounted jeunum lacteals from P13-P18 mice following postnatal TAM administration. (B) Quantification of zipper-like lacteal junctions in A. Each dot represents one lacteal (7 mice/group). Data are mean±SEM. ***, p<0.001. Mann-Whitney U test. (C) VE-Cad staining of whole-mounted jejunum BEC junctions from P7 mice. (D) Quantification of dextran leakage in P11 mice after Rhodamine-dextran and Alexa 647-IsoB4 i.v. injection for 10min. Each dot represents one villus (5-6 mice/group). Data are mean±SEM. ***, p<0.001. Mann-Whitney U test. (E) Western blots and quantifications of VEGFR2 phosphorylation in P7 jejunum lysates. n=5-6 mice/group. Data are mean±SEM. **, p<0.01. Mann-Whitney U test. (F-G) VE-Cad staining of villus BEC junctions and quantification of zipper-like lacteal junctions in jejunum from P18-P21 wildtype mice 30min after injection of growth factors or PBS. Each dot represents one lacteal (4-6 mice/group). Data are mean±SEM. n.s., not significant; **, p<0.01, ***, p<0.001. Mann-Whitney U test.

Fig.4.. Lacteal zipper junctions prevent chylomicron absorption.

(A-D) DC101 rescues fat absorption in Nrp1;Flt1iECko mice. (A) Experimental timeline of TAM injection and DC101 treatment (30μg/g, i.p., for 6h) and mesenteries of P7 Nrp1;Flt1iECko mice with or without DC101. (B) Quantification of chyle-filled lymphatics in A. Each dot represents one mouse (n=11-24/group). Data are mean±SEM. n.s., not significant; ***, p<0.001. Mann-Whitney U test. (C) Quantification of zipper-like lacteal junctions in P13-P18 Nrp1;Flt1iECko jejunum with or without DC101. Each dot represents one lacteal (4-7 mice/group). Data are mean±SEM. n.s., not significant; ***, p<0.001. Mann-Whitney U test. (D) Plasma triglyceride content in NC-fed adult mice after 6h fasting and lipid gavage. All mice (n=5-8/group) received TritonWR1339 (0.5g/kg, i.p., 30 min prior to gavage) and some received DC101 (30μg/g, i.p. 4h prior to gavage). Data are mean±SEM. *, p<0.05, **, p<0.01. Mann-Whitney U test. (E-F) BV13 rescues chylomicron uptake in Nrp1;Flt1iECko mice. (E) Quantification of chyle-filled lymphatics in P7 mice with or without BV13 treatment (10μg/g, i.p., for 4h). Each dot represents one mouse (n=5/group). Data show mean±SEM. n.s., not significant; **, p<0.01. Mann-Whitney U test. (F) VE-Cad and LYVE-1 staining of jejunum lacteals from P13 Nrp1;Flt1iECko mice with or without BV13. (G-I) ROCK inhibition prevents chylomicron uptake in wildtype mice. (G) Quantification of chyle-filled lymphatics in mesenteries of P10 mice treated with Y27632 (20μg/g, i.p., for 4h) or PBS. Each dot represents one mouse (n=5/group). Data are mean±SEM. *, p<0.05. Mann-Whitney U test. (H) Quantification of zipper-like lacteal junctions in P13-15 mice treated with Y27632 or PBS. Each dot represents one lacteal (4 mice/group). Data are mean±SEM. ***, p<0.001. Mann-Whitney U test. (I) TEM analysis of lacteals in P13 mice with or without Y27632 treatment. LL: lacteal lumen; LEC: lymphatic endothelial cell; CM: chylomicron. JNC: junction. (J) Model of NRP1;FLT1 effects on BECs and LECs in intestinal villi. VEGF-A binding to NRP1;FLT1 on BECs limits VEGF-A bioavailability for VEGFR2, resulting in continuous and discontinuous cell junctions in BECs and LECs, respectively. Discontinuous button-like LEC junctions allow lacteal chylomicron uptake. Note that NRP1;FLT1 are only highly expressed on BECs. Increased VEGF-A concentrations or Nrp1;Flt1 deletion in BECs result in excessive VEGFR2 activation, which disrupts BEC junctions while zippering up lacteal LEC junctions, thereby preventing chylomicron uptake. This phenotype can be rescued by inhibition of VEGFR2 signaling with DC101.