In this cross-sectional study conducted on community-dwelling elderly subjects at high cardiovascular risk, we report for the first time that low sTWEAK concentrations were associated with increased risk of MetS, independent of several confounders. Abdominal obesity, hyperglycemia and hypertriglyceridemia were the main contributors to this association. These findings add new knowledge to the current scientific literature, and suggest that sTWEAK may have a role in metabolic disorders such as MetS.
Overall, almost all the available studies have confirmed that patients with CVD or CVD-related diseases have decreased sTWEAK concentrations. In previous studies, a reduced concentration of sTWEAK has been described in patients with such pathological conditions as chronic heart failure , coronary and peripheral arterial disease [7–9], hypertension , T1D and T2D, and/or end-stage renal failure [10, 15, 16]. These data are in agreement with the findings of our study, which indicated a link between low sTWEAK concentrations and MetS, a cluster of metabolic disorders which predispose to the development of T2D and atherosclerosis, and increase the risk of CVD . In line with this, several authors have suggested that reduced levels of sTWEAK might serve as a novel biomarker of atherosclerosis [5, 10]. In fact, it has recently been reported that low sTWEAK levels were associated with long-term cardiovascular mortality in symptomatic PAD patients , and could be considered as a predictor of future cardiovascular outcomes in non-dialysis CKD patients . It is also important to mention that in our study as the number of MetS components increased, the sTWEAK values gradually decreased. In keeping with these observations, we found that individuals with lower sTWEAK levels were more likely to have more MetS components. Thus, the consideration of sTWEAK as an indicative biomarker of risk factors for CVD is reinforced by our findings in MetS patients. Although our data support the possible existence of etiological associations, the biological mechanisms through which low sTWEAK levels were related to MetS remain speculative. Obesity-induced inflammation might be one of the mechanisms explaining the observed association in our study.
Vendrell et al.  reported that TWEAK and Fn14 gene expression were higher in the adipose tissue of severely obese patients than in controls, and that inflammatory stimuli in vitro induced by lipopolysaccharide and TNFα up-regulated TWEAK in THP-1 macrophages and Fn14 expression in SGBS adipocyte cells, respectively . Despite the moderate inflammatory capacity of the sTWEAK cytokine over the adipocyte, a competitive interfering activity with TNFα signalling in the adipocyte has been described [27, 28]. An imbalance of sTWEAK forms (membrane bound mTWEAK and soluble sTWEAK) in the obese state may help potentiate the inflammatory effect of TNFα over the adipocyte.
Our finding that lower sTWEAK concentrations were related with abdominal obesity agrees with the data obtained by Maymó-Masip et al. , which show that sTWEAK levels in severely obese patients are lower than in controls, and that their concentration increases after weight loss. This may suggest that sTWEAK plays an anti-inflammatory role in this setting, although additional in vivo and in vitro studies are required. Moreover, in the case of severely obese individuals they found that the main determinant of the sTWEAK circulating levels was BMI, in an inverse-dependent manner .
There is no clear explanation for the mechanisms leading to lower levels of sTWEAK in individuals with MetS. The rationale that low levels of sTWEAK, unlike other cytokines, appear to protect against pathological conditions associated with increased chronic inflammatory activity is incompletely understood. Several conceivable explanations have been proposed. For instance, sTWEAK may have a beneficial effect on the regulation of the immune response because it has been shown that TWEAK-deficiency in mice leads to overabundant natural killer cells and hypersensitivity to bacterial endotoxin, with an excess of interferon-γ and IL-12 production from innate immune cells. Furthermore, sTWEAK inhibits the activation of the transcriptional stimulator signal transducer and activator of transcription (STAT)-1, and induces the association of p65 NF-κB with histone deacetylase 1, thus repressing cytokine production . A reduction of sTWEAK in serum, due to uptake by the Fn14 receptor, has also been postulated. Endothelial dysfunction is the initial pathophysiological step in the progression of vascular damage that precedes and leads to clinically visible CVD . Under these conditions, Fn14 expression is increased in the endothelium. Recently, we reported increased Fn14 expression in human adipocytes from severely obese subjects . These cells also showed an increase in Fn14 expression after inflammatory stimulation, thus increasing availability for the sTWEAK ligand, which could lead to a peripheral reduction in serum sTWEAK [26, 27]. An alternative hypothesis proposes the involvement of CD163, a monocyte-macrophage surface receptor which seems to act as a scavenger receptor for sTWEAK . Thus, the reduction in sTWEAK could be related to the presence of sCD163, which is up-regulated in patients with CKD and also in obese subjects [33–35]. This incremental increase could enable sTWEAK degradation by inflammatory macrophages, leading to decreased sTWEAK levels and the reduction in the sTWEAK/sCD163 ratio observed in such diseases as CKD . Thus, low sTWEAK levels may be related to the degree of macrophage activation.
In our study, we found that reduced levels of sTWEAK were not only related to abdominal obesity, but also to such other MetS components as hyperglycemia and hypertriglyceridemia. In this regard, we also found that in human studies, changes in circulating free fatty acids can influence circulating levels of sTWEAK, indicating that lipotoxicity could be an important factor that regulates sTWEAK levels in an inverse manner . Our findings with regard to hyperglycemia are in line with previous studies reporting an inverse association between sTWEAK and such glucose metabolism-related parameters as fasting glucose and HOMA-IR [5, 10, 15, 16]. In fact, impaired fasting plasma glucose levels are recognized risk factors for the development of T2D . Interestingly, in a large prospective nested case–control study we found that reduced sTWEAK levels were associated with T2D incidence . Consistent with our findings, in a study of 60 individuals with chronic hemodialysis (32 had T2D) and 60 controls (30 had T2D), the sTWEAK concentrations in T2D patients on hemodialysis were lower . Conversely, in vitro studies showed that NF-κB activation affects TWEAK-induced insulin resistance by attenuating the action of insulin in hepatocytes . Anyhow, in vivo experiments or animal models may help to clarify the effect of TWEAK on insulin resistance.
At the present time, how TWEAK binding to Fn14 can have such diverse cell-type specific biological effects is not understood, but the differential activation of intracellular signalling cascades is likely to be one explanation. TWEAK can have either positive (beneficial) or negative (detrimental) biological effects, depending on the type of cell, tissue injury and/or the stage of the disease [3, 4].
Thus, considering these observations as a whole, we suggest that TWEAK contributes to the inflammatory/anti-inflammatory imbalance observed in obesity, particularly abdominal-visceral obesity and the insulin resistance state, and largely explains the association of TWEAK with hyperglycemia  and the future development of T2D. The examination of the biological mechanisms through which sTWEAK improves insulin sensitivity has demonstrated that, in visceral adipocytes, treatment with sTWEAK ameliorates TNFα-induced insulin resistance on glucose uptake [39, 40]. This occurs because it abolishes the stimulatory effect of TNFα on JNK1/2 kinase, which is directly involved in the development of insulin resistance . This effect is at least partly produced by a reduction in the cellular concentration of TRAF2, leading to a curbing of TNFα intracellular signaling events. Furthermore, this modulation of TNFα-induced changes in insulin sensitivity was found to be associated with an increase in the activity of PP2A, a Ser/Thr phosphatase, known to negatively regulate cytokine signaling . Additionally, in human subcutaneous adipocytes, sTWEAK exerts a modulatory effect on TNFα-induced cytokine production by inhibiting the MAPK and NF-κB signalling cascades commonly used by TNFα .
This protective/modulatory effect of sTWEAK on TNFα activity has been observed in such pathologies as rheumatoid arthritis , ischemic stroke  and several tumour epithelial cell lines , suggesting general competitive behaviour between sTWEAK and TNFα.
Lower levels of sTWEAK have been reported in young individuals with primary hypertension . However, our study conducted in an older population did not provide any evidence to support that reduced levels of sTWEAK are associated with hypertension. The fact that the majority of subjects (90%) were hypertensive might partly explain the lack of associations observed between sTWEAK and the hypertension component of the MetS.
Notwithstanding the results observed, we should point out that our study has several limitations that should be kept in mind. Firstly, a major limitation is that the population studied consisted of elderly Mediterranean Caucasian individuals at high risk for CVD, so our findings cannot be extrapolated to other age groups or ethnicities. Secondly, although rare, we could not completely discard the presence of coronary heart disease in our population explaining the associations observed, given that neither echocardiography nor stress testing were systematically performed in our cohort. Finally, another limitation of our study is inherent to the study design. Because of the cross-sectional nature, conclusions on a cause/effect relationship cannot be drawn.
In conclusion, in the present study we have demonstrated that low TWEAK concentrations are associated with increased risk of MetS, and this relationship was mainly mediated by abdominal obesity, hyperglycemia and hypertriglyceridemia. These findings suggest that sTWEAK may have a key role in the development of these metabolic disturbances, supporting the concept that sTWEAK may be considered as a potential novel biomarker of MetS with a putative protective effect. Further longitudinal studies are warranted to confirm these results in different populations.