In this study, we investigated whether a specific plasma FA profile and/or modification in eCBome-related mediators are associated with the exceptional longevity of the HLZ population of Sardinia. Our data show a characteristic plasma FA profile and modifications in ECs-related molecules that may be consistent with the maintenance and increased capacity to regulate homeostatic balance against environmental challenges in octogenarians from the HLZ.

Higher levels of plasma POA may be a distinctive feature of the HLZ and a sign of a favourable metabolism since it is associated with enhanced insulin sensitivity, decreased lipid accumulation in the liver, and significant amelioration or prevention of diabetes [63, 64]. In addition, POA derived from adipose tissue DNL protects the adipose tissue against the deleterious effects of dietary lipid exposure, strongly stimulates muscle insulin action and suppresses hepatosteatosis in mice [63]. Moreover, adipose tissue DNL has been associated with caloric restriction [65], which has been shown in numerous mammalian species to prolong life span and delay the development of ageing-associated diseases such as diabetes and atherosclerosis [66]. It is therefore highly plausible that enhanced DNL in adipose tissue improves glucose homeostasis and may mediate favourable metabolic effects, possibly including the release of POA into the circulation [67]. On the contrary, PA levels did not differ amongst the groups; this POA precursor is implicated in various physio-pathological conditions such as atherosclerosis, neurodegenerative diseases and cancer [68, 69]. The fact that Old-HLZ participants displayed circulating levels of POA similar to those in Young-HLZ leads us to hypothesise that more efficient DNL in adipose tissue leading to a general improvement in insulin sensitivity and consequent amelioration of muscle efficiency occurs in Old-HLZ and not Old-LLZ. This is in agreement with the exceptional physical functionality in relation to an high total energy expenditure that we observed in octogenarians from the HLZ [57]. The increase in SA levels in Young-HLZ may be also derived from adipose tissue DNL.

Moreover, we found significantly higher circulating levels of CLA in Old-HLZ subjects compared to both Old-LLZ and Young-HLZ, which might reflect a greater intake of dairy products in Old-HLZ [70] or the presence of specific CLA-producing gut microbes [9, 12, 13] such as Bifidobacterium spp., known to be higher in centenarians [71]. This could be relevant because a significant number of studies report CLA effects [19, 20, 22] including enhanced fat utilization in muscle via FA beta-oxidation [22] and the capacity to prevent age-associated obesity and muscle loss as demonstrated in mice models, with the potential to prevent sarcopenia and sarcopenic obesity [72–74]. Based on the biohydrogenation pathway observed in the rumen, some authors have proposed that a similar pathway can also occur in the human gut due to the presence of an extensive number of bacteria highlighting their metabolic potential in host physiology [75–77].

The small but significant differences found in the circulating levels of LA may be due to either slight changes in dietary intake and/or enhancement of LA beta-oxidation. In any case, while this increase was mirrored by the level of its delta 6 desaturated metabolite GLA, the other important metabolite arachidonic acid did not change significantly.

OCS-FA origin has long been attributed to the diet because they are produced by rumen microbial fermentation and microbial DNL [26, 78, 79] and then incorporated into fat depots. OCS-FA have been shown to have beneficial effects [27, 80] and may exhibit protective properties against several age-related ailments such as Alzheimer’s disease [25], cancer [26], coronary heart diseases [27] and metabolic diseases [27, 28]. The distinctively higher levels of 17:0 in Old-HLZ and 15:0 in Young-HLZ may result from different metabolic pathways. In fact, the lack of correlation between 15:0 and CLA in Old-HLZ and the trend toward negative correlation found between 17:0 and CLA in Old-HLZ may suggest that they do not derive from a common dietary source, i.e., dairy products, as it has been previously suggested for 15:0 [26, 81] but not for 17:0 [78, 82]. In addition, we did not find any correlation between 17:0 and LC-PUFA n-3 (data not shown), which rules out a possible association between 17:0 and the intake of fish as previously demonstrated in a large cohort study that identified a strong positive correlation [82].

An exclusive association of dietary 15:0 and 17:0 and their tissue levels remains elusive but there have not been investigations into the contributions from non-ruminant gut microbiota or from biosynthesis such as alpha-oxidation, a pathway that utilizes the elimination of the alpha-carbon, through the conversion of 16:0 or 18:0 (end products of DNL) to a hydroxyl FA followed by decarboxylation to produce either 15:0 or 17:0, respectively [10, 83, 84]. Thus, we explored whether alpha-oxidation could explain the presence of circulating OCS-FA, particularly 17:0. The observed strong positive correlation between 15:0 and 16:0 and between 17:0 and 18:0 only in Young-HLZ participants and the concomitant higher plasma accumulation of 15:0 strongly suggest that rather than originating from a dietary source; this lipid may derive from an enhanced 16:0 alpha-oxidation. Indeed, this pathway has been shown to be induced by the activation of the PPAR-alpha [85]. PPAR-alpha activation is known to improve lipid and energy metabolism [86]. However, we observed a significant negative correlation between 17:0 and 18:0 in Old-HLZ. Therefore, this rules out the putative origin induced by an alpha-oxidation pathway but rather suggests derivation from the gut microbiota. In fact, 17:0 could be derived by the elongation of propionic acid (3:0), a volatile short chain FA derived from food fermentation that could be trapped by bacteria and used to produce OCS-FA. Indeed, due to its influence on human metabolism and immunology, the gut microbiota has been proposed as a possible determinant of healthy ageing [87, 88]. Thus, the apparent discrepancy found in the literature on the significance of circulating OCS-FA levels may be ascribed to their different origin. In the present study, with correlation analysis, we were able to shed some light on whether they originate from metabolic alpha-oxidation or gut microbiota.

Furthermore, besides its impact on human health and immunity, higher gut microbial diversity and enrichment of several potentially beneficial bacterial taxa have been linked to healthy ageing. For instance, this is the case with well-known beneficial microbes such as Akkermansia muciniphila and Bifidobacterium spp. that are found to be present in higher levels in long-lived people, possibly revealing a link between healthy ageing and gut microbiota [89, 90]. Interestingly, recent strong evidence obtained in humans suggests a link between the microbiota and CLA in the human body [9, 11–13]. Druart et al. have found a positive correlation of CLA tissue level with specific faecal bacteria (Bifidobacterium spp., Eubacterium ventriosum and Lactobacillus spp.) and an inverse correlation with serum cholesterol (total, LDL, HDL). These correlations suggest a potential beneficial effect of some of these metabolites, but this remains to be confirmed by further investigation [13]. Importantly, the bacteria that are found to be linked with CLA production are also known as beneficial microbes (e.g., Bifidobacterium spp., Faecalibacterium prausnitzii) [91]. A recent study shows that alteration of the microbiota via the use of prebiotics was associated with a reduction in low-grade inflammation, improvement in cardiometabolic profile and an increase in the specific taxa known to produce CLA [92]. Therefore, we now have clear evidence that changes in the tissue levels of specific FA, such as CLA, may not only be derived from dietary sources, but can also clearly be produced by the gut microbiota and accumulate in host tissues. Therefore, we can speculate that the microbiota of Old-HLZ, rich in CLA-producing bacteria, can partly explain the higher overall-health profile apparent in the Old-HLZ group.

Interestingly, in Young-HLZ, the observations that (1) the higher circulating levels of retinol, the increase of which has been previously shown to be linked to PPAR-alpha induction [93] and (2) the higher DHA/EPA ratio, previously shown to be a marker of increased peroxisomal beta-oxidation induced by PPAR-alpha [94], together support the hypothesis of enhanced PPAR-alpha activity.

The balance between ageing processes and counteracting homeostatic mechanisms is important in the progression of ageing and there are numerous studies demonstrating that the activity of eCBome can modulate this balance [95]. However, very few data are available concerning the eCBome modifications during age-related processes. We therefore measured a panel of eCBome-related mediators to investigate about a possible association with the exceptional longevity of the HLZ of Sardinia. The significantly elevated plasma levels of the two ECs in Old-HLZ subjects respect to Young-HLZ could modulate several functions through the activation of the cannabinoid receptors CB-1 and CB-2 and non-cannabinoid receptors [96]. Studies in experimental animals showed that the absence of CB1 receptors in specific neuronal types accelerates the appearance of brain ageing indicators, including neuronal loss and chronic neuroinflammation [97]; at the same time, mice lacking CB2 receptors showed a phenotype that is also reminiscent of accelerated ageing [98, 99]. Therefore, the increase of ECs observed in our study could positively prevent the disruption of cannabinoid receptors activity in enhancing the age-related decline in several tissues in which they have important physiological functions [100]. The same ECs can, at submicromolar concentrations, modulate other targets and therefore either reduce or enhance their effects on the cannabinoid receptors. For example, activation of TRPV1 channels, whose activation can both exacerbate and counteract some of the major symptoms in animal models of Parkinson’s and Huntington’s diseases, is the best-established non-CB1, non-CB2-receptor-mediating action of AEA [101–105]. 2-AG can directly enhance GABA_A receptor activity [106], influencing several aspects of ageing and neurodegeneration, the development of neuroinflammation and the establishment of synaptic plasticity. Finally, both AEA and 2-AG may also activate PPAR-gamma, a player in the control of neuronal activity and neuroinflammatory lipids [107, 108]. Notably, the same receptors, following their ligand-dependent activation, can exert important physiological functions also in peripheral tissues, further complicating the already intricate mechanism at central level. Noteworthy, the levels of the two endocannabinoids AEA and 2-AG increased both significantly only in Old-HLZ subjects respect to Young-HLZ and the Old groups respectively. Therefore, the putative beneficial effects, described above, of the combination of these two ECs may regard only the Old-HLZ.

Important findings of our study are the higher OEA, 2-LG and 2-OG levels in Old-HLZ group compared to Young-HLZ (as for OEA and 2-LG) and both the Old-LLZ and Young-HLZ for 2-OG. These molecules, which are congeners of the ECs, are known to be agonists (OEA with higher potency than 2-LG and 2-OG) of the GPR119 receptor [109]. This receptor, once activated, controls glucose homeostasis by enhancing insulin secretion in pancreatic β-cells and by stimulating, by boosting cAMP levels, secretion of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1(GLP-1) and peptide YY (PYY), all gut hormones which in turn induce additional increases in insulin secretion and improve hepatic glucose metabolism [110–113]. All these processes are known to deteriorate during ageing [114], thus leading us to hypothesise a positive metabolic rearrangement in elderly subjects of the HLZ. Conversely, PEA, which is also a potent agonist of GPR119, exhibited lower levels in the Old-HLZ compared with the Young-HLZ subjects, leading us to speculate about a possible counterbalancing mechanism. Notably, both OEA and PEA are also ligands of the PPAR-alpha receptor [115].

Interestingly, previous studies from Everard and Cani [116] demonstrated that A. muciniphila administration significantly increased intestinal levels of 2-OG, which stimulates GLP1 secretion, and 2-AG which was shown to reduce metabolic endotoxemia, peripheral and brain inflammation and circulating inflammatory cytokines. Furthermore, a bioinformatically based analysis on GPCR activity identified a clear overlap in structure and function between bacterial and human GPCR-active ligands for the receptor GPR119. This study leads to the isolation of the palmitoyl and oleoyl analogues of N-acyl serinol, differing from 2-OG by the presence of an amide instead of an ester and from OEA by the presence of an additional methanol substituent [117]. Based on this, we speculate that the changes we observed in the EC-related ligands could directly derive from the gut microbiota. However, more studies to define the role of microbiota-encoded small molecules in host–microbial interactions and in the endogenous mammalian signalling are needed.

Our study also highlighted in Old-HLZ significantly higher levels of the endogenous metabolite of DHA, DHEA, also known as synaptamide [118], which is known to bind the neurotrophic orphan receptor GPR110 (ADGRF1) recently identified as new GPCR target for immune regulatory function by upregulating cAMP-dependent signalling in microglia and innate peripheral immune cells under LPS- or TNF-α-stimulated conditions [119]. Considering the ageing process the result of the occurrence of a low grade chronic pro-inflammatory status called ‘inflammaging’, a term indicating that ageing is accompanied by a mild degree of chronic inflammation and an upregulation of inflammatory response [120], DHEA high levels found in the Old-HLZ group represent a sign of favourable metabolism since it has been shown to improve synaptogenesis, but also to possess a strong anti-inflammatory property [121].