The sweet taste receptors comprised of T1r2 and T1r3, sense glucose concentrations in the gastrointestine. While hyperglycemia was reported to decrease the T1R2 and T1R3 tanscript levels in healthy subjects, no change was observed in type 2 diabetes patients. We investigated which glucose level and nutrients affect those transcript levels in MIN 6 and primary cultured taste buds cells using quantitative Reverse Trancription Polymerase Chain Reaction. High glucose diminished T1r2 transcript levels in MIN 6 and primary cultured taste buds cells. Resveratrol and its analogue augmented transcript levels of T1r1 and T1r2 above normal levels in MIN 6 cells in the medium with 25 mM glucose. Adenine, but not guanine, augmented T1r2 transcript levels of MIN 6 cells in the medium with 25 mM glucose. These results imply that nutrients in meals could affect sweet taste sensitivity by modulating T1r2 transcript levels in response to blood glucose levels.
The sweet taste receptors comprised of T1r2 and T1r3, sense glucose concentrations in tongue, pancreas and intestine. During a meal, the receptor on the taste cells in tongues transduces the sweet taste response to brain through sensory nerves [
Young et al. reported that the duodenal expression levels of T1R2 and T1R3 in subjects with type 2 diabetes was inversely correlated with their blood glucose levels [
In this report, we first examine which glucose concentrations affect the transcript levels of T1r1, T2r2 and T1r3 using the MIN 6 cell lines and primary cultured taste bud cells from mice tongues.
In southern European countries, wheat bread―one of staple food with higher glycemic index―is usually taken with red wine. Resveratrol is a plant-derived antioxidant, which is contained in grapes and red wine. In INS-1-1E β-cells―another insulin secreting cell lines upon glucose stimulation―resveratrol was reported to potentiate insulin-secretion in the medium with the 15 mM, 20 mM and 30 mM glucose concentrations through the adenine mononucleotide-activated protein kinase (AMPK) pathway [
In Japan, rice―another staple food with higher glycemic index―is usually taken with soybean, especially as miso-soup. Adenine is known to be rich in soybean [
Glucose, adenine, guanine, thymine, and cytosine were purchased from Wako Pure Chemicals (Osaka, Japan). RSVA314 was purchased from EMD Millipore (Billerica, MA). Resveratrol and 5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside (AICAR) was purchased from Sigma (St. Louis, MOs). The animals used in this study were 8 to 16 weeks old male C57BL/6N mice fed with CE-2 containing 20% lactose, 2% calcium and 1.25% phosphate (Nihon CLEA, Tokyo, Japan) under the 12 h light: 12 h dark condition. Mice were purchased from Charles River Japan (Yokohama, Japan) and bred at the National Food Research Institute, Tsukuba, Japan. We followed the guidelines of our organization for the care and their use of experimental animals. Dissected tongues were used in the following experiments.
MIN 6 cells were grown in Dulbecco’s modified Eagle’s medium (25 mM glucose, Gibco, Grand Island, NY) equilibrated with 5% CO2 and 95% air at 37˚C. The medium was supplemented with 10% fetal bovine serum (Sigma), 0.25 μg/ml fungizone (Gibco), 50 mg/ml streptomycin (Meiji Seika, Tokyo, Japan) and 50 U/ml penicillin sulphate (Meiji Seika). MIN 6 cells were passaged with trypsin-EDTA and used between 22 - 30 passages.
Primary cell culture of taste buds cells from mice circumvallate and foliate papillae was performed as previously described [
MIN 6 cells (1 × 106) were cultured on a 12-well plate (BD Falcon) in the medium with 25 mM glucose for 16 h. The cells were treated with various glucose concentration (5, 10 and 25 mM) DMEM in the presence of stimulatory substances for 24 h. The cells were subjected to the RNA extraction.
For primary cultured taste buds cells, culture medium was changed with the QM12 medium (5 mM Glucose) after 60 - 65 h when the cells had attached to the collagen-coated plates. Sixteen h later, the medium was replaced with QM12 medium (5, 10, and 25 mM glucose) for 3 h in the absence of stimulatory substances―for 24 h in the presence of stimulatory substances. The cells in each well were subjected to the RNA extraction.
Total RNAs from MIN 6 and primary cultured taste buds cells were isolated with TRI Reagent (Molecular Research Center, Cincinnati, OH) or with FavorPrep total RNA Extraction Column (Favorgen Biotech Corp, Ping-Tung, Taiwan) following the manufacturers’ instructions. The total RNAs were transcribed into cDNA using ReveTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). For Real-time PCR analysis, reactions were performed in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) using THUNDERBIRD SYBR qPCR Mix (Toyobo). Each reaction was run in triplicate with 200 nM primers in a volume of 20 ml. Reaction conditions were as follows: an initial denaturation for 1 min at 95˚C, followed by 40 cycles of 15 sec at 95˚C, 45 sec at 60˚C. In each experiment, melting curve analysis was carried out at the end of the PCR program to confirm specific amplification of target genes. Relative gene expression value was calculated by the comparative Ct method. Primers used in this study are listed in
TATA-box binding protein (Tbp) was used as a reference gene for MIN 6. IP3r3 was used for primary cultured taste buds cells to minimize difference of each well in T1r2 expressing cell population.
All the data are averages of, at least, three independent experiments and bars represent standard deviation of the means. For statistical analyses, multiple comparisons were performed by Dunnett’s test. Statistical analyses were conducted with the statistical programming language R. Statistical significance was considered when the p-value was less than 0.05 or 0.01.
MIN 6 cells were maintained and passaged in high glucose (25 mM) medium. We thus analyzed what medium glucose concentrations affect T1r1, T1r2 and T1r3 mRNA expression in MIN 6 cells. As shown in
Gene | Primer sequence (strand) | Product size (bp) | Cycle (times) |
---|---|---|---|
IP3r3(qPCR) | 5'-TGTGCGTGTCCAACCGTATC-3' (+) | 124 | 40 |
5'-TGAGCCATCTCCTTCACTGG-3' (−) | |||
Tbp (qPCR) | 5'-ACCCTTCACCAATGACTCCTATG-3' (+) | 190 | 40 |
5'-ATGATGACTGCAGCAAATCGC-3' (−) | |||
T1r1 (qPCR) | 5'-CTGCCAAAGGACAGAATCCTC-3' (+) | 178 | 40 |
5'-GAACCGCATGGCTTGGAAG-3' (−) | |||
T1r2 (qPCR) | 5'-TGGCAGCTACTCAGGGAGAT-3' (+) | 155 | 40 |
5'-GAGTAGGAGGCGATGCTTTG-3' (−) | |||
T1r3 (qPCR) | 5'-TGGGGGCCTCTTTGTGTCT-3' (+) | 117 | 40 |
5'-TGGGTTGTGTTCTCTGGTTGA-3' (−) |
glucose. To confirm the down regulation of T1r2 transcript levels in the presence of high glucose, we conducted additional experiments with the primary cultured taste buds cells.
We analyzed the effect of increasing glucose concentrations on T1r1 and T1r2 mRNA expression in the primary cultured taste buds cells. While T1r1 transcript levels showed a tendency to decrease after 3 h of incubation as glucose levels were increased (
During meal digestion, locally high glucose levels in tongues and in intestines might lower sweet sensitivity. Hypothesizing that the nutrients in meals could neutralize the above down-regulating effect of glucose concentrations during meal digestion, we analyzed nutrients to determine whether they could influence the effect of high glucose on T1r2 expression.
We found that resveratrol and its analogue RSVA 314, which was reported to regulate energy flow via mitochondrial AMPK pathways, augmented T1r2 mRNA transcript levels (
In the murine Neuro 2a cell line, resveratrol was reported to increase adenine nucleotide pools [
The duodenal expression levels of T1R2 and T1R3 in type 2 diabetic subjects were inversely correlated with their blood glucose levels [
In a previous report, we used 4.75 μg/ml insulin for the primary taste buds cells culture [
Hypothesizing that other nutrients in meals may modulate T1r2 expression during acute glucose rise, we analyzed the effect of resveratrol on T1r2 expression in MIN 6 cells, because resveratrol is known as effective against oxidative stress and diabetes [
Adenine is rich in chicken liver (63.0 mg/100g), soybean (38.5 mg/100g), and dried mushroom (105.0 mg/100g) [
In podocytes, high glucose negatively regulated AMP activated kinase (AMPK) [
High glucose concentration (25 mM) significantly diminished T1r2 transcript levels in MIN 6 and primary cultured taste buds cells with RT-PCR methods. Adenine and resveratrol augmented T1r2 transcript levels above normal levels in MIN 6 cells in the medium with 25 mM glucose. These results imply that nutrients in meals could affect sweet taste sensitivity by modulating T1r2 transcript levels in response to blood glucose levels.
We are deeply grateful to Dr. Nancy E. Rawson for critical reading of the manuscripts and to Dr. Jun-ichi Miyazaki for providing us the MIN 6 cell lines. This work is in part supported from JST A-step grant to T. O.
Shin-ichi Nakmura,Tetsuya Ookura, (2016) Nutrients Modulate T1r2 Transcript Levels in MIN 6 and Primary Cultured Taste Buds Cells under High Glucose Condition. Food and Nutrition Sciences,07,312-319. doi: 10.4236/fns.2016.74033