Physical decline and survival in the elderly are affected by the genetic variability of amino acid transporter genes

Introduction

One of the most dramatic modifications associated with human aging is the progressive decline in skeletal muscle mass and function, known as sarcopenia. Sarcopenia causes physical function impairment, leading to a loss of functional independence and to an increased incidence of adverse health outcomes [1,2]. The prevalence of this condition, which is progressively increasing due to the prolonged life expectancy, has led in the recent decades to increased needs for health care services and resources to support older people [3]. The rate of skeletal muscle loss is estimated at 8% per decade from the 4th until 7th decade, with about 15% lost each decade after 70 years of age and loss of strength is estimated to be even longer [4].

The cause of sarcopenia is widely regarded as multifactorial, with chronic diseases, hormonal and inflammatory changes, mitochondrial dysfunction, physical inactivity, and malnutrition, being the main risk factors [5]. In addition to these causes, loss of muscle has been related to the imbalance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB), associated with alterations in muscle anabolic responses to nutritional stimuli and physical activity, the so-called “anabolic resistance” according to which in the elderly process the “anabolic threshold” required to maximize anabolic pathways is increased [6,7].

Amino acids (AAs) availability is among the most important anabolic signals for MPS [8], and essential amino acids (EAAs), leucine in particular, has been shown to be critical in the regulation of skeletal muscle protein synthesis and degradation in the elderly [9]. There is growing evidence that AA transport into muscle cells may be a rate-limiting step in the process of AA induced stimulation of skeletal muscle protein metabolism, and that AA transporters may have a key role in such process as AA sensors [10]. This is likely linked to the ability of AA transporters to act as both transporter and receptor (transceptor) [11,12] able to transduce a signal reflecting AA availability and leading to activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), the major nutrient-sensitive signalling pathway [13]. Thus, AA transporters may be an important link in the ability for AAs to stimulate MPS. This has been supported by studies in human skeletal muscle showing that the expression of AA transporters is highly dynamic and responsive to different anabolic stimuli [14]. In particular, in 2010, Drummond and colleagues [15] characterized the expression of selected AA transporters in the human skeletal muscle following AA ingestion in a group of healthy young individuals. They found increased mRNA expression of L-type AA transporter SLC7A5 (LAT1), SLC3A2 (CD98), sodium-coupled neutral AA transporter SLC38A2 (SNAT2), and proton-coupled amino acid transporter SLC36A1 (PAT1), which are transporters thought to have key roles in mTORC1 signalling regulation and muscle protein synthesis and muscle growth. These authors argue that, changes in the expression levels of AA transporters, possibly mediated by mTORC1 activity, could serve as an adaptive response for improving AA intracellular delivery and for regulating the rate of muscle protein synthesis in response to anabolic stimuli or during periods of decreased muscle protein synthesis [15]. Consistently with this hypothesis, the same authors showed that an up-regulation of the above transporters occurred after resistance exercise in both young and older adults, likely regulated in an age-dependent manner [16], as well as in older persons in conditions of short-term bed rest [17]. Soon after that, Dickinson and co-workers [14] reported that aging differentially affected the expression of SLC7A5 and SLC38A2 in the skeletal muscle when EAAs were ingested after exercise, suggesting that aging may influence the function of specific AA transporters, and possibly age-related phenotypes, such as sarcopenia [14,18].

Based on these evidences, to shed a light on the impact of the above AA transporters on the age-related loss of muscle strength and physical performance, we screened genetic variants occurring in their genes in a population of elderly subjects (< 90 years), analysing them in combination with parameters of physical status. Moreover, beside these four genes [SLC7A5 (LAT1), SLC3A2 (CD98), SLC38A2 (SNAT2) and SLC36A1 (PAT1)], we also included variants located in SLC7A8 (LAT2) and SLC43A1 (LAT3) as leucine transporters like LAT1, SLC1A5 (ASCT2) as a glutamine transporter like SNAT2, SLC38A3 (SNAT3) and SLC38A7 (SNAT7), preferentially transporting glutamine and arginine, SLC38A9 (SNAT9), as an arginine transporter [19,20].

To investigate whether variants in the selected genes also affect the chance to survive and/or reach very advanced ages, we exploited both the cross-sectional and longitudinal approach by taking advantage of the available dataset, which also included very old subjects (≥90 years), and survival data within 10-year follow-up period from the baseline visit.

Results

Demographic, clinical, and anthropometric characteristics of the analysed sample are presented in Table 1. Among the selected SNPs, 10 did not pass the QC phase. In particular, four SNPs were excluded due to a MAF (Minor Allele Frequency) lower than ten percent per locus (rs17112008, rs7968173, rs1175, rs7735053), while six were excluded because they showed a significant deviation from HWE in control subjects (rs11749532, rs7736177, rs2897968, rs17794251, rs7193392, rs8058969).

Association with survival

Next, using 10-year of follow-up survival data, we investigated the association with survival of the analysed SNPs, estimating their quantitative effects. An increased risk of death was found for subjects carrying the C allele at rs14160 (SLC36A1) compared to those with the TT genotype (adjusted HR = 1.59, 95% CI: 1.025–2.47; p=0.038) (Figure 1a). In SLC38A9, the presence of the A allele for the rs10056358 variation confers a survival advantage before 90 years of age, compared to those with the TT genotype (adjusted HR=0.48, 95% CI 0.27-0.84, p=0.009) (Figure 1b), and a disadvantageous effect later, (adjusted HR = 1.43, 95% CI 1.01-2.03; p= 0.045) (Figure 1c).

Figure 1. Survival function of carriers of minor allele (black) vs non carriers (grey). (A) rs14160 (SLC36A1) in subjects aged <90 years; (B) rs10056358 (SLC38A9) in subjects aged ≥90 years; (C) rs10056358 (SLC38A9) in subjects aged > 90 years. Time is expressed in months, where 0 is considered the time of recruitment, and each individual is followed up for survival status till death. HR value, confidence interval and p-value from Cox regression analysis are reported inside the figure.

Analysis of all other SNPs showed no statistically significant or suggestive differences in survival between genotypes.

Discussion

The goal of the current study was to investigate the impact of SNPs in selected AA transporters genes on physical performance and survival at old age.

Our analysis of a group of elderly individuals in the age ranges of 50-89 years, showed that genetic variants in SLC7A5 (rs4329925) and SLC7A8 (rs999165 and rs12588118), negatively affected HG strength, a reliable marker of the muscle performance and an indicator of quality of aging [21]. Additionally, different SNPs in the same genes (rs731710 in SLC7A5 and rs3783436 in SLC7A8) were significantly associated with ADL levels. SLC7A5 and SLC7A8 code, respectively, for the plasma membrane proteins LAT1 and LAT2. Each of them forms with CD98 (SLC3A2) a heterodimeric bidirectional antiporter that regulates the simultaneous transport of leucine into cells and efflux of glutamine out of cells. Intriguingly, we found that two variants in SLC3A2 (rs12804553 and rs4726) were associated, with opposite effects, with walking ability. These findings point to a role of the LAT1(LAT2)/CD98 complex in affecting muscle strength and physical performance in the elderly, likely through regulation of the anabolic signal mediated by the intracellular leucine/glutamine levels, and downstream activation of mTORC1. On the other hand, it is well known that leucine provides direct anabolic stimuli to skeletal muscle, so that the post-exercise leucine assumption is considered a potential treatment in numerous muscle wasting conditions [18]. Similarly, glutamine status is considered a hallmark of catabolic states and muscle loss [22].

Further supportive evidence to the above hypothesis is provided by the association with genes that mediate the transport of glutamine, such as SLC38A2 (SNAT2) and SLC38A7 (SNAT7). We found, indeed, significantly worse ADL scores associated to rs1873793 (SLC38A2), a finding supported by a disadvantage in attaining longevity for subjects carrying the same allelic variant, and a better walking performance associated to rs9806843 (SLC38A7). For the above transporters an important role in mTORC1 activation was documented. For instance, a reduction of SNAT2 activity in muscle cells is associated with reduced levels of glutamine and leucine and impaired protein synthesis through mTORC1 [23]; furthermore, it was found that the expression of system SNAT2, LAT1 and CD98 was upregulated after leucine availability in L6 myotubes [24]. The importance of these AA transporters is further bolstered by the fact that the heterodimer LAT1-CD98 also regulates the leucine flux into the lysosome [25], the major cellular compartment for mTORC1 activation [26], and that SNAT2 may act as a transceptor involved in both amino acid transport and signal transduction [27].

Although the mechanisms by which aging alters leucine-induced protein synthesis are currently under investigation, our data are in accordance with the hypothesis that a concerted activation of AA transporters may occur differently in young and older adults, finally influencing the anabolic response of skeletal muscle to AA availability. This hypothesis is supported by Drummond and Dickinson’s work showing that aging differentially affects the expression of SLC7A5 and SLC38A2 in the skeletal muscle, in response to resistance exercise and essential AA supplementation [14,17]. These evidences are also in keeping with our findings regarding SLC36A1 (PAT1), which has been reported to function as transceptor on the lysosomal membrane [28]. For this gene, we found the minor alleles at rs357618 and rs357629 (in LD with each other) associated with a better ADL performance. A different variant of SLC36A1 (minor allele of rs14160), was found, instead, to increase the risk of death along a 10 year follow up of survival in our younger sample. Similarly, different SLC3A2 variants showed cross-phenotype associations on different age-related phenotypes [29]. As to SNAT9 (SLC38A9), another lysosomal transceptor [30–32], we found a complex and dynamic association with survival chance. In fact, while a survival advantage was found for carriers of the rs10056358 A allele in the sample group aged 50-89 years, 90-plus subjects carrying this allele showed a lower probability to survive. Accordingly, the same minor allele A negatively influenced the probability to achieve longevity. Thus, this SNP manifests an antagonistic pleiotropic effect on survival at old ages, with the rs10056358-A allele conferring a positive effect on survival at ages before 90 and negative effect afterwards. The absence of correlation between SLC38A9 gene and the parameters of physical performance suggests that it functions as a pleiotropic gene with age-dependent effects, affecting survival and longevity but not quality of aging [33,34]. On the contrary, conditional effects of these genes on quality of aging but not on longevity are shown for the most part of the genes studied in this work.

Overall, our data provide evidence that the genetic variability of these genes may impact on muscle performance and/or physical decline in adulthood, as well as on the probability to survive at old age. Thus, taking into account that AA transporters may act as sensors of AA availability and that this availability is closely related to skeletal muscle metabolism, AA transporter genetic variability may act at forefront of individual susceptibility to anabolic resistance experienced with age, acting as risk factor for the onset of muscular decline in the elderly population.

Hence, understanding the functional implications of the associated SNPs may represent an important indication for elucidating the possible molecular mechanism underlying the associations found. However, the majority of the associated SNPs are non-coding variants, except two SNPs located in the coding regions. Our bioinformatic analyses identified, for some of them, a number of features (histone modifications, DNase I hypersensitivity clusters and transcription factor binding sites) consistent with a possible regulatory function, although we cannot exclude the possibility that the associated variants represent proxies for unknown causal variants as a result of LD, apart from those not showing any LD in their genomic region (rs12794763 and rs999165).

We are aware that our study has some weaknesses that should be addressed. A first limitation of the study is the lack of proper correction for multiple testing. Since this study was exploratory, a Bonferroni correction would have eliminated potentially important findings if applied. Another possible limitation could be the time of the survival follow up, not sufficient to draw long-term conclusions on the effect of genetic variants with minor effect on survival. Furthermore, the sample size could be increased and further explorations in additional study populations are needed before conclusions can be drawn.

Notwithstanding, and considering that this is the first study reporting genetic variants in AA transporter genes associated to the age-associated decline of muscle performance, we believe that our findings can open future investigations on the role of AA transporters in the quality of aging and longevity. This could provide valuable insights into potential targets for risk stratification in the population, and for therapeutic interventions aimed at increasing muscle mass and strength at old age.

Methods

Supplementary Materials

Supplementary File