Research ArticleMetabolic Disease

Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin’s glucose-lowering effects

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Science Translational Medicine  13 Sep 2017:
Vol. 9, Issue 407, eaad4000
DOI: 10.1126/scitranslmed.aad4000
  • Fig. 1. APLNR expression is restricted to the endothelium of adult tissues.

    (A) Representative in situ hybridization of adult human skeletal muscle with adjacent sections stained for von Willebrand factor (vWF) (white) and with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 50 μm. (B) Representative green fluorescent protein (GFP) expression in the tissues of adult AplnrCreER:RosamTmG mice. DAPI nuclear stain (blue), GFP expression (green), VE-cadherin endothelial stain (red), and merged images are shown for skeletal muscle (Sk. musc.), WAT, and BAT. Scale bars, 100 μm. (C) Relative mRNA expression of Aplnr in tissue homogenates versus isolated ECs from various mouse adult tissues (n = 3 mice per group). **P < 0.01.

  • Fig. 2. Endothelial APLNR is critical for apelin signaling.

    (A) Representative immunoblot of AKT and AMPKα phosphorylation in response to apelin stimulation in skeletal muscle with or without EC depletion (n = 3 replicates per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.s., not significant. (B) Representative immunoblot of AKT, AMPKα, and eNOS phosphorylation in response to apelin stimulation in skeletal muscle of AplnrECKO mice and control littermates (n = 3 replicates per group). (C) IPGTT of AplnrECKO mice and control littermates [n = 10 (control) and 14 (AplnrECKO)]. (D) IPGTT in AplnrECKO and control mice 30 min after intravenous apelin injection [n = 5 (control) and 7 (AplnrECKO)]. (E) Intraperitoneal insulin tolerance testing of AplnrECKO mice and their control littermates under basal conditions (n = 6 per group). (F) Representative apelin expression in skeletal muscle, WAT, and BAT, as assessed by lacZ staining of Apln+/lacz reporter mice fed a normal chow or a high-fat diet (HFD). Scale bar, 100 μm. (G) Relative mRNA expression of apelin in various tissues of mice fed a normal chow or a high-fat diet (n = 5 per group). *P < 0.05 and **P < 0.01.

  • Fig. 3. Apelin induces endothelial FOXO1 inactivation and inhibits FABP4 expression.

    (A) FOXO1 phosphorylation in response to apelin stimulation (1 hour at the indicated concentrations) of HUVECs. (B) Time course of FOXO1 phosphorylation in HUVECs treated with apelin. (C) Subcellular localization of FOXO1 in response to apelin stimulation. Scale bar, 5 μm. (D) Apelin-induced FOXO1 phosphorylation in conjunction with inhibition of phosphoinositide 3-kinase by pretreatment with either Wortmannin or LY-294002. (E) Representative immunoblot of FABP4 in HUVECs in response to apelin knockdown (APLN si), FOXO1 knockdown (FOXO1 si), or both. (F) Representative immunoblot for FABP4 expression in isolated skeletal muscle ECs from the indicated mouse strains. *P < 0.05, **P < 0.01, and ††P < 0.01.

  • Fig. 4. Apelin-FABP4 signaling regulates endothelial FA uptake and transfer.

    (A) Representative image of BODIPY uptake in HCMECs subjected to apelin pretreatment. Scale bar, 5 μm. a.u., arbitrary units. (B) Representative image of BODIPY uptake in HCMECs subjected to APLNR knockdown (APLNR si). Scale bar, 5 μm. (C and D) Assessment of the FA transfer across a confluent HCMEC layer, as measured by determination of BODIPY in the bottom well of a Transwell plate, in response to apelin stimulation (C) or APLNR knockdown (D). (E) Fluorescent BODIPY transfer in response to APLN and APLNR knockdown in HCMECs and in conjunction with treatment with the FABP4 inhibitor BMS309403. *P < 0.05 and **P < 0.01 (for 1 μM); P < 0.05 and ††P < 0.01 (for 10 μM). (F) Tissue uptake of 14C-OA in the liver, WAT, BAT, heart, and skeletal muscle of Apln−/− mice at 2 and 24 hours after oral gavage (n = 5 for each time point). FM, femoral muscle; DM, dorsi muscle. (G) Representative images and quantification of oil red O staining of skeletal muscle from Apln−/− mice and wild-type littermates (n = 5 per group). Scale bar, 20 μm.

  • Fig. 5. Apelin-FOXO1 signaling regulates tissue FA uptake in vivo.

    (A) IPGTT in Apln−/− and Apln−/−:Foxo1ECKO double-knockout mice (n = 5 per group). (B) Representative images and quantification of oil red O staining of skeletal muscle from Apln−/− and Apln−/−:Foxo1ECKO double-knockout mice (n = 5 per group). Scale bar, 20 μm. (C) Assessment of 14C-OA uptake in Apln−/− and Apln−/−:Foxo1ECKO double-knockout mice at 24 hours after oral gavage (n = 5 per group). (D) Assessment of 14C-OA uptake in Apln−/− mice and wild-type (WT) littermates treated with the FABP4 inhibitor BMS309403 (n = 5 per group for each time point). DMSO, dimethyl sulfoxide. Data are means ± SEM. *P < 0.05, **P < 0.01, P < 0.05, and ††P < 0.01.

  • Fig. 6. Impaired glucose uptake of AplnrECKO mice can be rescued by FABP4 inhibition.

    (A) Tissue uptake of 14C-OA in the liver, WAT, BAT, heart, and skeletal muscle of AplnrECKO mice at 24 hours after oral gavage (n = 5 per group). (B) Representative images and quantification of oil red O staining of skeletal muscle from AplnrECKO mice and control littermates (n = 5 per group). Scale bar, 20 μm. (C) IPGTT in AplnrECKO mice after 7 days of treatment with BMS309403 by oral gavage (n = 7 per group). (D) Representative images and quantification of oil red O staining of skeletal muscle from AplnrECKO mice treated with BMS309403 (n = 5 per group). Scale bar, 20 μm. (E) Proposed mechanism of endothelial apelin/APLNR metabolic signaling. *P < 0.05, **P < 0.01, and ***P < 0.001

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/407/eaad4000/DC1

    Fig. S1. APLNR expression profiling.

    Fig. S2. Fat and muscle content of AplnrECKO mice.

    Fig. S3. Plasma insulin concentrations in Apln−/−, Aplnr−/−, and AplnrECKO mice.

    Fig. S4. Adiponectin concentration in AplnrECKO mice.

    Fig. S5. Hindlimb blood flow in AplnrECKO mice.

    Fig. S6. Effect of apelin on transendothelial glucose transfer.

    Fig. S7. Metabolic effects of high-fat diet.

    Fig. S8. Effect of high glucose on APLN.

    Fig. S9. Gene expression profiling of ECs subjected to APLN and APLNR knockdown or FOXO1 knockdown.

    Fig. S10. FABP4 expression in response to APLN or FOXO1 knockdown.

    Fig. S11. FABP4 expression in response to APLN and PPARG knockdown.

    Fig. S12. FABP4 immunostaining in Apln−/− and Apln−/−:Foxo1ECKO mice.

    Fig. S13. Effect of continuous apelin infusion on glucose tolerance testing.

    Fig. S14. FABP4 expression in mice receiving apelin infusion.

    Fig. S15. FA uptake in HUVECs subjected to modulation of apelin/APLNR and FABP4 activity.

    Fig. S16. FA uptake profile of Foxo1ECKO mice.

    Fig. S17. Serum FA concentrations in Apln−/− and Foxo1ECKO mice.

    Fig. S18. Circulating serum FA concentrations in AplnrECKO mice.

    Fig. S19. FA and triglyceride content of skeletal muscle of AplnrECKO mice.

    Fig. S20. Endothelial permeability in the lung and skeletal muscle of AplnrECKO mice.

    Fig. S21. Endothelial permeability in response to APLN and APLNR knockdown.

    Fig. S22. Effect of pharmacologic FABP4 inhibition on glucose tolerance test in wild-type mice on normal chow.

    Fig. S23. Efficacy of Aplnr deletion.

    Fig. S24. Efficacy of Foxo1 gene deletion.

    Fig. S25. Efficacy of EC purification from mouse tissues.

    Fig. S26. Efficacy of APLN and PPARG knockdown.

    Fig. S27. Efficacy of APLNR knockdown.

    Table S1. Comparison of APLN/APLNR or FOXO1 knockdown to previous FOXO1/FOXO3A study.

  • Supplementary Material for:

    Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin's glucose-lowering effects

    Cheol Hwangbo, Jingxia Wu, Irinna Papangeli, Takaomi Adachi, Bikram Sharma, Saejeong Park, Lina Zhao, Hyekyung Ju, Gwang-woong Go, Guoliang Cui, Mohammed Inayathullah, Judith K. Job, Jayakumar Rajadas, Stephanie L. Kwei, Ming O. Li, Alan R. Morrison, Thomas Quertermous, Arya Mani, Kristy Red-Horse, Hyung J. Chun*

    *Corresponding author. Email: hyung.chun{at}yale.edu

    Published 13 September 2017, Sci. Transl. Med. 9, eaad4000 (2017)
    DOI: 10.1126/scitranslmed.aad4000

    This PDF file includes:

    • Fig. S1. APLNR expression profiling.
    • Fig. S2. Fat and muscle content of AplnrECKO mice.
    • Fig. S3. Plasma insulin concentrations in Apln−/−, Aplnr−/−, and AplnrECKO mice.
    • Fig. S4. Adiponectin concentration in AplnrECKO mice.
    • Fig. S5. Hindlimb blood flow in AplnrECKO mice.
    • Fig. S6. Effect of apelin on transendothelial glucose transfer.
    • Fig. S7. Metabolic effects of high-fat diet.
    • Fig. S8. Effect of high glucose on APLN.
    • Fig. S9. Gene expression profiling of ECs subjected to APLN and APLNR knockdown or FOXO1 knockdown.
    • Fig. S10. FABP4 expression in response to APLN or FOXO1 knockdown.
    • Fig. S11. FABP4 expression in response to APLN and PPARG knockdown.
    • Fig. S12. FABP4 immunostaining in Apln−/− and Apln−/−:Foxo1ECKO mice.
    • Fig. S13. Effect of continuous apelin infusion on glucose tolerance testing.
    • Fig. S14. FABP4 expression in mice receiving apelin infusion.
    • Fig. S15. FA uptake in HUVECs subjected to modulation of apelin/APLNR and FABP4 activity.
    • Fig. S16. FA uptake profile of Foxo1ECKO mice.
    • Fig. S17. Serum FA concentrations in Apln−/− and Foxo1ECKO mice.
    • Fig. S18. Circulating serum FA concentrations in AplnrECKO mice.
    • Fig. S19. FA and triglyceride content of skeletal muscle of AplnrECKO mice.
    • Fig. S20. Endothelial permeability in the lung and skeletal muscle of AplnrECKO mice.
    • Fig. S21. Endothelial permeability in response to APLN and APLNR knockdown.
    • Fig. S22. Effect of pharmacologic FABP4 inhibition on glucose tolerance test in wild-type mice on normal chow.
    • Fig. S23. Efficacy of Aplnr deletion.
    • Fig. S24. Efficacy of Foxo1 gene deletion.
    • Fig. S25. Efficacy of EC purification from mouse tissues.
    • Fig. S26. Efficacy of APLN and PPARG knockdown.
    • Fig. S27. Efficacy of APLNR knockdown.
    • Table S1. Comparison of APLN/APLNR or FOXO1 knockdown to previous FOXO1/FOXO3A study.

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