Physiology & Behavior 105 (2011) 413

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Physiology & Behavior

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b

Gustatory and extragustatory functions of mammalian taste receptors

Maik Behrens , Wolfgang Meyerhof
Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114116, 14558 Nuthetal, Germany

a r t i c l e

i n f o

a b s t r a c t
An ever increasing number of reports about taste receptors in non-gustatory tissues suggest that these molecules must have additional functions apart from taste. Of the extraoral tissues expressing taste receptors, the gastrointestinal tract received particular attention since evidence is mounting that tastants after being ingested might exert important regulatory roles in digestive and metabolic processes. At present, the G protein-coupled taste receptors for sweet, umami and bitter stimuli along with taste-related signaling molecules have been investigated in various parts of the alimentary canal. While the mechanism linking the gastrointestinal activity of sweet compounds via the activation of sweet taste receptors to metabolic adjustments has been worked out in some detail, other taste receptor mediated gastrointestinal activities are less well understood. The present article summarizes current knowledge on mammalian G protein-coupled taste receptors as well as various aspects of their proposed role in gastrointestinal tissues. 2011 Elsevier Inc. All rights reserved.

Article history: Received 21 December 2010 Received in revised form 1 February 2011 Accepted 7 February 2011 Keywords: Taste perception G protein-coupled receptors Gastrointestinal tract

1. Introduction The rst reports on the discovery of G protein-coupled taste receptors already provided hints that these receptors, although prominently expressed in taste tissue, are not restricted to the oral cavity [13]. This statement can be extended to taste signaling elements such as -gustducin [4], which was soon after its discovery in taste tissue detected in cells of the upper digestive tract [5]. Over the recent years the number of reports about taste signaling components found in extraoral tissues has grown steadily, including respiratory epithelia [6], testis [7] and, as already mentioned, the gastrointestinal (GI-) tract [5]. Hence, it is evident that molecules that act as tastants in the oral cavity through the activation of taste receptors may serve as agonists for the very same receptors also in extraoral tissues. Although it is not always possible to avoid terms such as tastant, taste-like signaling, etc. when describing nongustatory mechanisms involving those molecules, it is understood that taste perception per se is restricted to the oral cavity. Within the oral cavity of mammals the initial evaluation of the quality of consumed food prior to its ingestion takes place. The gustatory system distinguishes the ve basic taste qualities sour, salty, sweet, umami (the taste of L-amino acids and ribonucleotides), and bitter (for a recent review see Ref. [8]). In addition to the mentioned basic taste qualities the existence of further, perhaps in part speciesspecic, taste qualities such as fatty, metallic, or the tastes of maltodextrine, carbon dioxide, etc. are discussed in the literature. Three of the basic taste qualities, sweet, umami, and bitter, are

Corresponding author. Tel.: + 49 33200 88 545; fax: + 49 33200 88 384. E-mail address: behrens@dife.de (M. Behrens). 0031-9384/$ see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.02.010

mediated by G protein-coupled receptors (GPCRs) belonging to the TAS1R and TAS2R gene families. In fact, most reports on taste receptor gene expression outside the oral cavity focus on TAS1R and TAS2R genes and/or downstream signaling molecules associated with them, whereas the presence of acids and (sodium-) salts in the alimentary tract has not resulted in a comparably high number of studies trying to associate the sensing of these stimuli with taste-related mechanisms. The apparent bias towards the investigation of sweet, umami and bitter sensing taste receptors in GI tissues may be explained by certain difculties associated with in particular the sour and salty taste receptor molecules: 1.) although a number of candidate sour-taste transduction mechanisms have been proposed in the past [916], the exact identity of the sour taste receptor is still not established. 2.) In case of the salt taste receptor it appears that, nally, after many years of speculation one of the candidate receptors proposed for rodent [17] and human [18] salt taste perception, the epithelial sodium channel (ENaC), was unequivocally veried [19]. This channel (see Ref. [20] for review) is involved with numerous transepithelial sodium transport reactions so that its mere presence in non-gustatory tissues does not provide hints to speculate about taste-like sensory function. Therefore, we have not included these surely interesting taste qualities in the present manuscript. Within the GI tract presence of sweet, umami, and bitter taste receptors as well as taste-related signaling molecules has been demonstrated in the past years [5,2128]. The cellular localization of these molecules then allowed the identication of types of GI cells suspected to utilize taste cell related mechanisms for nutrient sensing. It turned out that the cell population expressing taste-related molecules is, perhaps partly due to species-related differences [29], rather heterogeneous including enteroendocrine cell types as well as brush cells/solitary chemosensory cells [21,23,24,27,30,31].

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Therefore, several researchers turned their attention to the investigation of model cell lines that allowed the characterization of homogeneous cellular models for functional consequences of GI nutrient sensing [2224,2628,30,32,33]. As one would predict for a research eld involving so many different types of tissues, cell types, and taste receptors putatively involved in nutrient sensing, the proposed physiological roles of taste receptor signaling in the gut are complex. They range from the modication digestive processes such as the speed of gastric emptying [34] to metabolic adjustments like the regulation of blood glucose levels [24]. The mechanisms by which taste stimuli present in the gut lumen may involve local paracrine, humoral, and neuronal transmission events [24,26,35,36]. 2. Taste system The mammalian sense of taste plays an important role in the evaluation of the quality of the consumed food. Food components within the oral cavity are monitored for caloric content, ripeness, bacterial spoilage, presence of minerals, and potential poisonous substances prior to ingestion. For their characterization the main nutritionally relevant food components are associated with ve basic taste qualities: sweet taste is elicited by carbohydrates in the form of mono- and disaccharides, an important energy source for the organism. Similarly, umami taste is associated with L-amino acids (in humans predominantly L-glutamate and ribonucleotides) indicative of protein-rich foods. Salt taste, which is mainly elicited by sodium ions, is important for electrolyte homeostasis of the body. Sour indicates unripe fruits or food that is potentially spoiled. Finally, bitter taste is elicited by numerous toxic plant metabolites and thus, believed to protect animals from the ingestion of poisonous substances. The ve taste qualities are detected by specialized taste receptor cells located in the oral cavity. Here, taste receptor cells occur in morphological structures of 50100 cells, called taste buds. The majority of taste buds are found on the tongue surface embedded in gustatory papillae, on the soft palate and on the throat [37]. Within each taste bud a variety of specialized cells are found, which can be classied by functional and morphological characteristics. The type II cells express taste receptors of the GPCR type, type III cells form synapses with afferent nerve bers, type I cells are believed to have supporting functions and type IV cells, located at the basal parts of taste buds, are a pool of undifferentiated precursor cells devoted to replace the other cell types throughout life [38]. It is well established that type II cells can be subdivided into three different subpopulations each specialized to the detection of a single taste quality either sweet or umami or bitter depending on the taste receptor genes expressed [8]. The type III cells (also called presynaptic cells), which are the only cell type forming synapses with afferent nerve bers and therefore are thought to fulll a higher order integrative function, are the sour sensors [39]. Also salt taste is mediated by a distinct subpopulation of intragemmal cells, the exact cell type, however, needs to be established [19]. 2.1. Signal transduction cascade in sweet, umami, and bitter taste The signal transduction pathway of taste receptors belonging to the GPCR family has been characterized in detail [40]. Although various components of the taste signaling cascade are predominantly expressed in taste receptor cells, the overall composition is typical for GPCR mediated signal transmission (Fig. 1). The activated taste receptor proteins interact with a heterotrimeric G protein complex consisting of a G-subunit such as Ggustducin [4], G-transducin [41], G14 [42], as well as G3 or G1, and G13 [43,44]. After activation by a taste receptor the complex dissociates and the G3/G13 heterodimer stimulates phospholipase C2 [4548] resulting in the generation of the second messengers
Fig. 1. Signal transduction cascade of G protein-coupled taste receptors. Activated taste receptors (here exemplied by a TAS2R) interact with heterotrimeric G proteins consisting of G-gustducin, G3 or G1, and G13. After dissociation of the G protein subunits, the G-subunits activate the phospholipase C2, which, in turn, produces diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). IP3 facilitates the release of intracellularly stored calcium ions from the endoplasmic reticulum (ER) via type-III IP3-receptor activation. Finally, the elevated intracellular calcium level results in the opening of a non-selective kation-channel, the transient receptor potential channel (TRPM5) causing cell depolarization. Please note that the cyclic AMP signaling, which is initiated by the activated G-gustducin subunit of the heterotrimeric G protein, has been omitted.

diacylglycerol and IP3. Next, the activation of the type III IP3-receptor residing within the endoplasmic reticulum membrane [49] results in an increase of the intracellular calcium ion level, which, in turn leads to the opening of a transient receptor potential channel, TRPM5, located in the plasma membrane [48,50,51]. The activation of this non-selective cation channel nally results in cell depolarization coinciding with elevated intracellular calcium levels causing the extrusion of ATP, the transmitter substance of type II taste receptor cells through pannexin-hemichannels [52,53]. The ATP released by type II taste receptor cells has several functions. Firstly, it stimulates afferent purinergic nerve bers terminating within the taste bud [54]. Secondly, ATP is also able to activate type III taste receptor cells resulting in the secretion of the neurotransmitters serotonin [53] and norepinephrine [55]. Thirdly, ATP acts in an auto- and paracrine fashion also on type II taste receptor cells [56]. The complex mechanism of signal integration within taste buds and the transmission of taste information to the brain are not fully understood and currently a matter of intense research. 2.2. G protein-coupled taste receptors Two gene families code for G protein-coupled taste receptors, the TAS1R gene family (taste receptor, type 1) with three members called TAS1R1, TAS1R2, and TAS1R3, and the TAS2R gene family (taste receptor, type 2) with numerous members called TAS2R(member number), depending on the species. The nomenclature, and connected to this, the approved gene symbols have been subjected to frequent changes in the past and present (Table 1). Especially the nomenclature of bitter taste receptors, which received in the early years after their discovery multiple gene symbols for the same genes, is difcult and even extends to the use of species specic rules (e.g. TAS2R for humans, Tas2r for mouse). Regarding their classication among the gene superfamily of G protein-coupled, heptahelical receptors, the three TAS1R genes are members of the class C GPCRs. The class C family of receptors that contains also the metabotropic glutamate receptors is characterized by their large extracellular amino termini. The TAS2R genes are either not grouped together with other GPCRs because they lack sufcient amino acid sequence homologies or loosely clustered with the frizzled receptor family [57]. 2.2.1. Sweet taste receptors The mammalian sweet taste receptor is composed of the two subunits, TAS1R2 and TAS1R3, forming a functional heteromer

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Table 1 Approved gene symbols and previous symbols and aliases for human and mouse G protein-coupled taste receptors. Sources: Hugo Gene Nomenclature Committee (www.genenames.org), International Committee on Standardized Genetic Nomenclature for Mice (www.informatics.jax.org/ mgihome/nomen/). Human Approved Previous symbols symbols and aliases Taste receptor, type 1 TAS1R1 T1R1, TR1, GPR70 TAS1R2 T1R2, TR2, GPR71 TAS1R3 T1R3 Taste receptor, type 2 TAS2R1 TAS2R3 TAS2R4 TAS2R5 TAS2R7 TAS2R8 TAS2R9 TAS2R10 TAS2R13 TAS2R14 TAS2R16 TAS2R19 TAS2R20 TAS2R30 TAS2R31 TAS2R38 TAS2R39 TAS2R40 TAS2R41 TAS2R42 TAS2R43 TAS2R45 TAS2R46 TAS2R50 TAS2R60 T2R1, TRB7 T2R3 T2R4 T2R5 T2R7, TRB4 T2R8, TRB5 T2R9, TRB6 T2R10, TRB2 T2R13, TRB3 T2R14, TRB1 T2R16 TAS2R48, TAS2R23, T2R19, T2R23 TAS2R49, T2R20, T2R56 TAS2R47, T2R30 TAS2R44, T2R31, T2R53 PTC, T2R61 GPR60 T2R59 T2R24, T2R55, hT2R55, TAS2R55 T2R52 ZG24P, GPR59 T2R54 T2R51 T2R60 Tas2r102 Tas2r103 Tas2r104 Tas2r105 Tas2r106 Tas2r107 Tas2r108 Tas2r109 Tas2r110 Tas2r113 Tas2r114 Tas2r115 Tas2r116 Tas2r117 Tas2r118 Tas2r119 Tas2r120 Tas2r121 Tas2r122 Tas2r123 Tas2r124 Tas2r125 Tas2r126 Tas2r129 Tas2r130 Tas2r131 Tas2r134 Tas2r135 Tas2r136 Tas2r137 Tas2r138 Tas2r139 Tas2r140 Tas2r143 Tas2r144 T2R102, mT2R51, STC9-7 T2R103, T2R10, TRB2 T2R104, mT2R45 T2R105, T2R5, T2R9 T2R106, mT2R44 T2R107, mT2R43, T2R4, STC5-1 T2R4, T2R8 T2R109, mT2R62 T2R110, mT2r57, STC 9-1 T2R113, mT2R58 T2R114, mT2R46 Mouse Approved symbols Tas1r1 Tas1r2 Tas1r3 Previous symbols and aliases

GPR70 T1R2, GPR71 T1R3

Fig. 2. Schematic of G protein-coupled taste receptors. Two gene families code for G protein-coupled taste receptors, the Tas1r genes and the Tas2r genes. The corresponding receptors possess 7 transmembrane domains (cylinders) that are connected by three intracellular and three extracellular loops. Functional umami and sweet taste receptors (left) are obligatory heteromeric complexes consisting of either, TAS1R1 and TAS1R3 (umami) or TAS1R2 and TAS1R3 (sweet). All TAS1R subunits have large extracellular domains at their amino termini forming ligand-binding venus ytrap motifs. In contrast to the TAS1Rs TAS2Rs (right) possess short amino termini and do not require heteromerization for function.

T2R116, mT2R56, TRB1, TRB4 T2R117, mT2R54 T2R16, T2R18, mt2r40 T2R119, T2R19 T2R120, mT2R47 T2R13, T2R121, mT2R48 T2R123, T2R23, mT2R55, STC9-2 T2R124, mT2R50 T2R125, mT2R59 T2R41, T2R12, T2R26 T2R129, mT2R60 T2R7, T2R6, T2R30, STC7-4, mT2R42 T2R134, T2R34 T2R135, T2R35, mT2r38 T2R136, T2R36, mT2r52 T2R3, T2R137, T2R37, mT2r41 T2R38, T2R138, mT2R31 T2R39, mT2R34 T2R140, T2R40, T2R8, T2R13, mT2r64, mTRB3, mTRB5 T2R143, T2R43, mT2R36 T2R40, mT2R33

synthetic origin. By functional heterologous co-expression of TAS1R2 and TAS1R3 in cell lines it was shown that indeed the TAS1R2/TAS1R3-heteromer was activated by all compounds known to taste sweet to humans (or attractive to rodents) including sugars, articial sweeteners, D-amino acids and glycine, sweet proteins and plant metabolites such as stevioside [60,6470]. However, it needs to be noted that species-related differences for sweet taste receptor activation have been reported. The most pronounced species difference was observed for the sweet taste receptor of cats, which has lost its function due to the pseudogenization of the gene coding for the TAS1R2 subunit [71]. Other species differences affect responses to a subgroup of sweet compounds such as aspartame, cyclamate, neotame, neohesperidin dihydrochalcone (NHDC) and the sweet protein monellin, which fail to activate the rat sweet taste receptor [66,69,70,72]. Similarly, the mouse sweet taste receptor is not responding to monellin, thaumatin, aspartame, glycyrrhizic acid and NHDC [73]. Thus, the human sweet taste receptor responds to a considerable number of additional compounds compared to its rodent counterpart. This observation is not limited to activators, but also observed for the sweet taste receptor antagonist, lactisole, which binds to the TAS1R3 subunit resulting in the inhibition of sweet and umami receptors [65,69,72]. Although the pharmacology of the TAS1R2/TAS1R3-heteromer sufciently explains most of the mammalian sweet taste perception, it should be mentioned here that some evidence suggests that also the homomeric TAS1R3 could act as lowafnity receptor for mono- and disaccharides [73] and that rodents may possess a receptor distinct from Tas1r2/Tas1r3 for the detection of polycose and other maltodextrins [74]. 2.2.2. Umami taste receptors As mentioned already in the previous section the umami taste receptor is composed of the TAS1R subunits TAS1R1 and TAS1R3. In contrast to the sweet taste receptor, additional umami taste receptors are proposed. These further umami receptors represent truncated forms of the metabotropic glutamate receptors, mGluR1 and mGluR4. In fact, the truncated variant of brain mGluR4, called taste mGluR4, was the rst candidate receptor for umami taste perception identied in taste tissue of rats [75]. Later, also a variant of mGluR1 was found to be present in individual cells of rat taste buds [76]. Although, as mentioned above, an involvement in umami taste sensation was proposed for several receptors, strong evidence supports the TAS1R1/ TAS1R3 heteromer as the predominant umami taste receptor. Of all candidates, the TAS1R1/TAS1R3 is not only activated by L-glutamate, but this activation is strongly increased in the presence of 5-ribonucleotides, a fact that is considered to be a hallmark of umami taste as for example explored in human psychophysical analyses [77]. Another well

(Fig. 2). Historically, the gene coding for the TAS1R2 subunit was identied rst, together with the gene for the TAS1R1 subunit, which is part of the umami receptor discussed in the next section [2]. It then took a few years to identify the gene for the third missing subunit, TAS1R3, and to elucidate the function of the heteromeric sweet taste receptor [5863]. On a cellular level, the TAS1R3 gene is coexpressed with both, TAS1R1 and TAS1R2, in subsets of taste receptor cells, however, TAS1R1 and TAS1R2 genes are expressed in a mutually exclusive fashion [2,59,60]. This expression pattern generates distinct subpopulations of either sweet- or umami-responsive taste receptor cells. An important question after the identication of the TAS1R2/ TAS1R3 heteromer as a sweet taste receptor was, if additional receptors for sweet tasting substances may be required to facilitate perception of the numerous sweet compounds of natural and

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documented feature of umami taste is its activation by the mGluR agonist AP-4 [78], which is also potentiated by 5-ribonucleotides [79,80]. Indeed, the TAS1R1/TAS1R3 responds not only to L-glutamate and L-AP-4, but also exhibits synergistically enhanced activity to both compounds in the presence of 5-ribonucleotides [68]. Moreover, genetic ablation of the Tas1r3 gene selectively abolishes sweet and umami taste perception of knock-out mice [73]. As observed for sweet taste, umami taste also shows signicant species differences. Whereas humans preferentially perceive L-glutamate as an umami tastant, rodents respond to most L-amino acids. Heterologous expression of chimeric umami receptors consisting of mouse Tas1r3 and mouse or human TAS1R1 recapitulate this species difference [73]. However, an independently generated Tas1r3 knock-out mouse model [81] exhibited, although strongly reduced, residual responses for L-glutamate as well as sweet stimuli, again supporting the presence of further umami receptors such as mGluR4. 2.2.3. Bitter taste receptors All vertebrate species investigated so far possess several or even numerous bitter taste receptor genes (TAS2Rs). Whereas avian and amphibian TAS2R genomes with 3 and ~ 50 genes, respectively, demarcate thus far the extremes with respect to functional TAS2R gene numbers, mammalian species exhibit average TAS2R repertoires lying between 15 and 36 genes [82]. Whether the considerable deviation of TAS2R gene numbers reects a differential importance among species or different pharmacological properties is presently unknown. After the initial discovery of TAS2Rs [1,3,83] most studies focused on the characterization of rodent and, even more, human TAS2Rs. Even though mammals possess numerous TAS2R genes, it has been an eminent question how such few receptors may possibly sufce to detect hundreds, if not thousands, bitter compounds present in nature [84]. Hence, the identication of bitter compounds specically activating TAS2Rs and the following pharmacological characterization has been, and still is, an important task in the taste research eld. Over the past decade, however, there has been a considerable bias towards the deorphanization and characterization of human versus rodent TAS2Rs, which represents now a major obstacle for research focusing on physiological roles of TAS2Rs in animal models. Whereas 80% (20 of 25) of the human TAS2Rs are deorphaned, only for 6% (2 of 33) of the mouse Tas2r genes agonists are known [83,85]. Therefore, it remains to be seen in the future, if the general pharmacological features established for the human TAS2R repertoire, may be applicable to other mammalian species or if modications are necessary. After the deorphanization of rst few TAS2Rs, it seemed as if they rather specically detect single or few bitter compounds such as cycloheximide (mouse Tas2r105; [83]), denatonium benzoate and 6-n-propyl-2-thiouracil (PROP) (mouse Tas2r108, human TAS2R4 [83]), strychnine (hTAS2R10, [86]) or groups of chemically related bitter compounds such as -D glucopyranosides (hTAS2R16, [86]). However, it soon became clear that some receptors are activated by various, chemically diverse bitter compounds (hTAS2R43, hTAS2R31, [87]) or even by a large proportion of all tested bitter substances (hTAS2R14, [88]). Although, during the recent years numerous receptors were added to the list of deorphaned TAS2Rs, and although some details needed to be modied, the overall picture for the human receptors was maintained. The recent screening of all 25 human TAS2Rs with more than 100 natural and synthetic bitter compounds conrmed that the breadth of tuning differs largely among the receptors [85]. The hTAS2R10 [86], hTAS2R14 [88] and hTAS2R46 [89] are broadly tuned receptor generalists recognizing about half of all tested substances if their activities are combined [85]. On the other end of the spectrum the specialist receptors hTAS2R3 [85], -R5 [85], -R8 [90], -R13 [85], -R20 [85], and -R50 [91] are found detecting only single or few compounds. Another group of receptors comprising of hTAS2R1 [92], -R4 [83], -R7 [93], -R9 [22], -R39 [85], -R40 [94], -R43

[87,95], -R31 [87], and -R30 [95] is activated by an intermediate number of bitter compounds. Two further receptors, hTAS2R16 [86] and -R38 [96,97], predominantly recognize compound groups with common structural motifs. Not only the breadth of tuning but also the sensitivity differs considerably among hTAS2Rs ranging from determined EC50-values in the mid nanomolar range as seen for the hTAS2R43 challenged with aristolochic acid [87] to the low millimolar range determined for hTAS2R16 responses to D-salicin [86]. Of course, one has to be aware that the pharmacological characterization of TAS2Rs is usually done in heterologous expression systems raising the question to which extend these data can be applied to the in vivo situation, e.g. the gustatory system, the GI system and beyond. From the analysis of functional gene polymorphisms in hTAS2R genes there is no doubt that on a qualitative level the function of an hTAS2R gene variant as measured in vitro mirrors the in vivo situation. The best example for this is the hTAS2R38 nontaster variant, which leads to a dramatic decrease in the ability to taste the bitter substances PTC and PROP of subjects homozygous for this variant [96]. This correlates with a loss-of-responsiveness observed in heterologous expression systems [97]. Additional examples are provided by functional polymorphisms in hTAS2R16 [98] as well as in hTAS2R43 and hTAS2R31 [90] or mouse Tas2r105 [83]. Owing to the fact that the human TAS2R gene family is highly variable [99,100], it appears likely that future research will provide additional examples, which may, however, sometimes become obscured by the considerable overlap in the pharmacological proles of hTAS2R genes. On a quantitative level the situation is more complex, since various possibly confounding inuences on hTAS2R function have been shown in vitro. The function of hTAS2Rs in vitro might depend on the presence of plasma membrane export-tags [101], protein glycosylation [102], or the presence or absence of co-factors [101,103]. Vice versa the situation in vivo might be different from the in vitro situation because in each taste receptor cell several receptor genes are co-expressed [104], form heterodimers [105] or the activation of TAS2Rs could be inuenced by perireceptor events such as the interaction with saliva [94,106]. Nevertheless, in general, there is a pretty good agreement between in vitro determined receptor responsiveness and human sensory data. For example the human detection threshold and EC50-values for D-salicin are 200 M and 1.1 mM, respectively. In vitro, the D-salicin-responsive receptor hTAS2R16 displays a threshold of 70 M and an EC50-concentration of 1.4 mM [86]. The recent discovery of a reasonable specic antagonist may provide novel tools for the analysis of TAS2Rs in vivo [107]. The compound 4-(2,2,3-trimethylcyclopentyl butanoic acid) (also named GIV3727) selectively inhibited six of 18 tested hTAS2Rs with different potencies, the most pronounced antagonism was observed for receptors hTAS2R40 and -R43. Such compounds may prove invaluable for dissecting the individual contributions of TAS2Rs in oral and extraoral bitter compound sensing. Moreover, a growing number of studies combining computational modeling of TAS2Rs with experimental approaches such as functional calcium imaging analysis of point-mutated receptor constructs, will provide an improved understanding of the structurefunction relationships underlying TAS2Ragonist interactions [108111]. This may ultimately pave the way for a rational design of more antagonists urgently needed for research. 3. Gastrointestinal system 3.1. Taste receptors in the gastrointestinal tract To date, most of the G protein-coupled taste receptor genes are reported to be expressed in the mammalian GI system as well. The rst receptors identied in GI-tissue were the Tas2rs in the stomach mucosa and duodenum of rats [27]. In fact, all of the randomly chosen rat Tas2rs analyzed by RT-PCR were found, indicating that not only a

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small subset of rat Tas2rs, but most likely the majority of these genes may have additional roles in the GI-tract. The expression of rat Tas2r genes appeared selective, since most, but not all of the tested Tas2r mRNAs were present in all of the different tissues. The same study demonstrated, again by RT-PCR analyzes the presence of mouse Tas2r genes in the corresponding GI-tissues and in mouse STC-1 cells, a mixed population of endocrine cells (see Section 3.4), indicating a conserved role in the gut. Subsequently, several additional rat and most mouse Tas2rs were detected at the RNA-level by RT-PCR in GItissues and cell lines of gastrointestinal origin [28,32,112,113]. Similar studies on human gut tissues and cell lines deriving from GI-tissues resulted in the identication of transcripts from most human TAS2R genes [22,30,112]. However, in the absence of in situ hybridization analyses specic cell types expressing the Tas2r genes have not been identied in these studies. To date, only one report provides a direct evidence for the type of GI-cells expressing TAS2Rs in vivo. In this study, the authors used an antibody apparently specic for mouse Tas2r138 for immunohistochemical staining of mouse proximal intestine [113]. Future work determining the specicity of this antibody in more detail such as staining of mouse gustatory papillae, mTas2r138 transfected cells, etc. should reveal whether this antiserum may represent the long missing tool allowing the characterization of bitter taste receptor expressing cell types in the gut in situ. Analog to the TAS2R gene family, also TAS1R genes were found in GI-tissues. The rst study already revealed the presence of all three Tas1r transcripts in the mouse intestinal mucosa as well as in the enteroendocrine cell line STC-1. The relative abundance, however, was shown to be different, with Tas1r2 and Tas1r3 being more abundant in the mid small intestine, whereas Tas1r1 mRNA levels were higher in distal portions of the intestine [23]. This would suggest a zonal segregation of sweet and umami receptors along the longitudinal axis of the small intestine. Results obtained by real time-PCR analyses of human GI-tissues, however, came to somewhat different conclusions. Although the apparent absolute amount of mRNAs for TAS1R1, TAS1R2, and TAS1R3, differed considerably among samples of stomach, duodenum, jejunum, ileum, and colon, the expression appeared rather stable along the longitudinal axis [21]. Interestingly, the authors of the latter study reported an approximately 100-fold difference each, among TAS1R3, the most abundant, TAS1R2, the least abundant, and TAS1R1, which is of intermediate abundance. Numerous reports corroborated and extended the initial observations with respect to the presence of TAS1R subunits [25,26,30,114116]. In addition to the TAS1R1/TAS1R3 heteromer, two further receptors for L-glutamate considered to play a role in umami taste, mGluR1 and mGluR4, are detectable in GI-tissues [114,117]. 3.2. Taste-related signaling components in the gut Long before taste receptor expression was investigated, the presence of the canonical taste signaling component -gustducin [4] in the stomach, duodenum and pancreatic ducts of rat was reported [5,31]. Later, -gustducin as well as the closely related transducin [21,25,27,116], which was shown to be expressed in taste buds in addition to -gustducin [41], was found in additional parts of the gut. More recently, additional molecules of the taste cell signaling cascade such as PLC2 [45,46] and TRPM5 [118] were localized in the GI-tract as well (Fig. 3). In view of the nding that most, if not all, taste signaling components are expressed within the GI-tract it appears that cells highly related to taste receptor cells are present in the mammalian gut. However, co-localization studies performed with various taste cell markers in GI-tissues revealed that cells expressing all taste transduction components together are rare. Using transgenic mice expressing green uorescent protein under the control of the TRPM5 promoter it was shown that colonic cells co-express TRPM5 and -gustducin, but not PLC2 [21]. Similarly, cells within the

Fig. 3. Expression of taste-related signal transduction components in gastrointestinal tissue. Cross-sections of mouse jejunum were stained for phospholipase C2. Positive, phospholipase C2 expressing cells (green, arrows) are scattered throughout the jejunal villi (A). Preabsorption of the primary antibody with the corresponding antigenic peptide suppress detection of PLC2 expressing cells conrming the specicity of the staining (B). Scale bar, 20 M. Microphotographs kindly provided by Mr. Simone Prandi.

duodenal villi, which co-express frequently TRPM5 and -gustducin, were rarely positive for PLC2. Although the overlap between TRPM5 promoter-driven GFP and PLC2 was high in duodenal glandular tissue, -gustducin seemed to be restricted to another population of GFP-positive cells [21]. 3.3. Cell types expressing taste-related molecules The majority of reports investigating taste-related molecules in mammalian GI-tissues associate expression of taste-related marker proteins with either one of two morphologically distinct cell types, enteroendocrine cells or brush cells. Brush cells possess, analogous to taste receptor cells of the tongue, a single apical tuft of rigid microvilli (for a review see Ref. [119]) and they were the rst cells outside the oral cavity shown to express -gustducin [5]. Enteroendocrine cells represent the second cell type commonly associated with taste signaling components in the gut. These cells occur throughout the GItract interspersed among mucosal cells [120]. Immunohistochemical staining of human colonic sections with -gustducin and chromogranin A, a marker for enteroendocrine cells, identied an gustducin-positive subpopulation of enteroendocrine cells [30]. Physiologically, enteroendocrine cells are by far not a homogeneous population, as they occur in numerous types distinguished by localization and the molecules they produce and secrete [120]. At present, it is rather difcult to rmly associate distinct cell populations with a taste cell-like function, e.g. relaying a signal provided by a cognate taste receptor agonist via taste receptor mediated signal transduction to a physiological response such as the secretion of a transmitter substance. There are multiple reasons for these difculties: 1. components of canonical taste-signaling (for a review see Ref. [40]) are used as surrogate markers for cells most

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likely expressing also taste receptors. While this, of course, reasonable assumption may apply to large fraction -gustducin- and TRPM5positive cells, the rather restricted overlap between PLC2-positive cells with other molecules characteristic for taste signaling [21] indicates that various subpopulations of GI-cells must exist, of which some, but not necessary all may express taste receptors. 2. Although numerous studies used RT-PCR analyses to investigate taste receptor expression in the gut, there is a lack of direct evidence for the expression of taste receptors in identied cells of GI-tissues. At present, only TAS1Rs [2426,115,116] and, by using an antibody with a, so far, unproven specicity, a single TAS2R [113] were detected in distinct cell populations of the rodent GI-tract. 3. For obvious reasons, experiments probing the physiological function of tastants within the GI-tract are performed in rodent models. However, in particular for bitter tastants an experimental strategy, which aims at concluding from the responses to well known bitter substances to the functional expression of the corresponding bitter taste receptor(s) is problematic. While the majority of human TAS2R has been deorphaned and, beyond that, several of these human TAS2Rs can be considered to be pharmacologically extensively characterized, this does not apply to rodent receptors (see Section 2.1). In view of the lack of data on the pharmacological properties of all but two rodent Tas2rs, it appears at present questionable to consider non-deorphaned rodent Tas2rs as functional orthologs to human TAS2Rs simply because they may represent the closest relative to the reference receptor. Nevertheless, there is a good evidence for the presence of the sweet taste receptor, TAS1R2/TAS1R3 in enteroendocrine L-cells, producing GLP-1 and PYY, and K-cells, which produce GIP. Within the gastric mucosa of mice Hass and colleagues identied cells expressing the common subunit of the sweet and umami receptors, TAS1R3, in brush cells as well as in closed enteroendocrine G-cells producing gastrin [115]. As shown by co-labeling experiments using an antibody raised against mouse Tas2r138 with chromogranin A, a subpopulation of enteroendocrine cells may express bitter taste receptors [113]. Because of the difculties associated with the detection of taste receptors, signaling components, and putative physiological consequences of tastant evoked the activation of GI-cells, many researchers turned to use various cellular models for their investigations. These model cell lines will be discussed in the next section. 3.4. Models of tastant-responsive gastrointestinal cells In order to study taste-responsive cells of GI origin in more detail, a number of cell lines were used. The mouse enteroendocrine cell line STC-1 originates from intestinal tumor material of transgenic mice, which have integrated a SV40-largeT antigen expressing construct under the control of the rat insulin gene promoter [121]. In a similar fashion, the GLUTag cell line was derived from the intestinal tumor tissue of transgenic mice expressing SV40-large T antigen under control of the rat glucagon promoter [122]. The cell line NCI-H716 was established from human colorectal carcinomas [123]. The HuTu-80 cells derive from human duodenal cancer [124] and the AR42J cell line [125] represents cells from chemically induced rat pancreatic acinar tumors. STC-1 cells were the rst cells characterized specically for the presence of taste signaling components and their responses to taste stimulation. It was shown that numerous Tas2r genes as well as Ggustducin and G-transducin 2, G3, G13, PLC2 and TRPM5 are expressed in STC-1 cells [27,28,32]. Stimulation with various bitter tastants led to increases in cellular calcium levels through the activation of L-type voltage-sensitive calcium channels [32] as monitored by functional calcium imaging experiments, whereas other bitter compounds did not induce such responses indicating specicity [27,32,126]. Furthermore, STC-1 cells which produce among other peptide hormones cholecystokinin (CCK) secrete CCK upon denatonium stimulation in a dose-dependent fashion [32].

However, the biological signicance of these observations remains to be seen, as it is presently impossible to match the functional responses to the Tas2r genes expressed. However, STC-1 cells do not only express Tas2r genes but also all three Tas1r genes [23,26], which should enable them to respond to sweet and umami stimuli as well. In fact, stimuli of all 5 basic taste qualities activate calcium responses in this cell line [127], although for the responses to umami tastants alternative pathways are discussed [128,129]. These observations are surprising and their biological relevance remains to be established since in the gustatory system cells expressing receptors for the ve basic tastes form strictly distinct populations. The NCI-H716 cell line is frequently used as a model for human enteroendocrine L-cells. Similar to STC-1 cells they express the canonical taste signaling components G-gustducin, G3, G13, PLC2, TRPM5, IP3 type III receptor [24]. In addition, the presence of all TAS1R genes [24] and of two bitter taste receptors, hTAS2R38 [30] and hTAS2R9 [22] was shown. On a functional level this model cell line responds to several natural and articial sweeteners with GLP-1 secretion indicating effective coupling of the sweet taste receptor with the intracellular signaling machinery [24]. Also the bitter compounds ooxacin, an agonist for hTAS2R9, and phenylthiocarbamide (PTC), activating hTAS2R38, induce responses in NCI-H716 cells as seen by GLP-1 secretion [22] and calcium-imaging assays [30], respectively. However, it has to be stated that NCI-H716 cells express the non-taster variant of the receptor hTAS2R38 [30] characterized by the presence of three amino acid residues alanine, valine, and isoleucin at receptor positions 49, 262, and 296, respectively [96]. Human subjects homozygous for the non-taster variant of hTAS2R38 show residual responsiveness for PROP and PTC [96,97], which may be due to the presence of a low-afnity bitter taste receptor for these substances such as hTAS2R4 [83], and at present non-deorphaned hTAS2Rs [85], or alternative, hTAS2R-independent mechanisms. However, in vitro experiments demonstrated the complete absence of activation of the non-taster hTAS2R38 even by high concentrations of PROP and PTC [97]. Therefore, it remains to be demonstrated whether PTC-stimulation of NCI-H716 cells is indeed mediated by one of the hTAS2Rs other than hTAS2R38. HuTu-80 cells have been shown to contain mRNA for Ggustducin, TAS1R3, and the majority of bitter taste receptors. Again, PTC was used to stimulate calcium signals, which would indicate that the expressed hTAS2R38 is functional [30]. However, as in the case of NCI-H716 cells (see previous paragraph), this cell line also carries the hTAS2R38 non-taster gene variant [30], hence, the testing of bitter compounds different from hTAS2R38 agonists would be desirable in future experiments. Most recently, it was demonstrated that HuTu-80 cells respond to stimulation with a bitter steroid glycoside derived from the plant Hoodia gordonii with secretion of CCK [33]. From the presence of hTAS2R14 and the absence of hTAS2R7 in HuTu-80 cells the authors concluded that this compound acts via hTAS2R14 activation, since hTAS2R14 and -R7 were the only receptors of all 25 hTAS2Rs screened by heterologous expression that responded robustly to this glycoside. Finally, AR42J cells were shown to express G-gustducin, Gtransducin 2, rTas2r16, -r34, and -r38. Their positive responses upon stimulation with bitter compounds denatonium, PTC and cycloheximide in calcium imaging experiments suggested functional coupling of taste receptors to the authors of this study [28]. The future pharmacological characterization of the corresponding orphane rat bitter taste receptors has to demonstrate whether the observed activity of the used bitter compounds indeed is linked to Tas2r activation. 3.5. Physiological function of taste signaling in the GI tract There are numerous reports on the effects of taste active substances on GI functions and metabolic changes. The question whether these observations are directly linked to the functional

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interaction of these compounds with taste receptors located in the gut is, however, less well understood. Almost 50 years ago it has been observed that orally administered glucose was more effective than glucose injected intravenously in elevating circulating insulin levels. Recent data [24,26] suggest that intestinal TAS1R2/TAS1R3 receptors interact with luminal sweet compounds resulting in this pronounced response called incretin effect [130]. The sweet taste receptor was demonstrated to be expressed in enteroendocrine L and K cells that secrete the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), respectively. Both peptides, GLP-1 and GIP, stimulate pancreatic insulin secretion resulting in blood glucose uptake. Moreover, although the enterocytes do not express the sweet taste receptor themselves, stimulation of enteroendocrine cells with sweet compounds result in the upregulation of the sodiumglucose cotransporter 1 (SGLT1) perhaps by a paracrine mechanism [26]. Thus, sweet tastants do not only cause the incretin effect, but also dynamically modulate the intestinal glucose-uptake capacity. The data that the observed effects are indeed mediated by the sweet taste receptor Tas1r2/Tas1r3 and not by another yet unidentied mechanism are convincing. Firstly, the sweet taste receptor subunits were detected together with other taste signaling components in enteroendocrine cells producing GLP-1, GIP, or both. Secondly, neither Ggustducin, nor Tas1r3 knock-out mice showed, in contrast to wild type controls, an upregulation of SGLT1 in the small intestinal brush border membrane in response to prolonged dietary carbohydrate exposure [26]. Thirdly, articial sweeteners known to activate the mouse sweet taste receptor resulted in SGLT1 upregulation as well, whereas aspartame, which is not an activator of mouse Tas1r2/Tas1r3 was ineffective. However, the presence of sweet taste receptors in the mammalian gut does not mean that it is the only sensor for carbohydrates. The observation that the initially reduced sweet preference of mouse strains with a less sensitive sweet taste receptor (129P3/J strain) can be improved by intragastric sucrose infusions [131] and that sucrose conditioned avor preferences are found even in Tas1r3 knock-out mice [132], argues for existing parallel sensing mechanisms. Moreover, several studies performed in rodents and human volunteers did not observe incretin release from the gut in response to stimulation with articial sweeteners [133135]. Similarly, gastric emptying in humans is not inuenced by the mere sweetness of ingested test solutions, since articial sweeteners are not able to replace glucose or sucrose [133,136]. Hence, future research is needed to clarify the exact contributions of gut sweet taste receptors and possible alternative sensing mechanisms for the above observations. Although the expression of the Tas1r1 as well as the mGluR1 and mGluR4 genes was reported in the GI tract and the infusion of L-amino acids, especially that of L-glutamate in the stomach results in an increased ring rate of afferent nerve bers, an association with a taste-related detection and signaling pathway is not as evident as for sweet and bitter. In fact, it seems that, at least in the gastric mucosa, L-amino acid sensing is achieved by mechanisms different from umami taste perception [137]. In view of the numerous reports on TAS2Rs expressed in the gut and cell lines originating from GI tissues, it is not surprising that several physiological consequences have been associated with the consumption of bitter compounds in mammals. One of the most frequently used bitter compounds to study potential physiological effects in GI tissues is denatonium benzoate. Straub and colleagues used denatonium to stimulate insulin secretion from clonal pancreatic -cells, HIT-T15, as well as from acutely isolated rat pancreatic islets [138]. At a concentration of 100 M a higher denatonium was able to stimulate insulin release signicantly from explanted rat islets in the presence of 8.3 mM glucose. Additional physiological and biochemical experiments demonstrated that denatonium acted most likely via an ATP-sensitive potassium channel to result in cell depolarization,

which in turn triggers the activation of voltage-gated calcium channels leading to calcium inux from the extracellular side. In fact, by a transducin breakdown assay [41] the authors demonstrated that the activity of the classical bitter compound denatonium was not mediated by signaling components present in taste cells [138]. Another example for taste receptor independent action of bitter compounds is provided by isohumulones. These substances are found in hops used for the brewing of beer. They seem to exert their activities independent of Tas2rs by directly interacting with transcription factors of the PPAR-family [139]. It was shown that the insulin-sensitivity of an obese and hyperglycemic mouse model improved by the addition of isohumulones to the diet. Therefore, the reader should be aware that some of the reported GI activities of bitter compounds do not necessarily involve taste-like signal transmission. One of the reported activities of bitter compounds is to delay gastric emptying. Whereas in humans contrasting evidence has arisen on the effectiveness of bitter substances on gastric emptying [136,140], gastric infusion of 10 mM denatonium robustly delayed gastric emptying in rodents [34]. This observation would be in agreement with the notion that bitter taste within the oral cavity protects animals against the ingestion of potentially harmful substances. Therefore, it would make sense that toxic bitter compounds, which escaped the oral quality assessment, delay gastric emptying to reduce the rate of ingestion. Especially for rodents lacking the vomiting reex, this would present an important survival strategy. Interestingly, there is evidence for an elevated anion secretion within the large intestines of human and rat by another bitter compound, 6-n-propyl-2-thiouracil [112]. This could indicate a bitter compound mediated mechanism to ush out potentially harmful colonic content faster. Whereas, as mentioned already before, the activity of 6-n-propyl-2-thiouracil cannot be tightly associated with a particular rat Tas2r because pharmacological data are lacking, for the experiments done with human tissue samples the concentration range tested (mid M to mid mM) appears to be rather high to indicate a direct involvement of hTAS2R38. Whereas the aforementioned activities t to a general aversion elicited by bitter compounds, various other reports indicate an involvement of gut bitter sensing in metabolic control. A direct involvement of TAS2Rs in human metabolic control was suggested recently for hTAS2R9. Dotson and colleagues showed that an inactive variant of this receptor is associated with altered glucose homeostasis in a human family study [22]. As this receptor is expressed in NCI-H716 cells, representing a model for human enteroendocrine L-cells, a direct effect on the secretion of the incretin hormone GLP-1 was suspected as potential mechanism underlying the metabolic disease phenotype. Indeed, it was demonstrated that the stimulation of NCI-H716 cells with the uoroquinolone antibiotic ooxacin, an agonist of hTAS2R9 results in an elevated GLP-1 secretion. Interestingly, TAS2Rs may not only affect metabolism, they may also be dynamically regulated by metabolic parameters. As shown by Jeon and colleagues low-cholesterol diets could activate the expression of the mouse Tas2r138 via the activation of a sterol-sensitive transcription factor SREBP-2 present in STC-1 cells of enteroendocrine origin [113]. As a consequence low-cholesterol could result in an upregulation of mTas2r138, which in the presence of a specic bitter agonist, would cause elevated CCK-release from enteroendocrine cells. In the absence of a known agonist for the orphan receptor mTas2r138, a mixture of phenylthiocarbamide and cycloheximide, indeed resulted in an elevated level of circulating CCK in mice treated to mimic a low-cholesterol diet. Once a specic agonist for mTas2r138 becomes available, it remains to be demonstrated if the observed effects are indeed initiated by activation of this receptor. Although a direct humoral effect of bitter compounds present in the mammalian GI tract appears plausible, at least a part of the bitter sensing mechanism seems to involve the activation of vagal nerve

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bers [27,28]. It was demonstrated that a mixture of bitter compounds administered intragastrically in mice led to an activation of brainstem neurons within the nucleus tractus solitarius (NTS), the rst relay station of taste information within the brain. This effect, visualized by c-fos expression was abolished by subdiaphragmatic vagotomy as well as inhibitors of CCK and PYY receptors [36]. Since vagal nerve bers contain CCK1 and Y2 receptors and terminate close to enteroendocrine cells, bitter compounds could stimulate the secretion of these peptide hormones, which in turn activate vagal nerve bers. As elevated c-fos expression is not limited to the NTS, but also to other brain areas associated with conditioned taste aversion, bitter compounds present in the gut might elicit complex behavioral patterns [35]. Whether these behavioral responses are indeed mediated by gastrointestinal bitter taste receptors remains to be demonstrated in the future. 4. Summary/Outlook Over the past years it became increasingly clear that taste receptors cannot be considered as being solely involved in oral taste perception. To date, it is evident that these receptors play important additional roles in the respiratory [6,141146] and GI systems, perhaps even in the central nervous system [147] as well as in the male reproductive system [7,148]. However, concerning the role of taste receptors in GI tissues important questions have arisen and (mostly) remained unanswered. When comparing the current status of research on the role of the sweet taste receptor and bitter taste receptors, respectively, it becomes clear that important experimental tools, which were available for the investigation of the function of the sweet receptor activation in the gut, have been missing for the evaluation of the role of bitter taste receptors. 1.) Specic antibodies raised against taste receptor proteins that allow unambiguous identication and characterization of the cell types expressing the receptors in vivo: such antibodies have been available for TAS1Rs and were successfully used in several studies, whereas TAS2R-specic antibodies were not at hand until recently and therefore evidence for GI cells expressing TAS2Rs in vivo is rare. Currently, a number of antibodies raised against TAS2R are commercially available to, hopefully, close this gap. Alternatively, transgenic animals expressing marker proteins under the control of Tas2r promoters as exemplied recently for Tas1r2-lacZ knock-in mice [148] or in situ hybridization to detect TAS2R mRNAs might be used to solve this problem. 2.) Both, the human and the rodent sweet taste receptors, have been extensively characterized in vitro. This has enabled researchers interested in dening the role of GI-resident sweet taste receptors to use pharmacological approaches as well. Unfortunately, similar strategies to investigate Tas2rs are hampered by the fact that predominantly human (a species inaccessible to most experiments) TAS2Rs are well characterized, but only two rodent TAS2Rs. 3.) An elegant way to demonstrate the involvement of receptors in physiological pathways is the use of specic antagonists to dissect the exact contribution of this receptor to the observed effects. Again, for TAS1Rs such an antagonist, in the form of lactisole, exists and enabled experiments that are currently not possible for the investigation of TAS2Rs. The recent report on a reasonable specic antagonist for some human TAS2Rs may indicate that in some years from now more bitter blockers will become available also for rodent receptors. Alternatively, knock-out mice for pharmacologically characterized bitter taste receptors could be generated to investigate the physiological consequences of bitter compound stimulation tightly linked to TAS2R function. Acknowledgements We thank the German Federal Ministry of Education and Research for funding (BMBF 0315669 A to M.B. and W.M.).

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