Timothy A. Gilbertson

The molecular physiology of taste transduction

Taste receptor cells use a variety of mechanisms to transduce chemical information into cellular signals. Seven-transmembrane-helix receptors initiate signaling cascades by coupling to G proteins, effector enzymes, second messengers and ion channels. Apical ion channels pass ions, leading to depolarizing and/or hyperpolarizing responses. New insights into the mechanisms of taste sensation have been gained from molecular cloning of the transduction elements, biochemical elucidation of the transduction pathways, and electrophysiological analysis of the function of taste cell ion channels.

Fatty acid responses in taste cells from obesity-prone and -resistant rats

One of the transduction mechanisms for the chemoreception of fat has been proposed to involve the inhibition of delayed rectifying potassium (DRK) channels by polyunsaturated free fatty acids (PUFAs). In the present study we have compared the responsiveness of fungiform taste receptor cells (TRCs) to fatty acids in obesity-prone (Osborne-Mendel; O-M) and obesity-resistant (S5B/Pl) rat strains using patch clamp recording. TRCs from S5B/Pl rats were markedly more responsive to PUFAs than those from O-M, yet with identical inhibition constants. Moreover, addition of PUFAs to subthreshold concentrations of saccharin enhanced preference for the mixture in two-bottle preference tests compared to the saccharin alone in S5B/Pl but not O-M rats. The correlation between electrophysiological and behavioral effects of PUFAs suggested that differences in fatty acid-sensitive DRK expression may underlie the phenotypic differences between S5B/Pl and O-M rats. Consistent with this hypothesis, O-M rats exhibit a greater DRK current density and express quantitatively more DRK channels as assayed using quantitative real-time PCR. No differences were found when comparing expression of fatty acid activated two pore domain potassium channels. We propose that the ratio of fatty acid-sensitive DRK channels to fatty acid-insensitive DRK channels may be important to contributing to overall peripheral fatty acid sensitivity and in that way influence the strength of the resulting chemosensory response to fat.

Glucose transporters and ATP-gated K+ (KATP) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells

Although the heteromeric combination of type 1 taste receptors 2 and 3 (T1r2 + T1r3) is well established as the major receptor for sugars and noncaloric sweeteners, there is also evidence of T1r-independent sweet taste in mice, particularly so for sugars. Before the molecular cloning of the T1rs, it had been proposed that sweet taste detection depended on (a) activation of sugar-gated cation channels and/or (b) sugar binding to G protein-coupled receptors to initiate second-messenger cascades. By either mechanism, sugars would elicit depolarization of sweet-responsive taste cells, which would transmit their signal to gustatory afferents. We examined the nature of T1r-independent sweet taste; our starting point was to determine if taste cells express glucose transporters (GLUTs) and metabolic sensors that serve as sugar sensors in other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we determined that several GLUTs (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium–glucose cotransporter (SGLT1), and two components of the ATP-gated K+ (KATP) metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, and SUR1 were expressed preferentially in T1r3-positive taste cells. Electrophysiological recording determined that nearly 20% of the total outward current of mouse fungiform taste cells was composed of KATP channels. Because the overwhelming majority of T1r3-expressing taste cells also express SUR1, and vice versa, it is likely that KATP channels constitute a major portion of K+ channels in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors and KATP may serve as mediators of the T1r-independent sweet taste of sugars.

Transient Receptor Potential Channel Type M5 Is Essential for Fat Taste

Until recently, dietary fat was considered to be tasteless, and its primary sensory attribute was believed to be its texture (Rolls et al., 1999; Verhagen et al., 2003). However, a number of studies have demonstrated the ability of components in fats, specifically free fatty acids, to activate taste cells and elicit behavioral responses consistent with there being a taste of fat. Here we show for the first time that long-chain unsaturated free fatty acid, linoleic acid (LA), depolarizes mouse taste cells and elicits a robust intracellular calcium rise via the activation of transient receptor potential channel type M5 (TRPM5). The LA-induced responses depend on G-protein-phospholipase C pathway, indicative of the involvement of G-protein-coupled receptors (GPCRs) in the transduction of fatty acids. Mice lacking TRPM5 channels exhibit no preference for and show reduced sensitivity to LA. Together, these studies show that TRPM5 channels play an essential role in fatty acid transduction in mouse taste cells and suggest that fatty acids are capable of activating taste cells in a manner consistent with other GPCR-mediated tastes.

Taste transduction: appetizing times in gustation.

&NA; Taste receptors cells sample the chemical composition of ingested material in order to provide the initial sensory information to facilitate decisions regarding its eventual acceptance or rejection. Ion channels, ionotropic and metabotropic receptors have been implicated in the initial events of transduction but until recently their identification has proven difficult. Recent advances in the identification and functional characterization of mammalian taste receptors has greatly increased our understanding of the pathways for the transduction of taste stimuli. This basic information will be critical to answer longstanding questions regarding the coding of taste information and may help elucidate the role of the taste system in the control of food intake.

Cell signaling mechanisms of oro-gustatory detection of dietary fat: advances and challenges.

CD36 and two G-protein coupled receptors (GPCR), i.e., GPR120 and GPR40, have been implicated in the gustatory perception of dietary fats in rodents. These glycoproteins are coupled to increases in free intracellular Ca²⁺ concentrations, [Ca²⁺](i), during their activation by dietary long-chain fatty acids (LCFA). The transient receptor potential type M5 (TRPM5) channel, activated by [Ca²⁺](i), participates in downstream signaling in taste bud cells (TBC). The mice, knocked-out for expression of CD36, GPR120, GPR40 or TRPM5 have a reduced spontaneous preference for fat. The delayed rectifying K⁺ (DRK) channels believed to lie downstream of these receptors are also important players in fat taste transduction. The trigeminal neurons by triggering increases in [Ca²⁺](i) may influence the taste signal to afferent nerve fibers. Why are there so many taste receptor candidates for one taste modality? We discuss the recent advances on the role of CD36, GPR120, GPR40, TRPM5 and DRK channels, in signal transduction in TBC. We shed light on their cross-talk and delineate their roles in obesity as a better understanding of the molecular mechanisms behind their regulation could eventually lead to new strategies to fight against this condition.

TRPM5 is critical for linoleic acid-induced CCK secretion from the enteroendocrine cell line, STC-1.

Fatty acid-induced stimulation of enteroendocrine cells leads to release of the hormones such as cholecystokinin (CCK) that contribute to satiety. Recently, the fatty acid activated G protein-coupled receptor GPR120 has been shown to mediate long-chain unsaturated free fatty acid-induced CCK release from the enteroendocrine cell line, STC-1, yet the downstream signaling pathway remains unclear. Here we show that linoleic acid (LA) elicits membrane depolarization and an intracellular calcium rise in STC-1 cells and that these responses are significantly reduced when activity of G proteins or phospholipase C is blocked. LA leads to activation of monovalent cation-specific transient receptor potential channel type M5 (TRPM5) in STC-1 cells. LA-induced TRPM5 currents are significantly reduced when expression of TRPM5 or GPR120 is reduced using RNA interference. Furthermore, the LA-induced rise in intracellular calcium and CCK secretion is greatly diminished when expression of TRPM5 channels is reduced using RNA interference, consistent with a role of TRPM5 in LA-induced CCK secretion in STC-1 cells.

Insulin activates epithelial sodium channel (ENaC) via phosphoinositide 3-kinase in mammalian taste receptor cells

Diabetes is a profound disease that results in a severe lack of regulation of systemic salt and water balance. From our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel (ENaC), we have begun to investigate the ability of insulin to alter ENaC function with patch-clamp recording on isolated mouse taste receptor cells (TRCs). In fungiform and vallate TRCs that exhibit functional ENaC currents (e.g., amiloride-sensitive Na(+) influx), insulin (5-20 nM) caused a significant increase in Na(+) influx at -80 mV (EC(50) = 7.53 nM). The insulin-enhanced currents were inhibited by amiloride (30 μM). Similarly, in ratiometric Na(+) imaging using SBFI, insulin treatment (20 nM) enhanced Na(+) movement in TRCs, consistent with its action in electrophysiological assays. The ability of insulin to regulate ENaC function is dependent on the enzyme phosphoinositide 3-kinase since treatment with the inhibitor LY294002 (10 μM) abolished insulin-induced changes in ENaC. To test the role of insulin in the regulation of salt taste, we have characterized behavioral responses to NaCl using a mouse model of acute hyperinsulinemia. Insulin-treated mice show significant avoidance of NaCl at lower concentrations than the control group. Interestingly, these differences between groups were abolished when amiloride (100 μM) was added into NaCl solutions, suggesting that insulin was regulating ENaC. Our results are consistent with a role for insulin in maintaining functional expression of ENaC in mouse TRCs.

Orosensory Detection of Fatty Acids by Obesity-Prone and Obesity-Resistant Rats: Strain and Sex Differences

A series of brief-access (15s) behavioral assays following the formation of a conditioned taste aversion (CTA) to linoleic acid were performed in order to follow up on observations showing differences in the chemosensory responses to dietary fat in obesity-prone (Osborne-Mendel [O-M]) and obesity-resistant (S5B/Pl) rat strains. Strong aversions to linoleic acid (conditioned stimulus 100 microM) were generated in both O-M and S5B/Pl rats to concentrations as low as 2.5 microM. Observed strain differences were in contrast to expectations based upon electrophysiological studies previously showing greater fatty acid-induced inhibition of delayed rectifying K+ channels in S5B/Pl rats. In the CTA assays, the O-M rats showed aversions at lower fatty acid concentrations with more resistance to extinction in brief-access orosensory tests, suggesting that the obesity-prone strain may be more sensitive in the detection and subsequent avoidance of linoleic acid than the obesity-resistant strain. The independent variable of sex produced even greater differences in the avoidance of linoleic acid following conditioning than the effects of strain. Female rats of both strains were significantly more sensitive to fatty acids, showed greater cross-generalization from linoleic to oleic acid, and showed greater avoidance of linoleic acid than male counterparts. These findings suggest genetic influences on yet to be identified mechanisms potentially within the gustatory system that affect the sensitivity to detect the fatty acid chemicals found in dietary fat during brief-access orosensory testing.

Biophysical assessment of single cell cytotoxicity: Diesel exhaust particle-treated human aortic endothelial cells

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs.