Our results strongly suggest that: a) it is possible to evaluate IS in blood cells such as lymphocytes, using a relatively simple and repeatable procedure, b) mTOR, which regulates replacement of damaged blood and endothelial cells with consequent maintenance of vasculature integrity and potential regulation of thrombotic phenomena and other molecules involved in the intracellular IS are significantly altered in patients with MetS. Consequently, mTOR cellular expression can be used to evaluate the disease status and the risk of vascular thrombosis.
Intracellular Insulin Signaling
Reduced mTOR has many important consequences for cell metabolism and life span. Indeed, mTOR is stimulated by insulin signaling, nutrients, catabolic hormones, cytokines and growth factors . It activates not only the protein synthesis through the phosphorylation of the enzyme p70S6K1 but also regulates important enzymes for cell life. Inhibition of mTOR reduces translocation of a subset of mRNAs and dramatically represses ribosomal mRNA and tRNA transcription. In addition, the use of specific mTOR blockers stop cell cycle progression in the early G1 phase of the cell cycle, driving cells into G0 state promoting apoptotic processes [14, 15]. Moreover, mTOR not only avoids blood/endothelial cellular apoptosis but also probably repairs and replaces damaged endothelial cells stimulating endothelial progenitor cells with consequent maintenance of vascular functions including blood coagulation .
The role of mTOR in regulating blood coagulation has been recently clinically demonstrated. Indeed, cases of important de novo thrombotic micro-angiopathy have been reported in renal recipients treated with the mTOR inhibitor Sirolimus and Everolimus [8, 9]. Treatment of micro-angiopathy is based on removal of these drugs. This would indicate that mTOR inhibition is crucial to decrease thrombotic events. Consequently, the significant reduction of mTOR found in MetS patients suggests that mTOR might be, in part, responsible for increased cardiovascular thrombotic diseases seen in MetS.
In addition, we know that mTOR and other molecules linked with this kinase, are influenced by circulating inflammatory cytokines and the nervous system. Indeed, cytokines such as TNF alpha - which are high in MetS-patient blood cause serine phosphorylation of IRS-1 and inhibit its tyrosine phosphorylation with consequent impairment of mTOR function .
Interestingly, Morisco et al. also recently demonstrated the presence of a cross-talk between β-adrenergic stimulation and IS by AKT, suggesting that there is an inter-relationship between the activation of the sympathetic nervous system and IS including AKT which influences mTOR function . The role of mTOR and cross-talk with inflammatory and sympathetic systems and insulin signaling are very new and interesting observations and deserve further study to understand the molecular pathophysiology responsible for the increased cardiovascular disease associated with MetS. Moreover, we have recently shown that maintenance of cellular mTOR function by anti-hypertensive drugs improves insulin signaling increasing GLUT 4 expression and prevents micro-vascular rarefaction in spontaneously hypertensive rats with insulin resistance. This effect was independent of the reduction of blood pressure but was mTOR-related. .
We also found impairment of intracellular insulin signaling in patients with MetS. Indeed, insulin signaling is a complex phenomenon where mTOR plays a fundamental role. [5, 7]. In detail, insulin binding to its specific receptor leads to the autophosphorylation of the trans-membrane β receptor sub-units and tyrosine phosphorylation of IRS-1 after their recruitment to the cell membrane. When IRS-1 is activated, it stimulates GLUT 4, with consequent regulation of glucose and lipid intracellular metabolism. In addition, activated IRS-1 modulates the phosphoinositide 3-kinase (PI3K) that in turn indirectly stimulates the activity of mTOR . As discussed before, mTOR is a central regulator of cellular responses to hormones, growth factors and nutrients [7, 20]. Current understanding of insulin signaling regulation considers IRS-1 to be a key protein in this cascade and mTOR activation.
The main cellular molecular mechanism of insulin desensitization, with consequent insulin resistance presents in MetS-patients, involves increased serine phosphorylation and decreased tyrosine phosphorylation of IRS-1. This is true in type 2 diabetic patients as well as in experimental models of insulin resistance. Phosphorylation of the tyrosine residues 608 on IRS-1 after insulin stimulation is necessary for propagation of the signal with consequent active-mTOR expression.
On the contrary, phosphorylation of serine residues leads to reduced insulin signaling [21, 22]. It has therefore been proposed that changes in the equilibrium between serine or tyrosine phosphorylation lead to pathological conditions of insulin resistance and diabetes.
IRS-1 function is also negatively regulated by other circulating molecules found in the MetS such as catabolic hormones and inflammatory molecules . Indeed, recent data has shown that the cytokine leptin promotes phosphorylation of serine 318 in IRS-1 in both skeletal muscle and in lymphocytes of obese and diabetic hyperleptinemic patients . This would suggest: 1) that cytokines impair IRS-1 activity, blocking anabolic insulin signaling cascade with less activated mTOR and 2) that the molecular mechanism of leptin-mediated impairment of insulin signaling is similar in both skeletal muscle and lymphocytes.
Surprisingly, in our study p-serine-636/639-IRS-1 was significantly less in patients with MetS while there was a slight increased total IRS-1, although this was not statistical significant. We can explain these findings by considering that serine phosphorylated IRS-1 is rapidly eliminated in the cell cytoplasm like many other activated or deactivated molecules involved in intracellular signaling. Therefore, we probably observed the amount of p-serine-636/639-IRS-1 in the cell after its degradation. Indirect confirmation was the slightly increased total IRS-1, suggesting the cell's attempt to maintain adequate total concentrations of IRS-1 which could be activated or inactivated in response to specific stimuli. All these intracellular molecular data explain the phenomenon of insulin resistance present in patients with MetS.
Patients with MetS, in particular those with overt diabetes mellitus, are disproportionately affected by cardiovascular disease, compared with those without diabetes, due to a particularly pronounced atherosclerosis progression. Evidences suggest that insulin-resistance, diabetes and coronary heart disease share in common a deregulation of ubiquitin-proteasome system, a major pathway for nonlysosomal intracellular protein degradation in eucaryotic cells . This might represent a common persistent pathogenic factor mediating the initial stage of the atherosclerosis as well as the progression to complicated plaque in diabetic patients .
In obesity and in diabetes mellitus, an increase in plasma free fatty acids, even still within the physiological range, might induces markers of endothelial activation, vascular inflammation and thrombosis . Even transient and modest increases in plasma free fatty acids, also seen in healthy subjects may initiate early vascular abnormalities that promote atherosclerosis and cardiovascular disease . Finally, also changes in the immune system might play a role in cardiovascular pathology. Biological processes altered in T cell aging are not only those typically associated with immune cells (T cell receptor signalling, cytokine-cytokine receptor interactions, etc.) but also some not specific of immune cells, such as peroxisome proliferator-activated receptors and mTOR signalling, as well as glucose and glutathione metabolism, suggesting that T cell aging may be representative of a more generalized aging phenomenon , with features characteristic also of the MetS.
Surprisingly enough, our patients with or without MetS do not differ significantly in the levels of HDL cholesterol, although patients with MetS tended to have lower values. We have no good explanation for this observation, apart from the relatively modest number of patients and subjects evaluated.
The clinical perspective
MetS has many different clinical signs which include obesity, hypertension, diabetes and alteration of lipid metabolism . Furthermore, recent research has shown that circulating molecules such as stress hormones and inflammatory cytokines increase in patients with MetS and they can influence and/or worsen IS including the central role of mTOR.
However, little is known about the intracellular molecular mechanisms present in MetS. We have demonstrated that IS is impaired in patients with MetS. Consequently, the observed molecular alterations can be used as biomarkers of this disease and its evolution.
We not only analyzed mTOR but also its downstream effectors p70S6K and 4EPB1 which stimulate anabolic pathway and other fundamental biochemical pathways such as the production of adhesion molecules, replace damaged cells and cell survival (including blood and endothelial cells with consequent regulation of blood coagulation). We also investigated the molecules which regulate important intracellular metabolic pathway such as cellular insulin stimulated molecules.
For mTOR evaluations we have developed a method that allows the study of IS in human peripheral mononuclear cells. We believe that our method has some relevant advantages; these are namely; 1) it is relatively easy to perform and may be repeated several time in the same subject, allowing the evaluation of time the time course of changes or the effect of treatment, 2) it avoids the pain or discomfort related to muscle biopsies, 3) it allows us to identify and quantify intracellular molecular damage and/or to study molecules which could link MetS, sympathetic activation and cell energy regulation. In addition, as it is repeatable, this method could be useful to assess the effects of interventions with specific therapeutic strategies such as drugs, weight reduction and/or physical training. Further investigation is needed to evaluate any correlations between intracellular molecular alterations and cardiovascular disease in a large scale study.