CD36- and GPR120-mediated Ca²⁺ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice

Abstract

Background & aims: It is important to increase our understanding of gustatory detection of dietary fat and its contribution to fat preference. We studied the roles of the fat taste receptors CD36 and GPR120 and their interactions via Ca(2+) signaling in fungiform taste bud cells (TBC).

Methods: We measured Ca(2+) signaling in human TBC, transfected with small interfering RNAs against messenger RNAs encoding CD36 and GPR120 (or control small interfering RNAs). We also studied Ca(2+) signaling in TBC from CD36(-/-) mice and from wild-type lean and obese mice. Additional studies were conducted with mouse enteroendocrine cell line STC-1 that express GPR120 and stably transfected with human CD36. We measured release of serotonin and glucagon-like peptide-1 from human and mice TBC in response to CD36 and GPR120 activation.

Results: High concentrations of linoleic acid induced Ca(2+) signaling via CD36 and GPR120 in human and mice TBC, as well as in STC-1 cells, and low concentrations induced Ca(2+) signaling via only CD36. Incubation of human and mice fungiform TBC with lineoleic acid down-regulated CD36 and up-regulated GPR120 in membrane lipid rafts. Obese mice had decreased spontaneous preference for fat. Fungiform TBC from obese mice had reduced Ca(2+) and serotonin responses, but increased release of glucagon-like peptide-1, along with reduced levels of CD36 and increased levels of GPR120 in lipid rafts.

Conclusions: CD36 and GPR120 have nonoverlapping roles in TBC signaling during orogustatory perception of dietary lipids; these are differentially regulated by obesity.

Keywords: GLP-1; Linoleic Acid; Lipids; Serotonin.

Figure 1. Characterization of human fungiform TBC

Images were acquired with Leica TCS-SP2 confocal laser scanning microscope (A, B & C). Immunoreactivity for CD36 (red in A, B; green in C), GPR120 (red in C) and PLC-β (green in A) and α-gustducin (green in B) was observed in cultured TBC.

Figure 2. Effects of LA and GA on Ca²⁺ signaling in human fungiform TBC

The cultured TBC (2×10⁵ cells/assay) were loaded with Fura-2/AM and the changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) were monitored. The experiments were performed in Ca²⁺-containing (A - F) and in Ca²⁺-free media (B, D). In A and C, the colored time-lapse images show the changes in intracellular Ca²⁺ ([Ca²⁺]i) evoked, respectively by LA and GA. In E and F, TBC before exposure to 20 μM LA (E) or GA (F) were preincubated (20 min) with: SU6656 (5 μM), PP2 (10 μM), genistein (30 μM), SKF96365 (15 μM), econazole (30 μM), 2-APB (30 μM), PTX (10 ng/ml), GDP-β-S (300 μM), or M-βCD (2.5 mM). Data are means ± SEM (n=7). *p<0.001) as compared to control.

Figure 3. Effects of siRNA targeting CD36 or GPR120 on LA- and GA-evoked Ca²⁺ signaling in human fungiform TBC

A: Western blots showing selective downregulation of CD36 and GPR120 by siRNA targeting CD36 and/or GPR120. Mock: non-targeting siRNA. β-actin is the loading control. B-E: Increases in [Ca²⁺]i induced by either LA or GA in TBC transfected by the various siRNA. Data are means ± SEM (n=4) conducted in triplicates.

Figure 4. Effects of CD36 deficiency on LA- and GA-evoked Ca²⁺ signaling in mouse fungiform TBC

Ca²⁺ imaging studies were performed on fungiform TBC from wild-type (WT) or CD36^−/− mice and the changes in [Ca²⁺]i (F₃₄₀/F₃₈₀) were monitored as for Figure 2. The data are representative of at 4-5 experiments conducted in triplicates.

Figure 5. Effects of LA and GA on Ca²⁺ signaling in STC-1 cells

The studies were performed on STC-1 cells that endogenously express GPR120 but lack CD36 (GPR120+/CD36-), that stably express human CD36 (GPR120+/CD36+) or that express CD36 and are transfected with GPR120 siRNA (GPR120-/CD36+). The changes in intracellular Ca²⁺ (F₃₄₀/F₃₈₀) are shown in response to 5 or 20 μM LA. The data are representative of at 4-5 experiments conducted in triplicates.

Figure 6. Effects of LA-exposure on the distribution of CD36 and GPR120 in raft fractions in human (A, B & C) and mouse (D, E & F) fungiform TBC

The TBC were treated or not (control) with 20 μM LA for 20 min A & D: Cells were homogenized in 1% Triton X-100 at 4°C, and the lysates were subjected to discontinuous 5–40% sucrose gradient centrifugation. Different fractions were collected from the top of the gradient, and equal volumes of aliquots from each fraction were subjected to western blot. Histograms show the relative band intensity (arbitrary units) measured by densitometry of protein content in raft (3-6) and soluble fractions (7-10). The data were normalized with respect to band intensity of caveolin-1 (Cav-1) for raft fractions and β-actin for soluble fractions, measured under similar conditions. B & C are derived from A and E & F are derived from D. Data are means ± SEM conducted in triplicates.

Figure 7. High fat diet-induced obesity modulates Ca²⁺ signaling, gustatory fat preference and CD36 and GPR120 raft distribution in mouse fungiform TBC

The fungiform TBC were isolated from mice fed a standard chow diet (Std. Diet) or a high fat diet (HFD) for two months. A: Changes in [Ca²⁺]i. B: Spontaneous fat preference of mice fed standard or HF diets. Values are means ± SEM (n=6). * (p<0.001) Oil as compared to control solution. C & D: Distribution of CD36 and GPR120 in raft (R, 3-6) and soluble (S, 7-10) fractions from mice fed Std. or HF diets. Caveolin-1 (cav-1) is a raft marker. E & F: Relative densitometry quantifying data (arbitrary units) derived from C and D after normalization for band intensity of caveolin-1 (Cav-1) for raft fractions and β-actin for soluble fractions. NS=non-significantly different. Data are means ± SEM conducted in triplicates.