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TAS2R38 bitter taste receptor and attainment of exceptional longevity

Bitter taste receptors play crucial roles in detecting bitter compounds not only in the oral cavity, but also in extraoral tissues where they are involved in a variety of non‒tasting physiological processes. On the other hand, disorders or modifications in the sensitivity or expression of these extraoral receptors can affect physiological functions. Here we evaluated the role of the bitter receptor TAS2R38 in attainment of longevity, since it has been widely associated with individual differences in taste perception, food preferences, diet, nutrition, immune responses and pathophysiological mechanisms. Differences in genotype distribution and haplotype frequency at the TAS2R38 gene between a cohort of centenarian and near-centenarian subjects and two control cohorts were determined. Results show in the centenarian cohort an increased frequency of subjects carrying the homozygous genotype for the functional variant of TAS2R38 (PAV/PAV) and a decreased frequency of those having homozygous genotype for the non-functional form (AVI/AVI), as compared to those determined in the two control cohorts. In conclusion, our data providing evidence of an association between genetic variants of TAS2R38 gene and human longevity, suggest that TAS2R38 bitter receptor can be involved in the molecular physiological mechanisms implied in the biological process of aging.

Given the implications of TAS2R38 bitter receptor in taste perception, food preferences, diet and nutrition 2 (which can affect human development and subsequently longevity [67][68][69][70], and those in an efficient immune response 48 and disease aetiology 9,45,49,66 (which modulate the physiological mechanisms involved in the biological process of aging 71,72 ), it is likely that TAS2R38 and its variants can play an important role in the attainment of longevity.
In this study we analysed the genotype distribution and allele frequency of the TAS2R38 gene in a cohort of centenarian subjects recruited in a genetically isolated area of the central-eastern Sardinia island (called the Longevity Blue Zone, LBZ). This area, which includes six mountainous villages of Ogliastra and Barbagia, has a total population of nearly 12,000 inhabitants on a land area of 888 km 2 ( Fig. 1) 73 . This population has remained isolated for many centuries, which made its genetic make-up one of the most homogeneous in Europe 74 , and its sociocultural and anthropological characteristics well preserved throughout history 73,75 . LBZ represents an interesting case study because it shows a value of the Extreme Longevity Index (ELI) 76 computed for generations born between 1880 and 1900 that is more than twice as high as that of the whole Sardinia. For this reason, data from this population were compared with data from ancestrally-diverse cohorts recruited in another area of Sardinia (area of Cagliari, in the south of Sardinia).
The association between longevity and taste genetics has been already investigated 77,78 . Campa and co-authors 77 analysed, in a population from Calabria (Italy), the association with longevity of the common genetic variants of three bitter taste receptor genes that are involved in food preferences, food absorption processing and metabolism. They compared the results from centenarian subjects with those from non-elderly controls with a wide age range (20-84 years). Results showed that the frequency of subjects who carried the genotype homozygote AA for the polymorphism, rs978739, in TAS1R16 gene increases gradually from 35% in subjects aged 20 up to 55% in centenarians. However, another study did not confirm this association, by comparing results from centenarian with those from young controls (age range 18-45 years), in a population from another area of South Italy (Cilento) which could be subjected to different demographic pressures 78 . Here, we decided to compare results from centenarian subjects of the LBZ with those of two control cohorts south of Sardinia differentiated based on their age, one of young adult subjects (age ranging from 18 to 35 years) and a second of middle-aged adults and older adults (age ranging from 36 to 85 years).

Materials and Methods
Subjects. Three hundred seventy-three subjects were included in the study. They were divided in three groups based on their age and area of Sardinia island (Italy) where they were recruited: the Longevity Blue Zone cohort (LBZ) (n = 94) (age ranging from 90 to 105 years) included subjects recruited in the central-eastern area (Ogliastra/Barbagia) of Sardinia; the Cagliari young cohort (CY) (n = 181) (age ranging from 18 to 35 years) included subjects recruited in the area of the city of Cagliari (Sardinia); the Cagliari cohort including middle-aged adults and elder adults (CMAE) (n = 98) (age ranging from 36 to 85 years) with subjects recruited in the same area of CY cohort. The demographic features of the three cohorts are summarized in Table 1. All subjects were recruited through public advertisements. Specifically, in the case of LBZ cohort, subjects aged 90 years or older were considered eligible participants for this study and were recruited through the Longevity Blue Zone Observatorya research center which is systematically collecting demographic and individual data of people ≥65 years old from this area 75 . After excluding residents born outside the LBZ, a blood sample of each eligible subjects was obtained during a home interview. All subjects were informed concerning the procedure and the purpose of the study. All participants provided a signed informed consent form. The study was performed according to the guidelines of the Declaration of Helsinki of 1975 (revised in 1983), and the procedures involving human participants were approved by the ethical committee of the University of Sassari. Molecular analyses. DNA was extracted from blood samples by using the QIAamp ® DNA Mini Kit (QIAGEN Hilden, Germany) according to the manufacturer's instructions. Its concentration was assessed by measurements at an optical density of 260 nm with an Agilent Cary 60 UV-Vis Spectrophotometer (Agilent, Palo Alto, CA). All subjects were genotyped for 3 SNPs at base pairs (bp) 145 (C/G), 785 (C/T), and 886 (G/A) of TAS2R38 and for SNP, rs1761667 (G/A), by using TaqMan SNP Genotyping Assay (C_8876467_10 assay for the rs713598; C_9506827_10 assay for the rs1726866 and C_9506826_10 assay for the rs10246939) according to the manufacturer's specifications (Applied Biosystems by Life Technologies Milano Italia, Europe BV). Replicates and positive and negative controls were included in all reactions.

Discussion
In the present work we studied the role of the bitter receptor, TAS2R38 and its genetic variants on longevity. Our results show that the genetically homogeneous cohort of subjects ranging in age from 90 to 105 years of the an area, which was recognised as one of the world's longevity hot spots (Longevity Blue Zone) 80 , differed based on the genotype distribution and haplotype frequencies of TAS2R38 gene from the two genetically heterogeneous cohorts from the South of Sardinia where the longevity level is distinctly lower. Conversely, no differences were found between these two latter cohorts. Specifically, the centenarian cohort showed an increased frequency of subjects carrying the homozygous genotype for the dominant haplotype (PAV/PAV) (34.04%) and haplotype PAV (55.32%) and a reduced frequency of subjects who had the homozygous genotype for the recessive haplotype (AVI/AVI) (18.08%) and haplotype AVI (40.42%). Otherwise, the frequencies determined in the two cohorts from the South of the island, which had a prevalence of subjects carrying the homozygous genotype AVI/AVI (26.52% and 35.71%) and haplotype AVI (51.10% and 58.16%), were similar to those already reported for the Caucasian population in this area 25,[81][82][83][84][85] .
In most populations, females have been reported to live longer than males, with geographical differences in the female/male ratio 86 . However, the population living in the Sardinian LBZ is an exception among the other long-lived populations since the female/male ratio in the oldest old is close to 1:1 80 . In addition, several studies have shown that PROP phenotype and genotype variations are associated with gender, with females being more responsive than males 14,87 . We did not find an effect of gender on the genotype distribution and haplotype frequencies of TAS2R38 locus in the three cohorts studied in this work. By considering specifically the centenarian cohort (which includes a balanced number of males and females), this result allowed us to exclude that the increased frequency of subjects carrying the genotype PAV/PAV and haplotype PAV and the reduced frequency of subjects with the genotype AVI/AVI and haplotype AVI, that we found in this cohort, can to be due to gender bias.
It is well known that an improving diet aimed at increasing intake of fruits and vegetables instead of fat-rich foods may control obesity and reduce the risk of several diseases 88,89 . A number of studies on human nutrition www.nature.com/scientificreports www.nature.com/scientificreports/ have suggested that the TAS2R38 variants and the related PROP phenotype may influence dietary behaviour and nutritional status 1 . The possible association between PROP responsiveness and perception and intake of fats has been extensively studied, but with controversial results 1,13,16,17,20,26,28,30,33,34,[38][39][40]90 . The widely accepted hypothesis is that PROP non-tasters, compared to PROP super-tasters, show a reduced ability to perceive dietary fat which could lead them to increase the consumption of high-fat foods to compensate the reduced perception 57 . In agreement with this assumption, the high frequency of the tasting homozygous genotype (PAV/PAV) and the low frequency of the non-tasting one (AVI/AVI), that we found in centenarian subjects, suggest that these individuals may have reached an exceptional longevity because of their genetic predisposition to a low-fat diet. On the other hand, the extreme bitterness intensity of PROP super-tasters has been shown to be the primary reason for avoiding bitter-tasting fruits and vegetables 57,91 . Since many bitter-tasting compounds in foods (e.g., flavonoids, phenols, glucosinolates) have benefit effects for health 92,93 , our results in the centenarian cohort seem to be in contrast with the possibility that TAS2R38 genotype is a genetic factor that favour an adequate intake of fruits and vegetables or other bitter foods recommended for a healthy life. However, only a few studies have investigated the relationship between TAS2R38 variants and vegetable intake obtaining controversial results 35,38,91,94 . Although, the notion that TAS2R38 might serve to govern food intake is interesting, eating behaviour is a complex phenomenon influenced by a broad range of environmental factors, including social cues, socioeconomic status, culture and education, as well as by individual features, such as gender, body weight, ethnicity and health status 57 . All these factors will be considered in future studies aimed at analysing the role of TAS2R38 variants in the diet of centenarians.
In conclusion, our findings providing evidence of an association between genetic variants of TAS2R38 gene and human longevity, highlight the role of the G protein-coupled receptor (GPCR), TAS2R38 in the molecular physiological mechanisms of the bitter perception associated with important factors involved in the biological process of aging, and suggest that individuals who have a pair of functional alleles (PAV/PAV) at TAS2R38 gene may have a favourable genetic condition for the attainment of exceptional longevity.