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Mesenteric lymphatic dysfunction promotes insulin resistance and represents a potential treatment target in obesity

Abstract

Visceral adipose tissue (VAT) encases mesenteric lymphatic vessels and lymph nodes through which lymph is transported from the intestine and mesentery. Whether mesenteric lymphatics contribute to adipose tissue inflammation and metabolism and insulin resistance is unclear. Here we show that obesity is associated with profound and progressive dysfunction of the mesenteric lymphatic system in mice and humans. We find that lymph from mice and humans consuming a high-fat diet (HFD) stimulates lymphatic vessel growth, leading to the formation of highly branched mesenteric lymphatic vessels that ‘leak’ HFD-lymph into VAT and, thereby, promote insulin resistance. Mesenteric lymphatic dysfunction is regulated by cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF)-C–VEGF receptor (R)3 signalling. Lymph-targeted inhibition of COX-2 using a glyceride prodrug approach reverses mesenteric lymphatic dysfunction, visceral obesity and inflammation and restores glycaemic control in mice. Targeting obesity-associated mesenteric lymphatic dysfunction thus represents a potential therapeutic option to treat metabolic disease.

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Fig. 1: HFD-induced obesity is associated with progressive mesenteric lymphatic remodelling.
Fig. 2: Mesenteric lymphatic remodelling in HFD-induced obesity results in lymph ‘leakage’ to VAT.
Fig. 3: Mesenteric lymph leakage into VAT promotes adipocyte differentiation, lipid accumulation and insulin resistance.
Fig. 4: COX-2–PGE2 and VEGF-C–VEGFR3 signalling regulate HFD-associated mesenteric lymphatic dysfunction and glucose intolerance.
Fig. 5: Lymph-targeted COX-2 inhibition reverses HFD-induced mesenteric lymphatic branching and leakage by reducing local VEGF-C release.
Fig. 6: Lymph-targeted COX-2 inhibition reverses insulin resistance through restoration of mesenteric lymphatic function, not modification of systemic metabolic function.

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Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information). Data that support the findings of this study are available from the corresponding authors upon request. The exception is lipidomic data, which will be available in the NIH Metabolomics Workbench repository at https://doi.org/10.21228/M8GH59. Source data are provided with this paper.

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Acknowledgements

We thank the Monash Animal Research Platform; the Imaging and FACS core at the Monash Institute of Pharmaceutical Sciences, particularly S. Fung for technical support; the Melbourne Mouse Metabolic Phenotyping Platform, particularly V. Haynes for technical support; T. Karnezis, St. Vincent’s Institute, Melbourne, for providing human adipocytes and T. Tsiloulis, J.C.Y. Lo, D. Senyschyn and G. Chen for technical assistance. We sincerely thank surgeons G. Beban, N. Evenett, P. Johnston and A. Bartlett for their assistance with human mesenteric adipose tissue collection. This work was supported by project grants from the National Health and Medical Research Council of Australia (NHMRC grant 1100036 and NHMRC grant 1177084) and the Health Research Council of New Zealand (HRC 16-036) and the Hugo Charitable Trust (New Zealand).

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Authors and Affiliations

Authors

Contributions

E.C. conducted the majority of the experimental work, wrote the main paper and designed and interpreted experiments; N.L.T. was the main supervisor of E.C., conducted some adipocyte studies, wrote the main paper and designed and interpreted the work; C.J.H.P. and M.J.W. substantially revised the paper, co-supervised E.C. and contributed to experimental design and interpretation; M.J.W. also assisted with some metabolic analyses; C.J.N. optimized imaging methods and analysis and revised sections of the manuscript and methods related to imaging analysis; T.Q. synthesized and helped design the celecoxib prodrug and wrote methods for prodrug synthesis; J.S.S. facilitated the design and synthesis of the celecoxib prodrug and revised methods for prodrug synthesis; L.H. co-supervised E.C. and contributed to experimental design and interpretation; S.A., G.G., V.D.M.F., H.C. and A.L. conducted analyses such as ELISA, flow cytometry and metabolic analyses; G.S. conducted light-sheet microscopy analysis of human adipose tissue samples; J.A.W., A.R.J.P., J.H., A.B.J.E. and K.L.P. collected human samples, assisted with data interpretation and revised the manuscript; N.L.H. assisted with design and interpretation of lymphatic analyses and revised the manuscript. A.S., D.J.C. and D.A. conducted the metabolomic analysis, assisted with interpretation and presentation of metabolomic data and revised sections of the manuscript and methods related to the metabolomic analysis.

Corresponding authors

Correspondence to Enyuan Cao, Christopher J. H. Porter or Natalie L. Trevaskis.

Ethics declarations

Competing interests

C.J.H.P., N.L.T., T.Q., J.S.S., E.C. and M.J.W. are inventors of the lymph-directing glyceride prodrug technology described in the text (PCT/AU2020/050997). This technology has been patented and licensed via a commercial agreement with PureTech Health. PureTech Health has subsequently entered into a collaboration agreement with Boehringer Ingelheim to explore the technology in immune modulation. J.S.S. and T.Q. are currently employed by PureTech Health. The remaining authors declare no competing interests.

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Peer review information Nature Metabolism thanks Stephan Herzig, Joseph Rutkowski and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt

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Extended data

Extended Data Fig. 1 HFD-associated changes to intestinal villi and lacteals, and adipocytes and FALC in VAT.

a, Representative immunofluorescence images of intestinal villi. Cell nuclei (Hoechst, grey), CD31 + blood vessels (pink) and LYVE-1+ lacteals (green). Scale bars, 100 µm. b-e, Quantification of the width and length of the intestinal villi and lacteals from immunofluorescence images. f, Representative immunofluorescence images of VAT tissue stained for lipid droplets (green, Bodipy C16 FA). Scale bar, 50 µm. g, Quantification of adipocyte size (µm2). Mean ± s.e.m, n = 4, 5 mice in (b-c), n = 4, 4 mice in (d-e) with 3-4 intestinal villi analysed per mouse, n = 3, 3 mice in (g). Statistical differences, *p < 0.05 from two-tailed Mann-Whitney test. h, Immunofluorescent images of podoplanin + (green) lymphatic vessels in mesenteric adipose tissue from lean patient 2 and 3 and obese patient 2. Images from lean patient 1 and obese patient 2 are in Fig. 1c. (i-m) Representative immunofluorescence images of lymphatic vessels (LYVE-1 + , pink), blood vessels (CD31 + , cyan), CD11b + myeloid cells (grey), CD4 + T cells (yellow), B220 + B cells (blue), and YFP particles (green) in FALCs from mice fed CFD or HFD for 32 weeks. i–j, Type 1 FALCs were rich in CD31 + blood vessels, T cells and B cells but lacked CD11b + and LYVE + cells (that is LECs and macrophages) and had relatively low uptake of YFP-nanoparticles. k, Type 2 FALCs were rich in T cells, B cells and LYVE-1+ cells (macrophages or LEC) but had no observable lymphatic vessels and moderate uptake of YFP-nanoparticles. l-m, Type 3 FALCs contained, and were in close proximity to, LYVE-1+ lymphatic vessels and CD31 + blood vessels. The lymphatic vessels appeared disorganised and lacked a clear orientation toward a collecting lymphatic vessel. Type 3 FALCs also contained high numbers of T cells, B cells and myeloid cells. The observed overlap in staining between the YFP nanoparticles and CD11b + myeloid cells is most likely due to phagocytosis of the particles by the cells. Scale bars, 100 µm (i-m). (i-m) Representative of images from 2-3 FALCs for n = 5 mice. P values and details of the statistical testing are provided as source data.

Source data

Extended Data Fig. 2 An examination of mesenteric lymphatic dysfunction in 15-17 week old CFD fed db/db mice in comparison to C57BL/6 mice fed a CFD or HFD for 6, 15, 23 or 32 weeks from 6-7 weeks of age.

a, Total body weight. Mean ± s.e.m for n = 9, 9, 5. b, Representative immunofluorescence images of the mesenteric lymphatic vessels (LYVE-1 (green)) in VAT of db/db and C57BL/6 mice. Note white arrow indicates the direction of lymph flow, arrow heads indicate point of vessel branching. Scale bar, 500 μm. c, Quantification of mesenteric lymphatic vessel branching in VAT of mice. Mean ± s.e.m for n = 4 or n = 5 mice for week 6, n = 6 or n = 7 mice for week 15, n = 4 or n = 5 mice for week 23, n = 4 or n = 5 mice for week 32 and n = 5 for db/db mice. d, Representative images of Evans blue dye passage through mesenteric lymphatic vessels and leakage to VAT 10 min after intramucosal dye injection to CFD and HFD fed C57BL/6 mice and db/db mice. Scale bar, 5 mm. White circle highlights a site of lymph leakage. e, Area under the curve (AUC) of the Evan’s blue dye intensity plots. Mean ± s.e.m., n = 7, 7 and 5 mice. f, Total cells in mesenteric lymph of mice fed CFD or HFD for 6, 15, 23, 32 weeks or db/db mice fed CFD. Mean ± s.e.m for n = 5 or n = 4 mice for week 6, n = 8 or n = 9 mice for week 15, n = 6 or n = 6 mice for week 32 and n = 3 for db/db mice. g, Weight of subcutaneous adipose tissue (SAT, inguinal adipose tissue depot) and mesenteric adipose tissue (MAT). Note comparisons across CFD, HFD and db/db mice in (b, d, e, g) are made between C57BL/6 mice fed CFD or HFD for 23 weeks and db/db mice age 15-17 weeks. Statistical differences, *p < 0.05, **p < 0.01, ***p < 0.005, or ****p < 0.0001 from (a, e) one-way ANOVA, (c) two-tailed Mann-Whitney test for week 6, 23 and 32 data, two-tailed Student’s t test was used for week 15 data, one-way ANOVA was used for comparison between db/db mice and week 23 and week 32 data, (f) a two-tailed Student’s t-test (except one-way ANOVA was used for comparison between db/db mice vs week 6 data), (g) two-way ANOVA. P values and details of the statistical testing are provided as source data.

Source data

Extended Data Fig. 3 Body weight, and composition and effects of mesenteric lymph from Sprague-Dawley rats fed CFD or HFD for 6-9 weeks.

a, Body weight and b, TG concentration. Mean ± s.e.m for n = 4, 4 (HFD) rats. c, Heatmap analysis of lipidomics data of lymph from HFD mice and rats, expressed as mean peak intensity relative to CFD mice and rats, respectively. Blue indicates decreased abundance, red indicates increase and yellow signifies no change. n = 4, 4 mice/rats. d-e, PGE2 and VEGFc concentration in mesenteric lymph of rats. Mean ± s.e.m, n = 4, 4 rats. Statistical differences, *p < 0.05 from two-tailed Mann-Whitney test. f, Real time PCR analysis of Fabp4, Lepr, Gapdh, Pparg, Pnpla2, and Il6 mRNA expression in 3T3-L1 adipocytes treated with control media (Ctrl) or 0.5 or 2% v/v CFD-lymph in media. Mean ± s.e.m. for n = 3 from N = 1 experiment. g, Lipolysis in mature 3T3-L1 adipocytes (from glycerol release) in basal and forskolin-stimulated conditions after treatment with control media (Ctrl) or 2% lymph chylomicron (CM), VLDL or lipid-free protein fraction (supernatant). Mean ± s.e.m. for n = 3 except Ctrl (n = 9 (basal) and n = 6 (forskolin)) from N = 1 experiment. Statistical differences, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 from Two-Way ANOVA. h, 14C-2-deoxyglucose (2DG) uptake into SPF3 human adipocytes in basal and insulin-stimulated conditions after treatment with control media or 2% HFD rat lymph. Mean ± s.e.m. for n = 8, 8 (basal) and n = 6, 7 (insulin) replicates from N = 3 experiments. Statistical differences, *p < 0.05 from two-way ANOVA. P values and details of the statistical testing are provided as source data.

Source data

Extended Data Fig. 4 The impact of EP4 inhibition on HFD-lymph induced LEC migration, adipocyte lipolysis and adipocyte insulin sensitivity.

a, LEC migration over time on incubation with control media or 2% v/v HFD-lymph from mice ± EP4 antagonist, L-161,982. Mean ± s.e.m for n = 9, 7, 10 from N = 2 experiments. b, Lipolysis in mature 3T3-L1 adipocytes (from glycerol release) in basal and forskolin-stimulated conditions on incubation with control media or 2% v/v HFD-lymph from mice ± EP4 antagonist. Mean ± s.e.m for n = 8, 8, 8 (basal) and 6, 9, 8 (forskolin) replicates from N = 2 experiments. c, 14C-2-deoxyglucose (2DG) uptake into mature 3T3-L1 adipocytes in basal and insulin-stimulated conditions on incubation with control media or 2% v/v HFD-lymph from mice ± EP4 antagonist. Mean ± s.e.m. for n = 4 (basal) and n = 4 (insulin) replicates of N = 2 experiments. Statistical differences (a) *p < 0.05 from one-way ANOVA and (b-c) *p < 0.05, **p < 0.01 from two-way ANOVA. d-e, PGE2 and VEGFc concentration in plasma of mice fed CFD or HFD for 22-23 weeks. Mean ± s.e.m., n = 6, 6 (plasma VEGFc) and n = 5, 5 (plasma PGE2) mice. Statistical differences, ****p < 0.001 from two-tailed Student’s t-test. P values and details of the statistical testing are provided as source data.

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Extended Data Fig. 5 Immune cell accumulation in mesenteric lymph fluid and lymph nodes across prevention and treatment groups.

a-b, Total immune cells in mesenteric lymph fluid and lymph nodes. Mean ± s.e.m., (a) n = 5, 4 (week 6), n = 8, 9 (week 15), n = 6, 6 (week 32). (b) n = 5, 5 (week 6), n = 5, 5 (week 15), n = 6, 6 (week 32). c, Percent of CD45 + cells that were T cells (CD3 + ), B cells (CD19 + ), dendritic cells (CD11c + ), macrophages (F4/80 + ) and NK cells (NK1.1), and d, Percent of T cells that were Th1 (IFNγ + ), Th2 (IL-4 + ) and Th17 (IL-17 + ) cells in mesenteric lymph nodes of mice fed CFD or HFD for 6, 15 or 32 weeks. (c-d) all data at week 15 with n = 5 (CFD), 7 (HFD, except n = 5 for CD11c + and NK1.1+ cells). e, f, Total immune cells in mesenteric lymph fluid or node of mice fed with CFD, HFD or HFD plus Celecoxib (Cele) for 15 weeks (prevention study, see timeline Fig. 4e). Mean ± s.e.m., (e) n = 5, 5, 4. (f) n = 9, 10, 7. g-h, Percent of CD45 + cells that were T cells (CD3 + ), B cells (CD19 + ), dendritic cells (CD11c + ), macrophages (F4/80 + ) and NK cells (NK1.1) in mesenteric lymph fluid or nodes of mice fed with CFD, HFD or HFD plus celecoxib or Cele-Pro (treatment study, see timeline Fig. 5a). Mean ± s.e.m., (g) n = 6, 7, 4, 5 and (h) n = 5, 7, 6, 5 (except n = 4 for CFD NK1.1+ cells, n = 6 for HFD CD3 + cells and n = 6 HFD NK1.1+ cells). Statistical differences from two-tailed Student’s t-test (a-d) or one-way ANOVA (e-h), *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. P values and details of the statistical testing are provided as source data.

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Extended Data Fig. 6 Systemic exposure of celecoxib in mice administered celecoxib (Cele) or celecoxib prodrug (Cele-Pro).

a, Plasma concentrations of celecoxib over time in individual mice, and b, Area under the curve (AUC) of celecoxib plasma concentration versus time profile from time 0-5 h following oral gavage of 29 mg/kg Cele or 29 mg/kg Cele-Pro to fasted mice in a lipid based formulation. Mean of n = 2.

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Extended Data Fig. 7 Lipid metabolites in mesenteric lymph fluid obtained from mice fed a CFD, HFD, HFD plus celecoxib or celecoxib prodrug (Cele-Pro).

a-b, PGI2 and 6 keto-PGF1a concentrations in mesenteric lymph from CFD, HFD, HFD + Cele and HFD + Cele-Pro fed mice. Mean ± s.e.m for n = 5, 6, 6, 7. c, Free fatty acid (FFA), (d) cholesterol (Ch), (e) phospholipid (PL) and (f) glucose concentrations in mesenteric lymph. Mean ± s.e.m., n = 4, 8, 6, 7 or 4. g, Lipidomic analysis heatmap of mesenteric lymph from mice in the prevention study (Fig. 4e,g timeline) or treatment study (Fig. 5a timeline). h, Fold change in concentration of significant lipid metabolites in lipidomic analysis including sphingolipids, fatty amides and acyls, glycolipids, sterols, glycerophosphoglycerols, glycerophosphocholines, glycosylphosphatidylinositol, glycerophosphoethanolamines, glycerophosphoinositols in mesenteric lymph from mice in the treatment study (Fig. 5a timeline). h, Mean ± s.e.m. of peak intensities from treatment samples multiplied by the fold change values compared to control. n = 4 (CFD), 7 (HFD), 5 (Cele), 3 (Cele-Pro) mice. P values and details of the statistical testing are provided as source data.

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Extended Data Fig. 8 Dose-dependent changes to blood glucose and plasma insulin in mice treated with celecoxib or celecoxib prodrug (Cele-Pro).

Blood glucose (a) and plasma insulin (b) area under the concentration-time curve (AUC) after an oral glucose challenge in mice that were fed with CFD, HFD, HFD + celecoxib (~3, 10, 30 mg/kg) or HFD + Cele-Pro (~3, 10, 30 mg/kg, celecoxib equivalent). Study outline is as described in Fig. 5a. Mean ± s.e.m for CFD (12), HFD (8), HFD + celecoxib (~3 (n = 6), 10 (n = 8), 30 (n = 7) mg/kg) or HFD + Cele-Pro (~3 (n = 7), 10 (n = 6), 30 (n = 8) mg/kg) in (a). CFD (10), HFD (11), HFD + celecoxib (~3 (n = 7), 10 (n = 8), 30 (n = 6) mg/kg) or HFD + Cele-Pro (~3 (n = 7), 10 (n = 6), 30 (n = 8) mg/kg) in (b). Statistical differences, **p < 0.01, ***p < 0.005, or ****p < 0.0001 from one-way ANOVA compared to CFD. P values and details of the statistical testing are provided as source data.

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Supplementary information

Supplementary Information

Supplementary Tables 1–5 and Figs. 1–7

Reporting Summary

Supplementary Video 1

Mesenteric lymphatic vessels in VAT of human patient 1 who was lean.

Supplementary Video 2

Mesenteric lymphatic vessels in VAT of human patient 1 with obesity.

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Cao, E., Watt, M.J., Nowell, C.J. et al. Mesenteric lymphatic dysfunction promotes insulin resistance and represents a potential treatment target in obesity. Nat Metab 3, 1175–1188 (2021). https://doi.org/10.1038/s42255-021-00457-w

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