Investigation of free fatty acid associated recombinant membrane receptor protein expression in HEK293 cells using Raman spectroscopy, calcium imaging, and atomic force microscopy.

G-protein-coupled receptor 120 (GPR120) is a previously orphaned G-protein-coupled receptor that apparently functions as a sensor for dietary fat in the gustatory and digestive systems. In this study, a cDNA sequence encoding a doxycycline (Dox)-inducible mature peptide of GPR120 was inserted into an expression vector and transfected in HEK293 cells. We measured Raman spectra of single HEK293 cells as well as GPR120-expressing HEK293-GPR120 cells at a 48 h period following the additions of Dox at several concentrations. We found that the spectral intensity of HEK293-GPR120 cells is dependent upon the dose of Dox, which correlates with the accumulation of GPR120 protein in the cells. However, the amount of the fatty acid activated changes in intracellular calcium (Ca(2+)) as measured by ratiometric calcium imaging was not correlated with Dox concentration. Principal components analysis (PCA) of Raman spectra reveals that the spectra from different treatments of HEK293-GPR120 cells form distinct, completely separated clusters with the receiver operating characteristic (ROC) area of 1, while those spectra for the HEK293 cells form small overlap clusters with the ROC area of 0.836. It was also found that expression of GPR120 altered the physiochemical and biomechanical properties of the parental cell membrane surface, which was quantitated by atomic force microscopy (AFM). These findings demonstrate that the combination of Raman spectroscopy, calcium imaging, and AFM may provide new tools in noninvasive and quantitative monitoring of membrane receptor expression induced alterations in the biophysical and signaling properties of single living cells.

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.

Activation of oral trigeminal neurons by fatty acids is dependent upon intracellular calcium.

The chemoreception of dietary fat in the oral cavity has largely been attributed to activation of the somatosensory system that conveys the textural properties of fat. However, the ability of fatty acids, which are believed to represent the proximate stimulus for fat taste, to stimulate rat trigeminal neurons has remained unexplored. Here, we found that several free fatty acids are capable of activating trigeminal neurons with different kinetics. Further, a polyunsaturated fatty acid, linoleic acid (LA), activates trigeminal neurons by increasing intracellular calcium concentration and generating depolarizing receptor potentials. Ion substitution and pharmacological approaches reveal that intracellular calcium store depletion is crucial for LA-induced signaling in a subset of trigeminal neurons. Using pseudorabies virus (PrV) as a live cell tracer, we identified a subset of lingual nerve-innervated trigeminal neurons that respond to different subsets of fatty acids. Quantitative real-time PCR of several transient receptor potential channel markers in individual neurons validated that PrV labeled a subset but not the entire population of lingual-innervated trigeminal neurons. We further confirmed that the LA-induced intracellular calcium rise is exclusively coming from the release of calcium stores from the endoplasmic reticulum in this subset of lingual nerve-innervated trigeminal neurons.

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.

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.

High-Fat Diet Alters the Orosensory Sensitivity to Fatty Acids in Obesity-Resistant but not Obesity-Prone Rats.

Gene-environment interactions play a role in the development of obesity but specific effects of diet on the orosensory detection of fatty acids have yet to be clarified. The objective of this study is to characterize the effect of prolonged (5-week) exposure to a high-fat (60%) diet on the behavioral sensitivity to the fatty acid linoleate following a conditioned taste aversion in obesity-prone and obesity-resistant rats. Exposure to the high-fat diet significantly enhanced the sensitivity of obesity-resistant (S5B/Pl) rats to linoleate while producing no effect on the fatty acid sensitivity for obesity-prone rats. Specifically, high-fat diet fed S5B/Pl rats showed stronger initial avoidance of linoleate and slower extinction rates than their normal diet cohorts. Our study suggests that prolonged dietary fat consumption may alter the behavioral sensitivity to fatty acids particularly in obesity-resistant animals.

Establishment and Dispersal of Puccinia thlaspeos in Field Populations of Dyer's Woad.

Dyer's woad rust is being intensively studied as a biocontrol agent for the noxious weed dyer's woad. These studies report on methods to establish this fungus in woad populations and to obtain information about disease incidence and dispersal of the rust subsequent to establishment. Inoculum dosages as low as 1 mg/plant established disease in dyer's woad rosettes. Dosages higher than 1 mg/plant resulted in better levels of infection, and inoculation was equally effective when the inoculum was placed directly on the plants or on the soil adjacent to the plants. The highest incidence of infection occurred when plants were inoculated in the spring, whereas summer or fall inoculations did not result in infections. Large-scale inoculation of woad was effective and resulted in significant levels of disease. However, it was not clear if artificial inoculation was effective in changing disease incidence in populations of woad where the rust was present prior to treatment because there were no significant differences between inoculated and check plots. Woad rust is systemic but apparently it is not seed dispersed, even though some infected plants occasionally produce seed. Two years after inoculation, dyer's woad rust had dispersed an average of 14.6 m from the focus of inoculation. After 3 years, the dispersal distance had not increased and infected plants averaged only 10 m from the point of inoculation. Disease was maintained naturally in woad populations after treatment, but the overall percent infection in the treated plots decreased by 3 years after inoculation.

Expression and characterization of delayed rectifying K+ channels in anterior rat taste buds.

Delayed rectifying K+ (DRK) channels in taste cells have been implicated in the regulation of cell excitability and as potential targets for direct and indirect modulation by taste stimuli. In the present study, we have used patch-clamp recording to determine the biophysical properties and pharmacological sensitivity of DRK channels in isolated rat fungiform taste buds. Molecular biological assays at the taste bud and single-cell levels are consistent with the interpretation that taste cells express a variety of DRK channels, including members from each of the three major subfamilies: KCNA, KCNB, and KCNC. Real-time PCR assays were used to quantify expression of the nine DRK channel subtypes. While taste cells express a number of DRK channels, the electrophysiological and molecular biological assays indicate that the Shaker Kv1.5 channel (KCNA5) is the major functional DRK channel expressed in the anterior rat tongue.