RNA-Seq analysis of enteroendocrine cells reveals a role for FABP5 in the control of GIP secretion

Abstract

In response to fat intake, enteroendocrine K cells release the hormone glucose-dependent insulinotropic polypeptide (GIP). GIP acts on adipocytes to increase lipid uptake and enhance adipokine secretion, promoting weight gain and insulin resistance. Modulation of intestinal GIP release could therefore represent a therapeutic strategy for the treatment and prevention of obesity and diabetes. However, the prospects of using drugs to effectively target specific enteroendocrine cell types have been tempered by the realization that these cells share similar transcriptional programs and frequently employ common mechanisms of hormone secretion. To gain novel insights into the regulation of GIP release, we generated knock-in mice expressing green fluorescent protein (GFP) under the control of the endogenous GIP promoter that enable the isolation of a purified population of small intestine K cells. Using RNA sequencing, we comprehensively characterized the transcriptomes of GIP(GFP) cells as well as the entire enteroendocrine lineage derived from Neurogenin3-expressing progenitors. Among the genes differentially expressed in GIP(GFP) cells, we identified and validated fatty acid-binding protein 5 (FABP5) as a highly expressed marker of GIP-producing cells that is absent in other enteroendocrine cell types. FABP5 promotes intracellular transport and inactivation of endocannabinoids, including anandamide, which inhibits GIP release. Remarkably, we found that circulating levels of GIP were significantly decreased in FABP5-deficient mice in the fasting state and in response to acute, oral fat diet administration. Our findings highlight the power of RNA sequencing to uncover molecular signatures of specific enteroendocrine cell types that can potentially be exploited for therapeutic purposes in the treatment of metabolic disorders.

Figure 1.. Generation of GIP-GFP reporter mice by homologous recombination in embryonic stem cells (ESCs).

A, Schematic representation of the targeting strategy. The protein-coding region of exon 2 and part of exon 3 of the GIP gene was replaced by a sequence encoding EGFP and the PGK1-Puro resistance cassette flanked by LoxP sites. In correctly targeted clones, the GIP promoter drives expression of EGFP. Gray boxes indicate coding regions; the numbers in parentheses indicate kb; DT-A, diphtheria toxin A fragment. B, Correctly targeted ESC clones (2D and 10G) were identified by Southern blot analysis and treated with Cre recombinase to remove the puro cassette. C, GFP expression is restricted to GIP-producing cells in the small intestine epithelium. Intestinal K cells expressing GFP stain positive with a GIP-specific antisera.

Figure 2.. GIP^GFP cells represent genuine intestinal K cells.

A, FACS of GFP⁺ cells of the small intestine enables enrichment of GIP-expressing enteroendocrine cells. B, TaqMan gene expression analysis of the GFP⁺ and GFP⁻ cell populations confirms the identity of GIP^GFP cells.

Figure 3.. RNA-Seq analysis of enteroendocrine cell populations isolated by FACS.

A, Scatter plots of normalized FPKM expression values comparing the transcriptomes of the enteroendocrine lineage (Ngn3^TOMATO cells) with either the GIP-producing enteroendocrine cell subset (GIP^GFP cells) or the nonenteroendocrine lineage (Ngn3⁻ cells). Transcripts for gut peptides showing up- and down-regulation in Ngn3^TOMATO cells are indicated in red and green, respectively. B and C, Validation of the RNA-Seq data in individual RNA samples using TaqMan assays specific for gut hormones and enteroendocrine cell markers. A strong correlation was found between the RNA-Seq data and the qPCR data (r = 0.942, P < .01), confirming the utility of pooling RNA samples for the construction of the RNA-Seq libraries. D, Frequency distribution of transcript abundances in the 3 libraries. r, Pearson correlation coefficient.

Figure 4.. Identification of FABP5 as a marker of GIP-producing enteroendocrine cells.

A, Normalized RNA-Seq coverage (ie, number of reads matching an annotated gene) in the 3 libraries is displayed above the FABP5 gene. B, Virtually every GIP^GFP cell in the small intestine of GIP-GFP mice but only a subset of enteroendocrine cells in the small intestine of Ngn3⁻ TOMATO mice expresses FABP5 (arrows). Arrowheads indicate Ngn3^TOMATO enteroendocrine cells that do not produce detectable levels of the FABP5 protein.

Figure 5.. FABP5 is required for GIP secretion.

A, Plasma GIP levels in FABP5 null mice and wild-type littermates after an overnight fast and at 1 hour after oral gavage with olive oil (n = 4–6). B, Isolated crypts efficiently form intestinal organoids after ex vivo expansion in Matrigel, regardless of genotype, but basal GIP secretion is attenuated in FABP5-deficient organoids. Data are mean ± SE. *, P < .05 and **, P < .01 by Student's t test.