Immunoglobulin-like domain containing receptor 1 mediates fat-stimulated cholecystokinin secretion

Cholecystokinin (CCK) is a satiety hormone produced by discrete enteroendocrine cells scattered among absorptive cells of the small intestine. CCK is released into blood following a meal; however, the mechanisms inducing hormone secretion are largely unknown. Ingested fat is the major stimulant of CCK secretion. We recently identified a novel member of the lipoprotein remnant receptor family known as immunoglobulin-like domain containing receptor 1 (ILDR1) in intestinal CCK cells and postulated that this receptor conveyed the signal for fat-stimulated CCK secretion. In the intestine, ILDR1 is expressed exclusively in CCK cells. Orogastric administration of fatty acids elevated blood levels of CCK in wild-type mice but not Ildr1-deficient mice, although the CCK secretory response to trypsin inhibitor was retained. The uptake of fluorescently labeled lipoproteins in ILDR1-transfected CHO cells and release of CCK from isolated intestinal cells required a unique combination of fatty acid plus HDL. CCK secretion secondary to ILDR1 activation was associated with increased [Ca²⁺]_i, consistent with regulated hormone release. These findings demonstrate that ILDR1 regulates CCK release through a mechanism dependent on fatty acids and lipoproteins and that absorbed fatty acids regulate gastrointestinal hormone secretion.

Cholecystokinin (CCK) is a classic gastrointestinal hormone that stimulates several gastrointestinal processes, including gallbladder contraction, pancreatic secretion, and satiety. Together, these actions coordinate the ingestion and digestion of food. Diminished CCK secretion or CCK receptor blockade is associated with impaired gallbladder contraction and pancreatic secretion, enhanced hunger, and increased food intake (1–4), whereas exogenous CCK administration stimulates gallbladder contraction and pancreatic secretion, delays gastric emptying, and reduces food intake (5, 6).

CCK is produced by discrete enteroendocrine cells (I cells) that reside as single cells scattered among enterocytes in the mucosa of the small intestine. Like most gastrointestinal hormones, CCK is released into the blood following ingestion of a meal, and despite the apical surface being exposed to the lumen of the intestine, the mechanisms by which foods stimulate hormone secretion are largely unknown. It has recently been demonstrated that CCK cells contain calcium-sensing receptors that mediate amino acid–stimulated CCK release (7, 8), supporting the concept that cell surface receptors regulate food-stimulated gastrointestinal hormone release. Of the food components that stimulate CCK secretion, fats are the most important. Fat-stimulated CCK release is readily apparent in humans but requires lipolysis of triglycerides to fatty acids of the chain length of dodecanoic acid [C12] or longer (9–11). In vitro, it has been shown that fatty acid aggregates (carbon chain length 10 or greater) can stimulate CCK secretion from the enteroendocrine cell line STC-1, and this release is accompanied by an increase in intracellular Ca²⁺ ([Ca²⁺]_i) (12, 13).

It was recently demonstrated that stimulation of the G protein–coupled receptor 40 (GPR40) present in CCK-EGFP cells by C18 fatty acids, resulted in elevation of [Ca²⁺]_i as well as release of CCK (14). However, CCK cells isolated from Gpr40^–/– mice also responded to C18 fatty acids, suggesting that GPR40 is not the sole mechanism by which fatty acids stimulate CCK cells. Another proposed mechanism for CCK release from the intestine is through the action of chylomicrons and other related lipoproteins on CCK cells (15). Fat-stimulated CCK release was inhibited by administration of Pluronic-L81, which blocks intestinal lipoprotein assembly, and intra-arterial infusion of chylous lymph collected from intralipid-fed rats decreased gastric emptying through a CCK-dependent mechanism, raising the possibility that lipoprotein molecules can act on the basolateral rather than the apical surface of CCK cells to stimulate hormone secretion (16).

Immunoglobulin-like domain containing receptor 1 (ILDR1) is a member of the lipoprotein remnant receptor family and shares 31% sequence identity with the lipolysis-stimulated receptor (LSR) (17). LSR was first identified as a membrane protein that mediated LDL uptake in the presence of fatty acids in homozygous familial hypercholesterolemia fibroblasts deficient in LDL receptor (18). Subsequent studies showed that LSR had a higher affinity for oleate-induced binding of chylomicrons and VLDL rather than LDL and was perhaps involved in the clearance of triglyceride-rich lipoproteins by the liver. Three alternatively spliced isoforms of ILDR1 have been described (17). Two isoforms (α and α′) contain a putative transmembrane domain and are targeted to the plasma membrane, while the third isoform (β) lacks the exons encompassing the transmembrane domain and is presumably cytoplasmic in location. Given the amino acid similarity between ILDR1 and LSR, we postulated that lipoprotein–fatty acid interactions of ILDR1 may be involved in fatty acid sensing by intestinal CCK cells.

In an attempt to identify novel receptors and signal transduction pathways regulating CCK secretion, we performed a microarray analysis of CCK cells. CCK cells were enriched to >90% purity by FACS of dispersed intestinal mucosal cells from transgenic CCK-EGFP mice, which express EGFP under the control of the CCK promoter (ref. 8 and Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI68587DS1). The gene expression profile of CCK-EGFP cells was compared with that of non-EGFP mucosal cells of the small intestine. This array identified overexpression of Ildr1 mRNA in CCK-EGFP cells. Using real-time PCR (RT-PCR), we confirmed that Ildr1 is expressed in CCK cells of the proximal small intestine. RT-PCR demonstrated that Ildr1 mRNA is highly expressed in fluorescent CCK-EGFP cells compared with nonfluorescent intestinal mucosal cells (Figure 1). Ildr1 mRNA levels in EGFP-positive cells were not significantly different from Cck mRNA levels, whereas they differed significantly from those of β-actin. The levels of β-actin were very similar between EGFP-positive and EGFP-negative cell populations and to those of Gapdh (normalizer). Thus, Ildr1 and Cck were coexpressed in CCK cells of the proximal intestine.

Figure 1

Relative quantitation of Ildr1, Cck, and Actb gene expression in FACS-sorted CCK-EGFP cells using RT-PCR. The fold change (base 2 logarithmic scale) in relative quantity of Ildr1, Cck, and Actb mRNA is shown. Gapdh was used as the normalizer, and RNA isolated from CCK-EGFP–negative cells was used as the calibrator. Ildr1 and Cck mRNA levels were compared with Actb. **P < 0.01, ***P < 0.001 (n = 3; RNA pooled from 1 to 3 mice for each value).

To determine whether ILDR1 behaved similarly to LSR in mediating uptake of lipoproteins in the presence of fatty acids, we used CHO cells transfected with an ILDR1-encoding plasmid (Supplemental Figure 2). ILDR1 expressed in these cells migrated on SDS-PAGE as a band with a M_r of approximately 100 kDa (Supplemental Figure 2A). Immunostaining of ILDR1-transfected CHO cells showed that the protein was located on the membrane (Supplemental Figure 2B) as well as in punctate intracellular compartments. The identity of this intracellular compartment(s) has not been determined.

To elucidate the function of ILDR1, we tested whether it could mediate uptake of lipoproteins in the presence of a fatty acid, as has been reported for LSR (19). Uptake of fluorescently labeled lipoproteins (HDL, LDL, VLDL, and chylomicrons) in the presence or absence of C12 was quantitated by automated FACS of fixed cells (ref. 20 and Figure 2). HDL was the only lipoprotein that produced an increase in cellular fluorescence in ILDR1-transfected cells in the presence of C12. The specificity of this effect was demonstrated by competition with unlabeled HDL. These results suggested that, like LSR, ILDR1 may be involved in the binding and uptake of lipoproteins (HDL but not chylomicrons, VLDL, or LDL) in the presence of a fatty acid (C12).

Figure 2

DiI-HDL uptake in CHO and ILDR1-transfected cells. CHO and ILDR1-transfected CHO cells were incubated with DiI-labeled lipoproteins, and cellular fluorescence was measured using FACS. Cells were incubated with 5 μg protein/ml of DiI-labeled lipoproteins for 2 hours in the absence or presence of a 100-fold excess of unlabeled lipoproteins and 2 mM C12. Nonspecific fluorescence (observed in the presence of 100-fold excess of unlabeled lipoproteins) was subtracted from all values. *P < 0.05, ***P < 0.001 (n = 3 experiments). CM, chylomicron.

In order to determine the role of ILDR1 in regulating CCK secretion, we generated Ildr1 knockout mice (Ildr1^–/– mice) using ES cells containing a disrupted Ildr1 gene (German Gene Trap Consortium). The targeting vector (Figure 3A) carrying β-Geo fusion protein with a 5′ splice acceptor was integrated in intron 2 of the Ildr1 gene. By X-gal staining, β-galactosidase expression was observed in several tissues as described previously (ref. 17 and R. Chandra and R.A. Liddle, unpublished observations). The expression of β-galactosidase in intestinal CCK cells was confirmed by coimmunostaining for CCK and β-galactosidase proteins (Figure 3B). CCK and β-galactosidase colocalized in 93% of the CCK-positive cells in the villus, suggesting cellular targeting of Ildr1 gene deletion in CCK cells of the duodenum. Using RT-PCR, we failed to amplify Ildr1 from RNA prepared from GFP-positive intestinal cells of Ildr1^–/– CCK-EGFP mice (after 40 cycles), whereas it could be amplified from comparable amounts of total RNA (50 ng) isolated from GFP-positive cells of wild-type CCK-EGFP mice. Cck was amplified from intestinal RNA isolated from both wild-type and knockout mice (data not shown).

Figure 3

Ildr1 gene deletion and CCK cell-specific expression. (A) The mouse Ildr1 gene was disrupted by insertion of a β-Geo fusion reporter in intron 2, resulting in the production of β-galactosidase in targeted cells. The transmembrane domain is located in exon 4. A cysteine-rich region is present in exon 5 and a dileucine motif is located in exon 6 of human ILDR1. Introns are not drawn to scale. (B) Expression of EGFP and β-galactosidase in Ildr1^–/– CCK-EGFP mouse duodenum was determined by immunostaining frozen sections with antibodies to EGFP (CCK cell reporter) and β-galactosidase (Ildr1^–/– reporter). The left image shows colocalization (yellow) of EGFP (green) and β-galactosidase (red) antigens; nuclear staining (DAPI) is shown in blue (scale bar: 20 μm; original magnification, ×40). The top right images show staining of EGFP (green) or β-galactosidase (red) (scale bar: 5 μm). The bottom right image is a 3D reconstruction of this cell using Imaris software. The isosurface of the EGFP (green) cytoplasmic staining was rendered transparent to reveal β-galactosidase staining (red isosurface) and nucleus (blue isosurface) located within the cell (scale bar: 5 μm).

The effect of Ildr1 deletion on CCK secretion was studied by two independent methods. First, fatty acids were administered to wild-type and Ildr1^–/– mice via orogastric gavage (0.075 μmol/g mouse in 400 μl), and plasma CCK levels were measured by bioassay (21) 30 minutes later (Figure 4A). With octanoate, dodecanoate, or oleate, wild-type mice demonstrated a typical postprandial increase in plasma CCK levels. However, no increase in plasma CCK levels was observed in Ildr1^–/– mice. Similar effects were noticed with decanoate and palmitoleate (data not shown). At the time of blood collection, tracer methylene blue dye was detected in the intestinal lumen of both groups of animals, suggesting that the low levels of CCK in Ildr1^–/– mice were not due to impaired gastric emptying. It is important to note that although dietary fat is the major stimulant of CCK secretion, proteins also cause CCK release through a mechanism that involves intraluminal proteases (22). To determine whether the effects of ILDR1 deficiency were selective for fat-stimulated CCK release, we tested the ability of soybean trypsin inhibitor (SBTI, type II) to stimulate CCK secretion in wild-type and Ildr1^–/– mice (Figure 4B). Orogastric administration of SBTI caused comparable elevations in plasma CCK levels in wild-type and Ildr1^–/– mice, indicating that Ildr1-deficient CCK cells are capable of responding to other secretagogues and that fatty acids trigger ILDR1-promoted CCK secretion.

Figure 4

Plasma CCK responses in Ildr1^–/– mice. (A) Blood was collected for measurements of plasma CCK concentrations 30 minutes after orogastric administration of octanoate (C8), dodecanoate (C12), or oleate (C18) in wild-type and Ildr1^–/– mice. The dose of each fatty acid was 0.0749 μmol/g body weight administered in a volume of 300 to 400 μl. (B) To determine whether Ildr1 gene deletion was specific for fatty acid–stimulated CCK release, CCK levels were measured in wild-type and Ildr1^–/– mice that were administered 0.32 mg/g mg SBTI. ^†P < 0.01, ^#P < 0.001, comparison with basal release within a genotype; ***P < 0.001, comparison with basal release between genotypes (n = 3 values; plasma from 2 to 4 mice was pooled for obtaining each value).

To determine whether impaired fatty acid absorption could account for reduced CCK secretion in Ildr1-deficient mice, we measured serum triglycerides after a lipid-rich meal in both wild-type and Ildr1^–/– mice (Supplemental Figure 3). We found that triglyceride levels increased to similar levels after feeding in both groups of animals, suggesting that lipid absorption may not be markedly altered in knockout mice. In addition, both wild-type and Ildr1^–/– mice have similar growth curves on either a normal or high-fat diet, making it unlikely that Ildr1-deficient mice have an alteration in gut permeability to lipids (Supplemental Figure 4).

The effects of fatty acids and lipoproteins on CCK secretion were evaluated in vitro in dispersed intestinal cells from wild-type CCK-EGFP mice and Ildr1^–/– CCK-EGFP mice (Figure 5). Based on fatty acid–promoted HDL uptake in ILDR1-expressing CHO cells, we expected that CCK secretion would be enhanced in the presence of both lipoproteins and fatty acids. Neither fatty acids nor lipoproteins, when administered separately, stimulated CCK release in vitro. However, at concentrations that had no effect individually, HDL in combination with C12 significantly increased CCK release from wild-type CCK cells. In contrast, HDL plus C12 did not stimulate CCK release from Ildr1-deficient CCK cells, indicating that ILDR1 mediated the effects of fatty acids and lipoproteins. In addition, the amount of CCK released from wild-type mouse intestinal cells by KCl stimulation was not significantly different from that induced by HDL plus C12. CCK release was triggered in wild-type (but not Ildr1^–/–) CCK cells by the combination of C12 and chylomicrons and in both wild-type and Ildr1^–/– mice by the combination of C12 and LDL. However, there was no statistical difference between the wild-type and Ildr1^–/– genotypes, as was observed for HDL plus C12.

Figure 5

Effects of fatty acids and lipoproteins on CCK secretion from dispersed intestinal mucosal cells. Mucosal cells were dispersed from intestines of wild-type or Ildr1^–/– mice and incubated with C12 (100 μM), lipoproteins (100 μg protein/ml), or the combination of lipoproteins and C12. CCK released into the media after 30-minute incubation was measured by RIA. ^#P < 0.001, comparison of secretion in the presence of C12 with KCl (within a genotype) (wild-type); ^##P < 0.05, comparison of secretion in the presence of C12 with KCl (within a genotype) (Ildr1^–/–); *P < 0.05, **P < 0.01, ***P < 0.001 (n = 6–14 mice).

Changes in intracellular calcium concentrations are critically important for regulated CCK secretion (23, 24). To determine whether [Ca²⁺]_i is elevated in ILDR1-stimulated CCK secretion and to confirm whether fatty acids and lipoproteins act directly on CCK cells, we examined the effects of lipoproteins and fatty acids on calcium fluorescence in FACS-purified CCK-EGFP cells from both wild-type and Ildr1^–/– CCK-EGFP mice loaded with the calcium-sensitive dye, X-rhod-1 (Figure 6, A and B). Minimal calcium transients were induced in wild-type and Ildr1^–/– CCK-EGFP cells by preparations of C12 (100 μM), HDL, LDL, VLDL, or chylomicrons (all lipoproteins were at 100 μg protein/ml). However, the combination of HDL and C12 caused a substantial increase in [Ca²⁺]_i in wild-type CCK-EGFP cells but not Ildr1^–/– CCK-EGFP cells. Under all conditions, cell viability was confirmed by an appropriate [Ca²⁺]_i response to depolarizing concentrations of KCl. The percentages of EGFP-positive cells (wild-type and Ildr1^–/–) that responded to lipoproteins, lipoproteins and C12, and KCl are shown in Figure 6C (HDL) and Supplemental Figure 5 (LDL, VLDL, and chylomicrons). Intestinal cells from Ildr1^–/– CCK-EGFP mice showed elevation of [Ca²⁺]_i when treated with l-phenylalanine (Figure 4 and Supplemental Figure 6), suggesting that these cells were capable of responding to amino acid stimulation. This result is in accordance with the increase of plasma CCK levels observed in Ildr1^–/– mice after orogastric gavage of SBTI.

Figure 6

Effects of fatty acids and lipoproteins on intracellular calcium fluorescence in CCK cells. CCK-EGFP cells were isolated from the intestines of wild-type CCK-EGFP and Ildr1^–/– CCK-EGFP mice and enriched by FACS. Cells were loaded with calcium-sensitive dye (X-rhod-1) and incubated first with lipoproteins (100 μg protein/ml) or C12 (100 μM), followed by lipoprotein plus C12, and finally with KCl (50 mM). (A) The responses of representative wild-type CCK-EGFP or Ildr1^–/– CCK-EGFP cells to lipoproteins and lipoprotein plus C12 are shown. Change in fluorescence from green to yellow to red indicates elevation in [Ca²⁺]_i. An increase in [Ca²⁺]_i with KCl was used to confirm the viability of cells at the conclusion of each experiment. Results are representative of at least 3 experiments. (B) Change in fluorescence intensity in wild-type CCK-EGFP and Ildr1^–/– CCK-EGFP cells to C12 alone, HDL alone, HDL plus C12, and KCl. (C) Percentage of wild-type CCK-EGFP and Ildr1^–/– CCK-EGFP cells that showed at least a 10% increase in Ca²⁺ fluorescence in response to HDL or HDL plus C12. The percentage of cells responding to C12 alone is represented by the dashed line. Scale bar: 5 μm.

CCK is released from intestinal cells by the actions of amino acids and fatty acids. In this study, we show that a receptor, ILDR1, with novel function is selectively expressed in the CCK-producing enteroendocrine cells of the small intestine and modulates fatty acid and lipoprotein-mediated CCK secretion. In the absence of ILDR1, fatty acids were unable to stimulate the release of CCK, suggesting that ILDR1 is essential for this process. Since GPR40 has previously been shown to mediate fatty acid–stimulated CCK release (14), it is possible that both GPR40 and ILDR1 play physiologic roles in CCK release. However, ILDR1 appears to be critical for fatty acid–mediated CCK release, as knockout mice exhibited only basal levels of CCK in response to oral administration of fatty acids. Ildr1^–/– mice retain the ability to release CCK in response to SBTI, suggesting that mechanisms for protein-induced signal transduction and exocytosis were unaffected in CCK cells lacking ILDR1. Impaired CCK secretion in Ildr1^–/– mice appears to be a direct effect on CCK cells and not through other mechanisms, such as delayed gastric emptying. Addition of methylene blue to the fatty acid solutions showed that the administered fatty acid solutions reached the ileum of wild-type and knockout mice within 30 minutes, suggesting that gastric emptying was qualitatively similar in both control and knockout mice.

Measuring CCK release from in vitro cell preparations has been challenging. Primary CCK cells do not grow well in culture, and CCK-producing cell lines such as STC-1 differ from CCK cells in many important ways. For example, STC-1 cells contain many other hormones besides CCK, and they do not express the same repertoire of receptors as primary CCK cells (R. Chandra and R.A. Liddle, unpublished observations). In particular, we discovered that STC-1 cells do not express ILDR1. In attempting to study CCK release in vitro, we were unable to reproducibly measure regulated CCK secretion from CCK-EGFP cells after FACS, presumably because sufficient numbers of healthy, responsive cells could not be obtained. As an alternative, we examined dispersed intestinal mucosal cells that contained approximately 0.5% CCK cells. In these studies, C12 and individual lipoproteins alone had little effect on CCK release, and it was only with the combination of lipoproteins and C12 (primarily HDL) that a significant increase in CCK release was observed. We also observed minor effects of the combination of C12 and LDL as well as chylomicrons and C12 on CCK release. Since these effects were not observed in DiI uptake experiments or during measurements of Ca²⁺ fluorescence in wild-type and Ildr1^–/– CCK-EGFP cells, the relationship with ILDR1 function remains to be determined.

Calcium signaling in CCK cells has been shown to be critically important for regulated hormone secretion (8, 25, 26). Several physiological secretagogues (such as monitor peptide and amino acids) as well as exposure to depolarizing concentrations of KCl increase [Ca²⁺]_i, which is linked to hormone release (7, 8, 23, 24). In this study, involvement of calcium signaling in ILDR1-stimulated CCK secretion was demonstrated by comparing [Ca²⁺]_i in wild-type and Ildr1^–/– CCK-EGFP cells. The combination of HDL and C12 induced an increase in [Ca²⁺]_i in wild-type mice but not knockout mice. Our findings differ somewhat from observations in which fatty acids (C18) alone stimulated CCK release from FACS-enriched CCK cells, an effect that was partially reduced in CCK cells from Gpr40-deficient mice (14). Perhaps these differences are due to the viability of cells following FACS. Nevertheless, it appears that ILDR1 plays a major role in the regulation of fatty acid–stimulated CCK secretion, since CCK release is completely eliminated in Ildr1^–/– mice.

Infusion of ³H-triolein in rats demonstrated that the maximum uptake of fats occurs in the duodenum and proximal jejunum of the small intestine (27) where a majority of the CCK cells are located. It has previously been shown that the major lipoproteins secreted by the intestine are chylomicrons and VLDL (28). Glickman and colleagues have demonstrated that the intestine secretes discoidal and spherical HDL particles of distinct composition (29, 30), and it is now known that about 30% of plasma HDL is synthesized by intestinal mucosal cells (31). The pathways for the synthesis and secretion of these 3 lipoproteins appear to be segregated (32, 33). Chylomicrons are produced during the “fed” condition, whereas VLDL is synthesized during fasting. In addition, these 2 lipoproteins are present in separate vesicles in enterocytes and have different rates of synthesis. While chylomicrons and VLDL are believed to be assembled in the endoplasmic reticulum and Golgi and exocytosed from enterocytes (34), recent evidence discovered during a study of Tangier disease suggests that nascent HDL particles are assembled extracellularly (33, 35). That HDL is synthesized under conditions of feeding suggests that HDL is available to stimulate the ILDR1 receptor located on CCK cells.

ILDR1 shares 31% amino acid identity with LSR (17), and this homology is distributed over the entire sequence of the receptor. ILDR1 behaves similarly to LSR, in that uptake of HDL in the presence of C12 occurred in stably transfected CHO cells. The mechanism by which ILDR1 interacts with HDL and C12 is yet to be elucidated. It is possible that the HDL particle is taken up inside the cell by endocytosis, as has been shown for LDL receptors and LDL receptor–related proteins (LRPs) (36, 37). Our finding that DiI-labeled HDL uptake is enhanced in ILDR1-transfected cells indicates that either ILDR1 undergoes internalization or HDL binding is of sufficient duration to allow exchange of DiI from the outside to the inside of the cell. LSR is highly enriched in the endosomal compartment of rat hepatocytes; endosomal membranes showed greater than 10-fold oleate-induced ¹²⁵I-LDL binding compared with the plasma membranes (38). A receptor internalization motif (Y-X-X-ϕ where ϕ is a hydrophobic amino acid such as F, I, L, M, V) is present in ILDR1 at amino acid 459. This motif has been shown to be important for clustering of receptors in clathrin-coated pits by the μ chain of AP2 (39).

Lipoprotein receptors, including the LDL receptor and lipoprotein remnant receptors, recognize specific apolipoproteins present on the surface of the lipoprotein particles. Although we do not know which apolipoproteins specifically interact with ILDR1, the main candidates are lipoproteins that are present in HDL. Our preliminary data, obtained by immunoblotting of HDL, show that it contains apo A-I and apo A-II but not detectable amounts of apo A-IV or apo E. If lipid feeding increases the production of HDL, then it is logical to postulate that the apolipoproteins associated with HDL in the intestine should also undergo increased synthesis. Magun et al. have shown that lipid feeding increases the amount of apo B and apo A-I in the intestine (40), while Tso and coworkers have demonstrated an increase in the levels of apo A-IV after feeding of intralipid (27, 41). Apo A-IV has been shown to play a role in the short-term control of food intake (42–44). Chylous lymph collected from triglyceride-fed rats as well as injections of apo A-IV reduced gastric motility (45), and this effect was lost after denervation of vagal afferents or by application of devazepide (CCK1 receptor antagonist) (15), suggesting that the effects were mediated by CCK. Apo A-IV–mediated effects could account for the increased CCK secretion observed in the presence of both chylomicrons and C12 in intestinal cells from wild-type mice. LSR has been shown to bind apo B and apo E, while apo C-III inhibits the binding of LSR to chylomicrons and VLDL but not LDL (19). The apolipoproteins involved in the interaction of ILDR1 with HDL will be the subject of future investigations.

ILDR1 is expressed in a variety of tissues (17) (R. Chandra and R.A. Liddle, unpublished data). Recently, Ildr1 mRNA has been localized to hair and supporting cells present in the organ of Corti, and it was found that mutations in Ildr1 lead to DFNB42, a type of nonsyndromic autosomal recessive hearing impairment (46). It was recently demonstrated that LSR is essential for the formation of tricellular tight junctions and targeting of tricellulin to these junctions (47). Tricellulin is expressed in the inner ear, and mutations in this gene have been linked to DFNB49 (48), suggesting that the pathogenesis of this impairment may involve aberrations in LSR-promoted tricellular tight junction formation, which require proper targeting of tricellulin. Similar to LSR, it now appears that ILDR is also involved in targeting of tricellulin to the tight junctions in the inner ear and elsewhere in the body (49). There is a growing body of evidence to suggest that the lipoprotein receptors possess multiligand, multifunctional capabilities (50). LRPs were originally identified for their role in the uptake of chylomicron remnants and lipoprotein metabolism in the liver. However, LRPs have now been shown to interact with 30 different ligands and perform diverse roles such as uptake of lipoproteins, glucose regulation, transport of ligands across the blood-brain barrier, and Wnt/β-catenin signaling (51–54). Similarly, the cell surface HDL receptor SR-BI is involved in cholesterol uptake, vitamin E transport, host defense, and removal of apoptotic cells (55, 56). It is therefore plausible that ILDR1 performs distinct functions in different tissues.

Our studies demonstrate that Ildr1^–/– mice do not lack CCK, and can secrete CCK in response to SBTI, but have impaired responses to fat-stimulated CCK release. The reason that CCK cells or other hormone-secreting cells of the gastrointestinal tract are distributed as single cells among absorptive cells of the gut is unknown. Our findings provide a possible explanation (Figure 7). Ingested fat (in the form of triglycerides) in the intestinal lumen is catabolized by lipases to 2-monoacylglycerol and fatty acids that are absorbed by enterocytes. Larger chain fatty acids are reesterified, packaged, and released from the basal surface of enterocytes, while medium-large chain fatty acids (C8–C12) are released directly from enterocytes (57). The intestine is also a major source of HDL (29, 30, 35). Therefore, CCK cells, which are surrounded by enterocytes, would be exposed to both HDL and fatty acids released from the basal surface of enterocytes. Although the apical surface of many CCK cells is exposed to the intestinal lumen, our findings suggest that fatty acids, by virtue of their requirement for HDL to activate ILDR1, act on the basal surface of CCK cells to stimulate hormone secretion. This hypothesis is in agreement with the observation that disruption of lipoprotein assembly with Pluronic-L81 inhibited CCK secretion (15) and that we were unable to detect HDL in the lumen of the intestine (Supplemental Figure 7). Immunostaining of ILDR1 in CCK cells was not sufficiently robust to localize ILDR1 to the apical versus basolateral surface, so visual proof of this hypothesis remains outstanding. Nevertheless, this study indicates that CCK secretion is a regulated process resulting from the combined actions of locally released lipoproteins and intestinally absorbed fatty acids to activate ILDR1.

Figure 7

Model of ILDR1 regulation by fatty acids and lipoproteins. Ingested lipids in the form of triglycerides are hydrolyzed by lipases within the intestinal lumen to emulsified 2-monoacyl glycerol and fatty acids (i), which are absorbed by enterocytes (ii) (shown in blue). Long chain fatty acids are reformed into triglycerides and secreted from the basolateral surface of enterocytes. Smaller long chain fatty acids such as C12 transit the enterocyte without modification and have ready access to the basolateral surface of CCK cells (shown in green). In addition to chylomicrons, HDL is also produced by enterocytes (iii); thus, CCK cells are exposed to both fatty acids and intestinal lipoproteins following a fat meal. The combination of fatty acids (e.g., C12) and HDL activates ILDR1 (iv), elevating [Ca²⁺]_i (v), which ultimately causes CCK secretion (vi). CCK is released from the basolateral surface into the paracellular space in which it is taken up into the blood to exert effects on distant organs (e.g., pancreas, gallbladder, and nerves).

View Supplemental data

This work was supported by NIH grants DK091946 and DK064213 and the Duke Comprehensive Cancer Center Core facility. We would like to thank Mirta Mihilovic for sharing her protocols for lipoprotein preparations, Cathy Bittner for managing the mouse colony, and Faraz Majeed for help with purification of bacterially expressed ILDR1 proteins for immunization into rabbits. The authors also thank Sam Johnson and Yasheng Gao for their help with image acquisition and acknowledge Duke University’s Light Microscopy Core Facility. Finally, we thank Mike Cook for standardizing the parameters for flow cytometric measurements of DiI uptake in CHO cells.

Address correspondence to: Rodger A. Liddle, Box 3913, Duke University Medical Center, Durham, North Carolina, 27710, USA. Phone: 919.681.6380; Fax: 919.668.0412; E-mail: rodger.liddle@duke.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Note regarding evaluation of this manuscript: Manuscripts authored by scientists associated with Duke University, The University of North Carolina at Chapel Hill, Duke-NUS, and the Sanford-Burnham Medical Research Institute are handled not by members of the editorial board but rather by the science editors, who consult with selected external editors and reviewers.

Reference information: J Clin Invest. 2013;123(8):3343–3352. doi:10.1172/JCI68587.

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