Research ArticleMOLECULAR BIOLOGY

Retinol-binding protein 2 (RBP2) binds monoacylglycerols and modulates gut endocrine signaling and body weight

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Science Advances  11 Mar 2020:
Vol. 6, no. 11, eaay8937
DOI: 10.1126/sciadv.aay8937
  • Fig. 1 Rbp2−/− mice gain more body fat than littermate controls as they age.

    (A) Six- to 7-month-old male Rbp2−/− mice fed a chow diet throughout life have a significantly greater body weight than matched controls fed the same diet. (B) Magnetic resonance imaging analysis shows that the increase in body weight is associated with significantly increased body fat. (C) A significant increase in fat mass was observed in both subcutaneous (SubQ) and VISC adipose tissue but not in brown adipose tissue (BAT). (D) Hematoxylin and eosin (H&E) staining of VISC (epididymal) adipose tissue establishes that adipocyte size in VISC fat was significantly larger for Rbp2−/− compared to control mice. (E and F) Adipocyte size distribution (E) and mean adipocyte size (F) for VISC adipose tissue from Rbp2−/− and matched WT mice. Adipocyte size was determined using multiple H&E-stained sections obtained from three Rbp2−/− and three matched WT mice. Calorimetry studies at room temperature undertaken on 6- to 7-month-old Rbp2−/− mice fed a chow diet throughout life establish that the Rbp2−/− mice consume significantly less O2 during the light but not dark cycle (G) and have a similar respiratory exchange ratio (RER) to WT mice (H). Rbp2−/− mice generate significantly less heat during the light cycle compared to matched WT controls (I). Food intake monitored over a 5-day period is significantly lower, both in the light and dark cycles, in Rbp2−/− compared to matched WT mice (J). All data are presented as means ± SEM. Statistical significance: *P < 0.05.

  • Fig. 2 Six- to seven-month-old Rbp2−/− mice maintained on a chow diet throughout life clear an intraperitoneal glucose challenge significantly less well than matched WT mice and have elevated fasting hepatic TG levels but lower plasma TG levels compared to matched WT mice.

    (A) Time course showing blood glucose levels in response to an intraperitoneal challenge of glucose for matched 6- to 7-month-old male Rbp2−/− and WT mice fed a chow diet throughout life. (B) The area under the glucose clearance curves (AUCs) for Rbp2−/− mice is significantly greater than that of matched WT mice. AU, arbitrary units. (C) Fasting plasma insulin levels are not different for matched Rbp2−/− and WT mice. (D) Pepck mRNA levels were elevated in fasting liver of Rbp2−/− mice compared to WT controls. (E) Fasting hepatic TG levels are significantly elevated in male Rbp2−/− mice. (F and G) Fasting plasma nonesterified fatty acid (FFA) levels are not different for matched Rbp2−/− and WT mice (F) but fasting plasma TG levels are significantly lower in the Rbp2−/− mice (G). (H) Livers of Rbp2−/− mice secrete significantly less TG bound to very-low-density lipoprotein (VLDL) than matched WT mice when P407 was used to block lipase action on newly secreted TG in VLDL. (I) Expression of Fabp1 mRNA is elevated in livers of Rbp2−/− mice but no differences in expression levels of Fabp2 or Fabp4 were observed. All data are presented as means ± SEM. Statistical significance: *P < 0.05 and **P < 0.01.

  • Fig. 3 When 7-week-old Rbp2−/− and matched WT mice are placed on a high-fat diet (60% of calories from fat), the Rbp2−/− mice gain significantly more body weight than WT.

    (A) Weight gain curves for Rbp2−/− and WT mice maintained on either a high-fat diet or a purified control diet (10% of calories from fat). (B) Upon 10 weeks of high-fat diet feeding, as determined by dual-energy x-ray absorptiometry, Rbp2−/− mice accumulate significantly more body fat than WT mice. (C and D) As seen from the plasma glucose clearance curves following an intraperitoneal challenge with glucose (C) and the resulting AUCs (D), the high-fat diet–fed Rbp2−/− mice respond significantly less well to the glucose challenge than WT mice. (E) The responses of matched Rbp2−/− and WT mice were not different following an intraperitoneal challenge with insulin. All data are presented as means ± SEM. Statistical significance: *P < 0.05 and ***P < 0.001.

  • Fig. 4 RBP2 binds tightly 2-AG and 2-OG but not NAEs.

    (A) Incubation of holo-RBP2 with 2-AG, 2-OG, 1-AG, or 2-LG but not N-acylethanolamides (LEA, MEA, or AEA) leads to displacement of retinol from the binding site of RBP2. After incubation with tested ligands, the protein samples were repurified on an ion exchange column. The chromatograms represent elution profiles of RBP2 monitored at 280 nm (protein scaffold, green trace) and 325 nm (retinoid moiety, red trace). The inset shows the ultraviolet-visible absorbance spectrum for holo-RBP2. (B) Analysis of ethanolic extracts reveals the presence of an intense ion peak at mass/charge ratio (m/z) = 379.4 [M + H]+ in the protein samples preincubated with 2-AG. The molecular identification of the m/z = 379.4 parent ion as 2-AG was achieved by comparing the MS/MS fragmentation pattern with a synthetic standard. (C) Ribbon diagrams of the RBP2 structure in complex with 2-AG. The position of the ligand within the binding pocket is indicated by a van der Waals surface model. (D) Molecular organization of the 2-AG–binding site with selected residues involved in formation of the internal cavity. Blue mesh signifies a refined 2FoFc electron density map for the ligand at 1.4 σ. Carbon atoms of the ligand are colored blue, whereas oxygens are shown in red. Water molecules (W) are depicted as light blue spheres. (E) Comparison of the spatial orientation of the side chains and position of all-trans-retinol and 2-AG within the binding pocket. The overlay of structures of RBP2 in complex with all-trans-retinol (PDB 4QZT) and 2-AG (PDB 6BTH) indicates that the 2-AG occupies the same binding cavity as the retinoid by adopting a ring-like configuration of the acyl chain. (F) Details of the hydrogen bond network between 2-AG (shown in blue) or all-trans-retinol (shown in orange) and the polar side chain inside the ligand binding pocket. Both of the hydroxyl groups and the carbonyl oxygen of 2-AG are involved in numerous interactions with polar side chains, whereas hydrogen bonding of all-trans-retinol is limited to single interaction with the side chain of Q108. Distances between interacting atoms are in angstrom (Å).

  • Fig. 5 Levels of 2-AG, 2-PG, 2-LG, and 2-OG in intestinal scrapings from the jejunum and plasma levels of GIP, GLP-1, CCK8, and insulin for Rbp2−/− and WT mice 2 hours after oral administration of a challenge of 100 μl of corn oil.

    (A and B) Levels of 2-AG, 2-PG, and 2-LG but not 2-OG were significantly elevated in mucosal scrapings obtained from Rbp2−/− mice. (C to F) Plasma levels of GIP (C) and insulin (F) but not GLP-1 (D) or CCK8 (E) were significantly elevated for Rbp2−/− mice receiving the corn oil challenge. (G and H) GIP levels present in plasma of both fed (G) and overnight fasting (H) Rbp2−/− mice were significantly elevated compared to those of matched WT mice. GIP levels were also significantly elevated in younger Rbp2−/− mice fed a high-fat diet for 8 weeks compared to matched WT mice in response to an oral fat challenge (I) but not in response to an oral glucose challenge (J). (K) Blood GIP levels were significantly elevated in Rbp2−/− mice when young (80 to 83 days old) Rbp2−/− and matched WT mice, which had been fed a chow diet throughout life, were challenged with 100 μl of corn oil. All data are presented as means ± SEM. Statistical significance: *P < 0.05 and **P < 0.01.

  • Fig. 6 The absence of Rbp2 alters expression of genes in the intestine and hypothalamus.

    (A) Expression of both Fabp1 and Fabp2 but not Fabp4 are significantly elevated in the proximal portion of the small intestine for Rbp2−/− mice compared to WT. (B) Expression levels in the proximal small intestine were assessed for the endocannabinoid receptors Cb1 and Cb2, enzymes involved in NAE synthesis [N-acylphosphatidyl ethanolamine phospholipase D (Nape-pld) and degradation (fatty acid amide hydrolase (Faah)] and 2-MAG synthesis [diacylglycerol lipase b (Dgl-b) and degradation (monoglyceride lipase (Mgl)] and a number of cell surface and nuclear receptors that are known to bind FFA, NAEs, or 2-MAGs (Cd36, Gpr119, Trpv1, and Ppara). mRNA expression of Dgl-b and Faah was significantly reduced for Rbp2−/− mice, but no statistically significant differences were observed for the other transcripts. (C) RBP2 is not expressed in the hypothalamus of WT mice fed a chow diet. However, both Rbp1 and Rbp4 are expressed in the hypothalamus. (D) The absence of Rbp2 expression in the small intestine is associated with significantly diminished expression of the Pomc gene in the hypothalamus but has no effect on either Agrp or Pyy gene expression. All data are presented as means ± SEM. Statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/11/eaay8937/DC1

    Supplementary Materials and Methods

    Fig. S1. Lack of differences in ATRA levels in tissues and in circulating RBP4 levels between basal control and high-fat diet–fed Rbp2−/− and WT mice.

    Fig. S2. Determination of Kd values for interaction of RBP2 with MAGs and AEA.

    Fig. S3. Position of AEA inside the binding pocket of RBP2 and comparison of the modes of binding for all-trans-retinol, 2-AG, and AEA.

    Table S1. X-ray data collection and refinement statistics.

    References (6170)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Lack of differences in ATRA levels in tissues and in circulating RBP4 levels between basal control and high-fat diet–fed Rbp2−/− and WT mice.
    • Fig. S2. Determination of Kd values for interaction of RBP2 with MAGs and AEA.
    • Fig. S3. Position of AEA inside the binding pocket of RBP2 and comparison of the modes of binding for all-trans-retinol, 2-AG, and AEA.
    • Table S1. X-ray data collection and refinement statistics.
    • References (6170)

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