Intestinal Enteroendocrine Cell Signaling: Retinol-binding Protein 2 and Retinoid Actions

Abstract

Retinol-binding protein 2-deficient (Rbp2-/-) mice are more prone to obesity, glucose intolerance, and hepatic steatosis than matched controls. Glucose-dependent insulinotropic polypeptide (GIP) blood levels are dysregulated in these mice. The present studies provide new insights into these observations. Single cell transcriptomic and immunohistochemical studies establish that RBP2 is highly expressed in enteroendocrine cells (EECs) that produce incretins, either GIP or glucagon-like peptide-1. EECs also express an enzyme needed for all-trans-retinoic acid (ATRA) synthesis, aldehyde dehydrogenase 1 family member A1, and retinoic acid receptor-alpha, which mediates ATRA-dependent transcription. Total and GIP-positive EECs are significantly lower in Rbp2-/- mice. The plasma transport protein for retinol, retinol-binding protein 4 (RBP4) is also expressed in EECs and is cosecreted with GIP upon stimulation. Collectively, our data support direct roles for RBP2 and ATRA in cellular processes that give rise to GIP-producing EECs and roles for RBP2 and RBP4 within EECs that facilitate hormone storage and secretion.

Keywords: enteroendocrine cells; glucagon-like peptide-1; glucose-dependent insulinotropic polypeptide; incretins; retinoids; retinol-binding protein 2.

Figure 1.

Expression of retinoid-related genes in human and mouse enteroendocrine cells. (A) Schematic representation of retinoid metabolism. (B-D) Heat maps plotting expression of retinoic acid (RA) metabolism-related genes in published single cell and bulk RNA sequencing (RNAseq) datasets for (B) enteroendocrine cells (EECs), (C) K-cells, and (D) L-cells. Top 3 rows label region of the gut (darker blue labels more distal gut, white = whole small intestine (SI), * = combined colon and rectum); sample type (dark green = primary tissue, light green = organoids); and organism (pink = human, purple = mouse). Letters at the bottom label publication (a = Glass et al 2017, b = Roberts et al 2018, c = Larraufie et al 2019, d = Billing et al 2019, e = Goldspink et al 2020, f = Beumer et al 2018, g = Gehart et al 2019). Gene expression is plotted in log2 (counts per million). Expression for single cell RNAseq datasets (a, d, f, g) were calculated as the average across the cell type of interest.

Figure 2.

RBP2 is expressed in enteroendocrine cells present in wild-type C57BL/6J mouse jejunum. (A) Immunofluorescence (IF) confocal imaging establishing colocalization of RBP2 in ChgA⁺ cells, a marker for all EECs. (B) IF confocal imagining establishing colocalization of RBP2 with GIP. (C) IF confocal imaging establishing colocalization of RBP2 with GLP-1. The proteins colocalize in crypts (center left with higher resolution insets to the left) as well as in villi (center right with higher resolution insets to the right). Selected cells shown in the insets are highlighted by rectangles in the main micrograph. Color code: red indicates ChgA, GIP or GLP-1 IF; green indicates RBP2 IF; blue indicates DAPI staining; and merged images (ChgA, GIP or GLP-1 with RBP2) are in orange. All bars = 50 µm. (D) Western blot of EECs lysates confirming the presence of RBP2. PP: purified protein at a concentration of 10 ng (lane 1), 1ng (lane 2) and 100 pg (lane 3); NeuroD1^YFP wild type: NeuroD1 expressing cells sorted by FACS using YFP fluorescence from proximal small intestine of a C57BL/6J mouse consisting of 1,500 YFP⁺ cells (lane 4) representing the total EEC population and 1,500 YFP^– cells (lane 5) representing mostly enterocytes (since goblet and Paneth cells do not present positive IF for RBP2); NeuroD1^YFPRbp2^–/–: NeuroD1 expressing cells obtained by FACS sorting using YFP fluorescence from proximal small intestine of a Rbp2^–/–mouse to serve as a negative control for RBP2 for 1500 YFP⁺ cells (lane 6) representing the total EEC population and 1500 YFP^– cells (lane 7). Bands from beta-actin are only present in the sorted cell samples (lanes 4-7) at 42 kDa.

Figure 3.

RBP4, ALDH1A1 and RARα are all expressed in ChgA⁺ cells within the jejunums of male wild-type C57BL/6J mice. (A) Immunofluorescence (IF) confocal imaging establishing colocalization of RBP4 in ChgA⁺ cells. RBP4 and ChgA colocalize in crypts (center left with higher resolution insets to the left) as well as in villi (center right with higher resolution insets to the right). (B) IF confocal imagining establishing colocalization of ALDH1A1 and ChgA. The proteins colocalize in crypts (center left with higher resolution insets to the left) as well as in villi (center right with higher resolution insets to the right). Selected cells shown in the insets of A and B are outlined by rectangles in the main micrograph. Color code for A and B: red indicates ChgA; green indicates RBP4 or ALDH1A1; blue indicates DAPI staining; and merged images (ChgA with RBP4 or ALDH1A1) in orange. (C) IF confocal imaging establishing colocalization of RARα with ChgA. The left panel gives the merged image showing RARα and ChgA expression in a DAPI-stained villus. The subsequent 3 images (left to right) show ChgA expression in a DAPI-stained villus tissue, ChgA and RARα overlap, and RARα expression in DAPI-stained villus tissue. Color code for C: red indicates RARα; green indicates ChgA; blue indicates DAPI staining; and merged images (RARα and ChgA) are in magenta. All bars = 50 µm.

Figure 4.

RBP2 and RBP4 are in close relationship with chromogranin A vesicles in EECs within the jejunums of male wild-type C57BL/6J mice. (A) 3D SIM images of an EEC showing partial overlap of ChgA-containing vesicles and RBP2 (left) and individual distribution of ChgA vesicles (center) and RBP2 (right). (B) 3D SIM images of an EEC showing overlap of ChgA-containing vesicles and RBP4 (left) and individual distribution of ChgA vesicles (center) and RBP4 (right). Color code for A and B: red indicates ChgA IF; green indicates RBP2 or RBP4 IF; and merged images (ChgA with RBP2 or RBP4) are in orange. Scale bars = 3 µm. (C) GIP and RBP4 were cosecreted from murine proximal small intestine cultures upon stimulation with forskolin/-isobutyl-1-methylxanthine/10 mM glucose (F/I/10G). Samples were obtained from mouse primary intestinal cultures prepared for 4 different animals, each treated in duplicate. (C) Data represent mean ± SEM (GIP in black, RBP4 in blue) showing increased media levels after treatment compared with basal (0G) levels. Statistical significance: *P < .05.

Figure 5.

Effects of RBP2 deficiency on EEC numbers and distribution in the jejunum compared with matched littermate controls. (A) Small intestines of Rbp2^–/– mice showed lower numbers of ChgA⁺ cells for both the total crypt–villus axis as well as for crypts and villi alone compared with matched control mice. (B) Quantification of the total percentage of ChgA⁺ cells that coexpress GIP and GLP-1. (C,D) Immunostaining of matched control (wild type) and Rbp2^–/– mouse jejunum identified both a lower GIP⁺ total cell number and a lower number for crypts of Rbp2^–/– mice compared with controls. (E,F) Immunostaining of Rbp2^–/– mouse jejunum showed no differences in GLP-1⁺ cell number compared with controls. A, B, D, and F provide mean values and error bars represent SEM for 4 mice in each panel. Black dots represent wild-type C57BL/6J littermate controls and blue squares represent Rbp2^–/– mice. Statistical significance: *P < .05. Scale bar = 50 µm.

Figure 6.

A diagram showing RBP2 and other retinoid-related proteins present in enteroendocrine cells. Proteins of interest are in magenta and their known roles in retinoid metabolism are described in the panels on the left (rectangles). A diagram showing the cell lineages in the small intestine and their location in the crypt-villus axis is shown on the right side. Since enterocytes are only located in the villi, RBP2 is only found in the crypts when associated with EECs.