In the proximal intestine peptide hormones are released by enteroendocrine cells (EEC) in response to ingested nutrients, and function to orchestrate digestion in order to maximize nutrient absorption and utilization. Several so-called gut hormones have independently been shown to act directly on enterocytes to modulate nutrient absorption. In particular, the gastrointestinal peptide, GLP-2, and the ethanolamide lipid, Oleoylethanolamine (OEA) have each been reported to modulate fatty acid (FA) uptake by enterocytes (Yang et al., 2007; Hsieh et al., 2009). Conversely GLP-1, a product of the same gene as GLP-2, has been shown to decrease intestinal FA absorption (Hsieh et al., 2009; Mellitzer and Gradwohl, 2011).

The mechanism responsible for GLP-2—stimulated absorption involves increased expression and activity of the FA-sensing receptors cluster of differentiation CD36 protein (CD36); a membrane bound protein that potentiates cellular fatty acid uptake (Hsieh et al., 2009; Mellitzer and Gradwohl, 2011). Interestingly, CD36 has also been reported to be involved in orosensory, intestinal, and neuronal sensing of FA (Laugerette et al., 2005; Schwartz et al., 2008).

Cholecystokinin (CCK), often referred to as the archetypal gut hormone, is a key intestinal peptide secreted by small intestinal enteroendocrine cells in response to ingested nutrients (McLaughlin et al., 1998, 1999; Dockray, 2012; Rehfeld, 2017). Release of CCK into the circulation is primarily triggered by the presence of fatty acids or peptides in the intestinal lumen (Liddle et al., 1986; Cordier-Bussat et al., 1997; McLaughlin et al., 1999). Membrane-bound G protein coupled receptors resident on enteroendocrine cells transduce nutrient signals resulting in activation of second messenger cascades and secretion of CCK (Liou et al., 2011).

CCK is synthesized as a 115 amino acid prepro hormone, processed, then released in several forms that vary in peptide chain length and which are derived from a 58 residue peptide. The reported bioactivity of CCK resides in the C-terminal heptapeptide (Rehfeld, 2017). CCK's several actions are geared to promoting digestion of nutrients in particular ingested fatty acids. Once released, CCK slows gastric emptying, stimulates secretion of pancreatic digestive enzymes, and stimulates the release of bile by triggering contraction of the gall bladder and relaxation of the sphincter of Oddi. Centrally, CCK induces satiety, an action mediated via stimulation of the vagus nerve (Dockray, 2012). These actions are mediated by CCK receptors, of which two have been characterized: CCK-R_A and CCK-R_B (Kopin et al., 1992; Noble et al., 1999). CCK-R_A has approximately 100-fold higher affinity for CCK over gastrin compared to the CCK-R_B (Kopin et al., 1992; Wank et al., 1992; Dufresne et al., 2006).

Surprisingly, there is a scarcity of published research pertaining to a possible action of CCK to promote nutrient uptake, although indirect evidence is apparent that may point to this action (e.g., King et al., 2015). Given that CCK is secreted in response to fats we reasoned that CCK, like GLP-2 or OEA, may induce effects that facilitate FA absorption (McLaughlin et al., 1999). Therefore, the aim of the research described herein was to determine whether CCK modulates FA uptake into mouse primary duodenal enterocytes and if so to determine the mechanism responsible for this action. To assess these proposals we developed a robust fluorimetric assay and measured uptake of labeled-FA into mouse primary enterocytes.

The aim of the current research was to determine whether CCK modulates FA uptake by primary duodenal cells and if so to determine the mechanism of this action. Exposing mouse duodenal primary enterocytes to a physiological dose of CCK reproducibly caused a robust increase in C12 FA uptake. Irrespective of the genetic background of the mice strain, CCK reproducible induced increased FA uptake. Therefore, like GLP-2 and OEA CCK has the capacity to potentiate fatty acid uptake into enterocytes. This newly discovered function compliments the established actions of CCK that are geared for optimal and efficacious absorption of FA. Furthermore, our findings suggest that CCK released from enteroendocrine cells in the duodenum has the potential to act locally, in a paracrine manner, on neighboring enterocytes (see below).

Intriguingly, CCK showed a narrow window of activity compared to that reported by others in different tissues to duodenum (e.g., Simasko et al., 2002). We employed a CCK concentration of 10 pM because our concentration-response experiments showed this gave a statistically significant increase in Bodipy-FA uptake. Although, we observed some dose dependency, the levels of apparent increase did not reach statistical significance. At 10 pM, CCK is at the bottom end of the CCK-R_A binding curve although this concentration is capable of inducing near maximal increase in intracellular Ca²⁺ (Dong et al., 2014). Currently, the reasons why higher doses of CCK did not give statistically significant increases remain obscure. Taking a methodological view, conducting independent experiments for each dose would result in more concentrations being deemed statistically significant. On the other hand and from a physiological standpoint, there is evidence of CCK receptor heterogeneity (Morton et al., 2002), and CCK receptor function is modified by other cellular factors including membrane lipids (Potter et al., 2012). These reports indicate that tissue and cellular heterogeneity may underpin observed functional nuances that require careful consideration when designing future experiments to investigate CCK receptor function.

Having revealed a hitherto undescribed action of CCK we sought to determine the mechanism responsible for this effect. To do so we first determined which of the two described CCK receptors, if any, were responsible for transducing the hormone signal. These experiments showed that the selective CCK-R_A receptor antagonist Loxiglumide abolished the CCK-induced increase in FA uptake indicating that the CCK-R_A receptor was necessary for the observed response. Taken on face value this finding would suggest that the CCK-R_A receptor is expressed on enterocytes and CCK binding to this receptor triggered an intracellular response leading to an increase in FA uptake. The currently available expression data contained within the public databases accessed through NCBI or proprietary applications such as, Genevestigator record “medium” levels of CCK-R_A transcripts in mouse or human duodenum (Laule et al., 2006). Also, in humans CCK-R_A mRNA has been shown to be present in duodenal mucosa (Weinberg et al., 1997; Funakoshi et al., 1999). Thus, mRNA encoding CCK-R_A has been detected in duodenum epithelial cells, therefore corroborating our functional studies indicating the presence of the CCK-R_A receptor. An alternative mechanism is also plausible: It is conceivable that CCK may have acted indirectly via cells other than enterocytes and triggered release of an intermediary message that in turn propagated the observed increase in FA uptake. Considering this second possibility, the primary duodenal cells used in the current study comprised of mainly enterocytes and relatively low levels of other cell types (enteroendocrine ~1%, goblet ~10% and tuft cells ~1% Gerbe et al., 2012; Simmons et al., 2015), therefore it is possible, but untested that CCK may act via the CCK-R_A to stimulate release of a factor that in turn stimulated enterocytes.

Throughout the course of the experiments described herein, OEA and GLP-2 were used as positive controls to ensure viability of the primary cells in the Bodipy FA uptake assay. OEA or GLP-2 also stimulated FA uptake into enterocytes and interestingly CCK was not required for this action because when FA uptake experiments were carried out in cells from CCK knockout mice the stimulatory activity of both agents remained intact. Therefore, CCK is not a requirement for OEA or GLP-2 to bring about increase in FA uptake.

CD36 is a major potentiator of cellular FA uptake, is expressed in the intestinal epithelia (Lobo et al., 2001; Abumrad and Davidson, 2012) and is implicated in FA sensing (Nassir et al., 2007). We therefore set out to establish whether CD36 was involved in CCK-induced increased FA uptake. Two known CD36 inhibitors were employed; phloretin, a relatively broad-spectrum inhibitor and SSO, a CD36 selective inhibitor. Both reagents blocked CCK-stimulated FA uptake indicating that CD36 played a critical role in observed response (Ibrahimi et al., 1996; Coort et al., 2002; Pohl et al., 2005; Schwenk et al., 2010). Our findings fit well with those of Hsieh et al. (2009) who reported GLP-2-induced lipid uptake required CD36. Previously, CD36 has also been reported to participate in OEA-stimulated FA uptake (Yang et al., 2007) which, when considered alongside the findings of the current study and those of Hsieh et al. (2009), raises the possibility that CD36 acts as a point of integration for several gut hormones.

A consistent observation in the current study was that phloretin reduced FA uptake to below control levels whereas SSO did not. Because phloretin is relatively broad spectrum inhibitor, for example phloretin inhibits both glucose transport by SGLT proteins (Wright et al., 1991) and urea transport by UT urea transporters (Smith et al., 1995), this finding suggests that it may inhibit other FA transporters. For example, fatty acid transport protein 4 (FATP4) is expressed in duodenum and inhibited by phloretin (Masson et al., 2010; Kazantzis and Stahl, 2012) and inhibition of this transporter by phloretin likely explains the reduced uptake seen in phloretin treated samples to levels below those observed in control cells. Additionally, because SSO did not reduce uptake to below control values, CD36 is likely to be solely responsible for mediating the observed CCK-induced increase in enterocyte FA permeability.

In summary, the present work suggests that CCK plays a role in duodenal FA absorption. This conclusion raises the interesting possibility that CCK secreted by enteroendocrine cells embedded within the intestinal epithelium acts in a paracrine manner to stimulate adjacent enterocytes resulting in increased FA uptake. This is not a new concept with respect to enteroendocrine cell hormones and is one that has been suggested by others (Newberry and Davidson, 2009). Importantly, recent morphological studies have revealed structures that may facilitate this action: By utilizing exquisite imaging methods in mouse Liddle et al. have characterized granule containing processes that emanate from ileal and colonic PYY cells. These projections protrude from the basolateral surface of enteroendocrine cells and appear ideally positioned to enable paracrine communication between cells in the local environment (Bohórquez and Liddle, 2011, 2015; Bohórquez et al., 2011, 2015). Whether these structures are present in duodenum or participate in paracrine signaling remain to be determined.

The proposition that CCK induces an increase in enterocyte FA to facilitate nutrient absorption is supported by reported findings in CCK-KO mice (King et al., 2015). In response to acute intraduodenal introduction of lipids mice lacking CCK were observed to have delayed secretion of Apo B48-chylomicrons and lipid transport to the lymphatic system. Both of these effects are indicative of impaired FA uptake into enterocytes compared to wild type animals and support a physiological role for CCK-induced lipid absorption (King et al., 2015).

In conclusion, to the list of established functions of CCK can be added stimulator of duodenal enterocyte FA uptake. This action is mediated by the CCK-R_A receptor and CCK induced FA uptake is carrier mediated by CD36. In part our findings help explain defective lipid uptake in CCK knockout mice and are worthy of future consideration along with the reported actions of GLP-1 and OEA when considering hormone induced intestinal lipid absorption.

CD: performed the majority of the experiments and processed data. Grants held by CS and JM funded the work. CS performed some experiments. JM and CS conceived the experiments. CS and JM prepared the manuscript.