Cellular mechanisms governing Glucose-dependent insulinotropic polypeptide secretion


Frank Reimann, Eleftheria Diakogiannaki, Catherine E Moss, Fiona M. Gribble

From the Wellcome Trust/MRC Institute of Metabolic Science (IMS), University of Cambridge, United Kingdom






Corresponding authors:
Frank Reimann and Fiona M. Gribble
Wellcome Trust/MRC Institute of Metabolic Science (IMS)
MRC Metabolic Diseases Unit 
University of Cambridge,
Addenbrooke's Hospital, Hills Road
Cambridge CB2 0QQ,
Tel: 0044-1223-746796 or 336746
email: fr222@cam.ac.uk or fmg23@cam.ac.uk



Abstract:
Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone secreted from the upper small intestine, which plays an important physiological role in the control of glucose metabolism through its incretin action to enhance glucose-dependent insulin secretion. GIP has also been implicated in postprandial lipid homeostasis. GIP is secreted from enteroendocrine K-cells residing in the intestinal epithelium. K-cells sense a variety of components found in the gut lumen following food consumption, resulting in an increase in plasma GIP signal dependent on the nature and quantity of ingested nutrients. We review the evidence for an important role of sodium-coupled glucose uptake through SGLT1 for carbohydrate sensing, of free-fatty acid receptors FFAR1/FFAR4 and the monoacyl-glycerol sensing receptor GPR119 for lipid detection, of the calcium-sensing receptor CASR and GPR142 for protein sensing, and additional modulation by neurotransmitters such as somatostatin and galanin. These pathways have been identified through combinations of in vivo, in vitro and molecular approaches.  

Introduction:

GIP was originally isolated from the proximal small intestine as a peptide inhibiting gastric acid secretion, and was accordingly named gastric inhibitory peptide [1]. However, based on its sequence homology to other secretin family members known to stimulate insulin secretion it was hypothesised that GIP might actually be an incretin, a hormone released from the intestine in response to oral, but not intravenous, glucose, that stimulates insulin secretion, and indeed it was subsequently shown that exogenous GIP administration together with glucose had an insulinotropic effect [2]. The effect on gastric acid production, whilst clear in dogs, was only seen at very high (supraphysiological) concentrations in man, so the peptide was renamed to Glucose-dependent insulinotropic polypeptide, thus keeping the GIP acronym (see [3] for a historical review). 
Soon after the amino acid sequence of the GIP peptide was published [1] (a minor correction to the sequence reducing it from 43 to 42 amino acids was published later [4]), antisera were raised and used to identify the cellular origin of this hormone. Enteroendocrine cells (EECs) found in the duodenum and slightly less frequently in the jejunum [5] staining for GIP were originally thought to be a population known as D1-cells, but were subsequently identified as K-cells, both named to distinguish them from other EECs which differ in their vesicular appearance in “ultrastructural” electron-microscopic preparations, with some other EECs already named after the hormones associated with them, such as the gastrin producing G-cell [6, 7]. In recent years the classification of EECs into many different cell types based on vesicular appearance after diverse staining techniques and correlation with one predominant hormone has, however, been challenged, based on at the time unexpected co-localization of hormones, such as GIP and the sister incretin glucagon-like peptide-1 (GLP-1) occasionally in the same cells [8, 9], and even more promiscuous hormone expression profiles observed in transgenic animals expressing genetic tags in specific hormone expressing cells [10, 11]. Indeed, EECs are now thought to change their expression profiles whilst maturing along the crypt-villus or crypt-surface epithelial axis [12-14]. Nonetheless, hormones have preferential expression profiles along the proximal to distal gut axis and GIP is found predominantly in the very proximal small intestine, the duodenum and jejunum [15, 16]. In mice the overlap of GIP with GLP-1 in the same cells is relatively limited, such that most duodenal GLP-1 expressing cells do not co-express GIP and vice versa [17, 18] and we will continue to use the K-cell terminology for GIP-expressing cells throughout this review, in which we will focus on the sensing of secretory stimuli by these cells. K-cells are also reported to secrete another hormone, xenin [19, 20], a 25 amino acid peptide thought to act as a neurotensin receptor agonist [21] and identical in sequence to the N-terminal end of coatomer subunit alpha, a cytosolic protein involved in Golgi-ER trafficking. We were unable to detect the active xenin-25 fragment in whole tissue extracts by mass spectroscopy (LC-MS/MS) even though we readily detected other known enteroendocrine hormones and neuropeptides expressed in the enteric nervous system [16]. Whilst we do not exclude that the method employed in our LC-MS/MS study, which involves digestion in guanidinium chloride and acetonitrile precipitation to enrich for small peptide fragments without the need of protease digestion, which might release non-physiological peptides from larger proteins, is unsuitable for xenin, we will not further discuss it in this review.  

Sensing of macro-nutrients by K-cells

Total GIP and active GIP1-42 levels in the circulation rise rapidly following food ingestion, or when nutrients are infused into the duodenum. This is likely reflecting secretion of active GIP1-42, as although this is rapidly inactivated to GIP3-42 by dipeptidyl-peptidase-4 (DPP4), most assays in common usage report a rapid rise in serum/plasma concentrations even though they do not distinguish between these two forms of GIP. Infusion studies in humans have demonstrated that GIP secretion is strongly determined by the rate of nutrient infusion (e.g. of glucose), suggesting that a rise in circulating GIP levels predominantly reflects the rate of nutrient delivery from the stomach into the duodenum, and hence the rate of gastric emptying [22] although the glucose absorption rate in the duodenum/jejunum will also affect the signal [23]. As described below, a range of different nutrient and non-nutrient components have been found to modulate GIP secretion in different model systems. 

Carbohydrates:

Oral, but not intravenous glucose was shown to be a strong stimulant of endogenous GIP secretion as early as 1974 [24], which led to its classification as an incretin, as exogenous GIP had also been shown to stimulate insulin secretion [2]. In the following decade it became clear that carbohydrates that were substrates for sodium-dependent glucose transporters (SGLT; Slc5a1), such as glucose, galactose and even non-metabolisable glucose analogues such as -methyl-glucopyranoside (MDG) were good GIP stimulants and that inhibition of active sodium-dependent glucose uptake with phloridzin prevented glucose stimulated GIP release [25]. We revisited this once we had established mixed intestinal epithelial cultures and fluorescently labelled K-cells [26]. When cultures were co-treated with forskolin and IBMX to elevate cytosolic cAMP levels, glucose and MDG were reliable stimulants of GIP secretion (2-3-fold stimulation compared to forskolin/IBMX alone) and these responses were abolished by phloridizin. SGLT1 is restricted to the apical surface of K-cells [26] and thus ideally placed to respond to luminal changes in glucose concentration – comparable to our previous observations in GLP-1 secreting L-cells [27, 28] - and we concluded that direct depolarization through coupled glucose and sodium uptake into K-cells resulted in activation of voltage gated Ca2+-channels, with the resulting rise in cytosolic Ca2+ triggering GIP secretion. This mechanism is supported by the fact that SGLT1 knock-out animals lacked the rapid rise in plasma GIP levels normally observed after glucose ingestion [29]. Interestingly, even at later time points (up to 2 h after oral glucose challenge), no GIP response was observable in SGLT-1 knock-out animals. This contrasts  with GLP-1 responses, which whilst compared to wild-type animals are reduced at early time points (<15 minutes after glucose ingestion) are exaggerated at later time points (1 and 2 h). Late GLP-1 responses probably reflect delivery of glucose to the more distal intestine where it could stimulate a greater number of L-cells presumably after being converted to SGLT-1 independent stimuli such as short-chain fatty acids by the microbiota [30]. This differential effect of SGLT1 knockout, causing GIP inhibition but GLP-1 elevation following an oral glucose tolerance test, is also observed in human volunteers pre-treated with a mixed SGLT-1/2 inhibitor [31]. 

Despite good evidence for a predominantly SGLT-1 dependent mechanism, other possible glucose sensing pathways have been suggested to play a role in GIP secretion. The finding that K-cells express all the machinery employed by the pancreatic beta cell, including glucokinase [32], which channels glucose into glycolysis to eventually result in closure of ATP-sensitive potassium (KATP) channels, that otherwise keep beta-cells hyperpolarized thus preventing activation of voltage-gated Ca2+-channels under conditions of low ambient glucose, suggested that this pathway is also employed for glucose sensing in K-cells. Indeed tolbutamide, a KATP-channel inhibitor, stimulated GIP secretion from mixed epithelial cultures, but this effect was lost in forskolin/IBMX treated cultures [26]. As tolbutamide also affects Epac2 [33], a cAMP sensor expressed in K-cells, stimulation of GIP-secretion might be an “off-target” effect with respect to the KATP-channel. In wild-type mice another sulfonylurea, glimepiride, failed to elicit a GIP-response [34], although the authors observed some role for KATP channels in GIP secretion in streptozotocin treated diabetic mice [34]. However, no evident change in the GIP response to an oral glucose tolerance test was observed in patients with type 2 diabetes after initiation of sulfonylurea treatment [35]. Work with STC-1 derived cell lines, which express high levels of GIP [36], has also disputed any role of KATP-channels in GIP secretion, but, although the particular cell lines employed in these studies lacked any glucose-stimulated GIP secretion, a metabolic sensing mechanism involving AMPK-dependent kinase and Ca2+-release from intracellular stores was postulated [37-39]. It seems the KATP-channel, whilst expressed in K-cells, plays at best a modulatory role in glucose-stimulated GIP secretion. 

GIP-secretion was also reported in response to sucralose, an agonist on the sweet taste receptor (Tas1R2/3), from GLUTag cells [40], which however do not express high levels of GIP [10, 36]. This effect was blocked by the Tas1R2/3 antagonist gurmarin, and the sweet taste receptor was thus put forward as a candidate luminal glucose sensor for incretin release [40]. In our laboratory we do not see any significant GIP response to sucralose from primary intestinal cultures [26] nor is there any significant contribution of this mechanism to incretin release in the perfused rat intestine [41], and most reports do not support an acute GIP (or GLP-1) secretory response to artificial sweetener intake in man (for example [42, 43]). A luminally facing sweet taste receptor seems also difficult to reconcile with the lack of a GIP response in SGLT1 knock-out animals, given that glucose would linger around at the luminal side for longer in these animals, providing greater opportunity to stimulate such a receptor. However, others have come to different conclusions, as sweet taste receptor activation can upregulate SGLT-1 expression and there might be a complicated interplay between these pathways [44]. Theoretically it remains possible that there is a complex interaction in which sweet taste receptor activation only stimulates GIP (or GLP-1) secretion when the EECs are concomitantly depolarized by SGLT1-dependent glucose uptake – however, this is very different from the idea that Tas1R2/3 encodes the critical glucose sensing moiety in K-cells, and the selective sweet-taste receptor antagonist gurmarin did also not reduce glucose stimulated GIP secretion in the perfused rat small intestine [41].

There is mixed evidence for fructose, a (sweet) monosaccharide that is not a substrate for sodium-dependent glucose transport, as a stimulant for GIP secretion. K-cells do express the fructose transporter GLUT5 and fructose did modestly stimulate secretion in primary intestinal epithelial cultures, possibly reflecting a metabolic component of stimulation observable at basal cAMP levels and comparable to the tolbutamide triggered response under these conditions [26]. However, no significant fructose stimulated GIP secretion was observed in perfused intestinal preparations from rats [25] or after oral ingestion in rats, mice and humans [45], although some response was seen when fructose was administered orally to diabetic mice (after streptozotocin treatment) [46] or very obese leptin-deficient ob/ob mice [47]. Whether this latter observation has anything to do with a reported increase in K-cell density in this model [48, 49] or the reported over-secretion of GIP in obese humans in response to other stimuli including glucose and fat [50], has to our knowledge not been fully investigated. However, and in contrast, reduced GIP-responses to a mixed meal have been reported in morbidly obese individuals when compared to lean subjects, which could be correlated to delayed gastric emptying [51]. 

Lipids:

Even though GIP is considered an incretin, boosting insulin secretion in the context of elevated plasma glucose, when isocaloric intraduodenal lipid and glucose infusions are compared head to head, more GIP is secreted in response to the fat stimulus[52]; as blood glucose is not elevated under these conditions, this does not result in an elevation of plasma insulin [52, 53]. This finding, whilst not reproduced in every study (e.g. [54]), presumably reflects an important role of GIP in the control of adipocyte metabolism discussed elsewhere in this issue. Early investigations into the mechanisms underlying lipid stimulated GIP secretion established that only longer chain fatty acids (>C10) were effective stimulants and that non-saturated fatty acids were more effective compared with their saturated C18-counterpart [55]. Once it became clear that K-cells express G-protein-coupled receptors for long-chain free fatty acids (GPR40/FFAR1 and GPR120/FFAR4) [26], research focused on these, as has been reviewed previously (e.g.[56]). Some controversy still exists about the importance of these receptors, especially FFAR4, for GIP secretion; whereas Ekberg et al. reported, consistent with previous publications [57], a GIP secretory defect in response to oral olive oil in FFAR1 single-knock out mice, they failed to observe a similar defect in FFAR4 single knock-out mice [58]. By contrast, Iwasaki et al. reported a 75% reduction of GIP secretion in response to lard oil in FFAR4 knock-out mice [59], although, subsequently the group reported that the FFAR4 effect likely resided in cholecystokinin (CCK)-secreting I-cells, as CCK-replacement was able to recover corn oil induced GIP-secretion in FFAR4, but not FFAR1 single knock-out mice [60]. The effect on GIP secretion was proposed to be a reduction in lipid digestion, as the reduced CCK secretion would result in reduced bile and pancreatic lipase delivery to the intestinal lumen. Both groups, however, showed that FFAR1 and FFAR4 are highly expressed in K-cells and that double knock-out of the two receptors had a more profound effect than either single knock-out on lipid stimulated GIP secretion [58, 61]. In our laboratory we did observe a reduced responsiveness to a mixed corn oil/olive oil gavage in FFAR4 single knock-out mice in vivo (Figure 1a). As free fatty acids proved to be a fairly poor stimulus of GIP secretion when tested in isolation in mixed epithelial cultures [26], we investigated this further in vitro using more complex mixtures of lipids, aiming to simulate the composition of post prandial micelles (PPM), which contain bile acids and mono-acyl-glycerides in addition to free fatty acids. This mixture triggered a profound secretory GIP response in mixed epithelial cultures (Figure 1b), probably reflecting simultaneous stimulation of free fatty acid receptors and receptors for the other PPM components, like GPR119 (activated by mono-acylglycerides) and GPBAR1 (also known as TGR5 and activated by bile acids). These predominantly Gs-coupled receptors are also expressed in K-cells and synergy between Gs- and Gq coupled receptors for incretin secretion has been demonstrated [58]. Note, however, that raising cytoplasmic cAMP with forskolin and IBMX was insufficient to enable strong responses to free fatty acids in mixed epithelial intestinal cultures [26], suggesting that additional signaling pathways other than adenylate cyclase stimulation downstream of Gs might play a role. Interestingly, in this experimental setup both FFAR1 and FFAR4 single knock-out significantly reduced PPM-stimulated GIP secretion (Figure 1b/c). As, in this in vitro system, no change in bile or lipase availability would occur, fatty-acid dependent signaling of both FFAR1 and FFAR4 within K-cells seems likely, although we cannot exclude that paracrine cross-talk between different cells within the culture contributes to the observed effects. In L-cells, FFAR1 has been linked to activation of TrpC3 [56, 62], whereas FFAR4 has been linked to TrpM5 activation in linoleic acid stimulated CCK-release from the STC1 cell line [63], which also produces GIP and GLP-1. Future research should address whether FFAR1 and FFAR4 can recruit different signaling cascades in K-cells. 

In GLP-1 secreting L-cells, fatty acids appear to stimulate the cells from the basolateral direction, rather than the intestinal lumen facing apical cell surface, as a small molecule agonist of FFAR1 only stimulated secretion when applied from the basolateral/vascular side [64]. We are unaware of similar studies showing a dependence of lipid stimulated GIP secretion on lipid absorption, but interference with chylomicron formation by co-application of the surfactant pluronic L-81 largely abolished GIP-secretion in response to intraduodenal lipid application in rats [65]. It has been suggested that free fatty acids might be locally released from chylomicrons produced and secreted by nearby enterocytes [66], stimulating both GIP and GLP-1 secretion, although there was clearly also an FFAR1-independent sensing mechanism for chylomicrons observable in mixed epithelial cultures [66]. A dependence of GIP-secretion on fat absorption is further supported by studies interfering with monocaylglyceride-acyltransferase-2 (MGAT2) and diacylglyceride-acyltransferase-1 (DGAT1), two enzymes involved in the step-wise re-esterification of mono-acylglycerides to tri-acylglycerides inside the intestinal epithelium during lipid absorption. Both MGAT2 and DGAT1 knock-out animals show reduced GIP-responses to an oil gavage at all time points [67] and similar blunted GIP-responses were observed after pretreating mice with a DGAT1 inhibitor [68]. Lipid stimulated GLP-1 and PYY excursions in these models were more complex, with elevated plasma levels at later time points, presumably reflecting increased delivery of un-absorbed lipids to the distal intestine, where more of these hormones are produced, similar to the observations in SGLT-1 knock-out animals after a glucose gavage.   

 
Despite the apparent importance of the G-protein coupled receptors for sensing of long-chain fatty acids (FFAR1/4) and mono-acylglycerides (GPR119), some other lipid sensing moieties have been proposed. K-cells express, compared to the surrounding epithelial cells, higher levels of fatty-acid binding protein 5 (FABP5), an intracellular chaperone for lipophilic compounds, and FABP5 has been implicated in responses to oral lard [69]. Although the exact intracellular mechanism, dependent on solubilization of lipids by bile, remains unclear, FABP5 knock-out animals are partly resistant to HFD induced obesity, if to a lesser extent than GIP knock-out mice [69]. The fatty acid transporters cluster of differentiation-36 (CD36), also known as scavenger receptor class-B member-3 (SCARB-3), and fatty-acid transporter-4 (FATP4), have been implicated in CCK, secretin [70] and GLP-1 [71] release and are thus likely to play some role in GIP-secretion, possibly indirectly through effects on chylomicron formation. The scavenger receptor class-B member-1 (SCARB-1), implicated in intestinal cholesterol absorption, is also highly expressed in K-cells, but we have not been able to demonstrate a role of this receptor for K-cell lipid sensing (unpublished observations).

Protein:

Although protein and its digestion products are considered the less important macronutrient stimulus for GIP secretion, an early study showed that a mix of amino acids stimulated GIP secretion in man when perfused intraduodenally, but not when given intra-venously [72]. Recently it has been reported that the GIP response to intraduodenally infused hydrolyzed whey protein, also containing a mix of amino acids and small peptides, is more pronounced in older compared with younger healthy men [73]. In mice, a range of different amino acids has been found to stimulate GIP-secretion either in vivo in the leptin deficient ob/ob mouse, which shows exaggerated GIP-responses (alanine, arginine, cysteine, histidine, lysine and hydroxyproline) [74] or in mixed epithelial cultures (glutamine) [26]. Intraduodenal glutamine infusion (without the addition of other amino acids) also stimulates GIP secretion in man [75]. The underlying cellular sensing mechanisms for amino acids in K-cells have not been widely studied, but it seems likely that mechanisms identified in GLP-1 secreting L-cells could also be present in K-cells. These include active transport of di/tripeptides by PEPT1 (Slc15a1), an electrogenic H+-dependent transporter [76], and electrogenic Na+-dependent uptake of amino-acids for example via BOAT1 (Slc6a19) and SNAT2 (Slc38a2) [77], which could, in analogy to the glucose sensing mechanism via SGLT1, explain the selective stimulation by luminal, but not intra-venously supplied amino-acids. Note, however, that Slc6a19 knock-out mice have been reported to have exaggerated rather than reduced GIP responses to refeeding [78], which seems unexpected if BOAT1 would act similarly to SGLT1. Alternatively, amino acids can be sensed by G-protein coupled receptors expressed on K-cells. These include the calcium-sensing receptor (CASR), shown to be important for amino-acid sensing in L-cells [76, 79], and GPR142, for which a synthetic agonist elicited a strong GIP response when given orally, which was absent in GPR142 knock-out mice [80]. 

Non-nutrient regulators:

The K-cells also express a number of receptors for luminal constituents that may rise postprandially, but are not macronutrients. These include the G-protein coupled receptors for bile acids (G-protein bile acid receptor 1 (GPBAR1), also known as Takeda G-protein coupled Receptor 5 (TGR5)), and bile triggers a strong GIP secretory response in the perfused intestine [81]. Similarly, short chain fatty acids, products of bacterial fermentation, can be sensed by K-cells via the G-protein coupled free fatty acid receptors FFAR2 and FFAR3, but interestingly, this has been linked to inhibition of GIP secretion, predominantly through FFAR3 [82], and stimulation of GLP-1 secretion [82, 83], predominantly through FFAR2. 
In parallel to the direct postprandial sensing of luminal constituents by K-cells, GIP secretion is also under the control of other gut hormones. When purifying canine K-cells by elutriation, Kieffer et al. noted that the enteroendocrine-enriched cultures had a strong inhibitory somatostatin (SST) tone and SST-immuno-neutralization resulted in elevated GIP-secretion [84]. We confirmed SST to be a potent inhibitor of GIP secretion in mixed epithelial cultures, which could be partly blocked by a SST-receptor-5 antagonist, that by itself had, however, no effect in the absence of exogenously added SST in these cultures [85]. In the perfused pig intestine it was shown that GIP stimulates SST release [86], so it seems likely that there is a complicated cross talk and signal integration at the level of the intact epithelium. SST is also a neurotransmitter in the enteric nervous system (ENS) and modulation of K-cells by ENS in contrast to D-cell derived SST is also likely. Other inhibitory signals converging on K-cells include endocannabinoids, which inhibit GIP, but not GLP-1 secretion through cannabinoid (CB1) receptors [85], and galanin [87], another common neurotransmitter in the ENS, which inhibits both GIP and GLP-1 release. Gastrin-releasing peptide, also a neurotransmitter in the ENS, was shown to stimulate elutriated canine K-cells [84], however, mouse K-cells lack the cognate bombesin 2 receptor. Consequently in mice secretion of GIP is not stimulated by bombesin application, which, however, stimulates GLP-1 secretion in the same preparation [17]. Although K-cells express muscarinic receptors such as M4, there is little evidence for cholinergic modulation of GIP secretion [88]. Whilst the apparent over-secretion of GIP observed in obesity and type 2 diabetes was at one stage thought to reflect a defective feed-back inhibition by insulin [50], this might be of minor relevance and more related to the difference in meal size in obese volunteers [89]. 

Summary and Outlook:
GIP-secreting K-cells, found at highest numbers in the duodenum, are well placed to respond to luminal macronutrient rises after a meal. However, they do not just detect the presence of these components in the intestine, but secretion of GIP is closely coupled to nutrient absorption (Figure 2). This is either achieved by the use of the same molecular mechanism used to extract the nutrients from the lumen for detection, as in the case of SGLT1, or by expressing nutrient sensitive G-protein coupled receptors on the basolateral side, shielded from the lumen, but presumably exposed to high nutrient concentrations being released from enterocytes in their vicinity, as exemplified by FFAR1. The predominant location of K-cells in the proximal intestine in contrast to the more distal location of the sister incretin GLP-1, can result in different postprandial profiles, especially when digestion and/or nutrient absorption is compromised (as discussed above for SGLT1 inhibition) or delivery to the more distal intestine is accelerated, as after bariatric surgery [90]. These conditions have been shown to have little impact or even reduce GIP-secretion, whilst they enhance GLP-1 secretion.


Figure legends:

Figure 1: Effects of FFAR knock-out on lipid stimulated GIP secretion
a) Plasma hormone changes in response to an oral lipid tolerance test in wild-type (WT) and FFAR4 knock-out (FFAR4-/-) mice were examined as previously described [91]: In brief, mice were fasted overnight (<16 hr). Intragastric gavage of a 1:1 mix of olive:corn oils or phosphate buffered saline (control) was administered at 10ml/kg body weight. 25 min later, mice were anaesthetized with isoflurane, and terminal blood samples taken at 30 min by cardiac puncture. 
b) Hormone secretion from primary mixed murine duodenal cultures were performed as previously described [85]: In brief, supernatants were collected after a 2 h incubation in either control buffer (containing in mmol/l: 138 NaCl, 4.5 KCl, 4.2 NaHCO3, 1.2 NaH2PO4, 2.6 CaCl2, 1.2 MgCl2, 10 HEPES, pH 7.4 with NaOH and supplemented with 0.1 % bovine serum albumin) or control buffer with the addition of post-prandial micelles (PPM; containing: oleic acid (200 μM), 2-monooleoyl-glycerol (70 μmol/l), L-α-lysophosphatidylcholine (70 μM), cholesterol (17 μM) and taurocholic acid (700 μM)), or control buffer with the addition of forskolin and 3-isobutyl-1-methylxanthine (Fsk/IBMX; 10 and 100 μM, respectively). Remaining cells were also lysed and both supernatant and lysate GIP content was measured, enabling calculation of the % secretion of the total well content. (Left) Responses in cultures established from FFAR4 knock-out and matched wild-type animals. (Right) Responses in cultures established from FFAR1 knock-out and matched wild-type animals.
Plasma and culture GIP (supernatant and cell lysate) were assayed by ELISA (GIP Total ELISA Kit; Millipore, USA). FFAR1 [92] and FFAR4 [93] knock-out and matching wild-type tissues were kind gifts from AstraZeneca. Statistical analysis was performed in Graphpad Prism version 8 with a one-way ANOVA and Sidak’s multiple comparison compensation. **** p<0.0001, *** p<0.001 compared to control in the same genotype; #### p<0.0001, ## p<0.01 compared to the same stimulus in the other genotype, as indicated.

Figure 2: Schematic of K-cell stimulation
Macronutrients, broken down by digestion in the intestinal lumen into monomeric components, either stimulate K-cells directly from the luminal side through electrogenic uptake into K-cells, via sodium-coupled glucose (SGLT1) or amino-acid (BOAT1) transport or proton coupled di/tri-peptide (PEPT1) transport, triggering changes in the membrane potential () and voltage gated Ca2+ entry. The same transporters also play key roles in absorption of these nutrients into the interstitial space, where they can stimulate G-protein coupled receptors, such as the calcium sensing receptor (CASR) or GPR142, both sensitive to amino acids. Triglycerides are absorbed as long chain free fatty acids (LCFA) and mono-acyl-glycerides (MAG) and released after re-esterification in chylomicrons. Free-fatty acids released at the basolateral side of enterocytes, possibly in chylomicrons, stimulate their respective G-protein coupled receptors (FFAR1 and 4) on the basolateral side of K-cells. See text for further explanation.

Acknowledgements:
Research in the Reimann/Gribble laboratory is currently funded by the Wellcome Trust (106262/Z/14/Z, 106263/Z/14/Z) and the Medical Research Council (MRC_MC_UU_12012/3). CEM was funded by a BBSRC-iCase PhD studentship in collaboration with Astra Zeneca. We would like to thank Carol Lenaghan and David M. Smith for provision of FFAR1 and FFAR4 knock-out- and corresponding wild-type tissue. 

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