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Inflammaging, Metabolic Syndrome and Melatonin: A Call for Treatment Studies

The metabolic syndrome (MS) is a collection of risk factors for cardiovascular disease, including obesity, hypertension, hyperinsulinemia, glucose intolerance and dyslipidemia. MS is associated with low-grade inflammation of the white adipose tissue, which can subsequently lead to insulin resistance, impaired glucose tolerance and diabetes. Adipocytes secrete proinflammatory cytokines as well as leptin and trigger a vicious circle which leads to additional weight gain largely as fat. The imbalance between inflammatory and anti-inflammatory signals is crucial to aging. Healthy aging can benefit from melatonin, a compound known to possess direct and indirect antioxidant properties, to have a significant protective effect on mitochondrial function, to enhance circadian rhythm amplitudes, to modulate the immune system and to exhibit neuroprotective actions. Melatonin levels decrease in the course of senescence and are more strongly reduced in diseases related to insulin resistance. This short review article analyzes the multiple protective actions of melatonin that are relevant to the attenuation of inflammatory responses and progression of inflammaging and how melatonin is effective to curtail MS in animal models of hyperadiposity. The clinical data supporting the possible therapeutic use of melatonin in human MS are also reviewed. Since attention has been focused on the development of potent melatonin analogs with prolonged effects (ramelteon, agomelatine, tasimelteon, piromelatine) and in clinical trials these analogs were administered in doses considerably higher than those usually employed for melatonin, clinical trials on melatonin in the range of 50-100 mg/day are needed to further assess its therapeutic value in MS.

There is impressive information indicating that the obesity in MS is associated with low-grade inflammation of the white adipose tissue, which can subsequently lead to insulin resistance, impaired glucose tolerance and diabetes [5,6] . Adipocytes actively secrete proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 as well as leptin, and trigger a vicious circle which leads to additional weight gain largely as fat. Increased circulating levels of C-reactive protein and other inflammatory biomarkers also support the occurrence of inflammation in obesity [7,8] .
Several studies have shown that altered production of proinflammatory cytokines modulates adipocyte size and number through paracrine mechanisms that exert an important role in the regulation of fat mass [9][10][11] . The amount of proinflammatory molecules derived from adipose tissue in obese patients diminishes after weight loss [12] . Therefore, the fat cells are both a source as well as a target for TNF-α, IL-1β and IL-6.
The imbalance between inflammatory and anti-inflammatory signals is also a hallmark of aging and contributes to its progression. The term 'inflammaging' was introduced to underscore the importance of inflammation in senescence and its role in the development of age-related diseases as MS [13][14][15] . The levels of inflammatory mediators typically increase with age, even in the absence of acute infection or physiological stress. Such stress leads to inflammatory damage of cellular components, including proteins, lipids and DNA, and contributes to the age-related decline in physiological functions particularly in neural, immune and endocrine cells that regulate homeostasis. Therefore, the functional losses observed during aging include a slowly progressing, persistent type of oxidative stress resulting from the increased production of reactive oxygen species and reactive nitrogen species, which is enhanced by damage to the mitochondria [16,17] .
An age-related proinflammatory tendency is mostly unavoidable because of thymic involution and extended germ exposure, which both lead to the exhaustion of various subforms and developmental stages of leukocytes (details in Hardeland [17] ). However, considerable interindividual differences exist in the velocity of these changes and the balance between proinflammatory and antiinflammatory cytokines [17] . This may in part be due to genetic predispositions [18] as well as to histories of viral load [19] , which contribute an immune risk profile (IRP) [20] . In some centenarians, either an 'inverted IRP' has been found or a combination of elevated pro-and antiinflammatory cytokines, two conditions which are believed to represent protective phenotypes [21,22] . On this background, it has been concluded that an increased tendency to inflammatory responses may place limits on lifespan [22] , and that a well-functioning immune system is the strongest predictor of human longevity and healthy aging [23][24][25] . Therefore, inflammaging is associated with a state of oxidative stress, defined as an excessive production of reactive oxygen species and reactive nitrogen species compared to the level of antioxidants that act in the natural defense systems. Among antioxidants, melatonin has a special place for its antioxidant and anti-inflammatory properties, and partly for its role as a metabolic regulator [26,27] . As melatonin modulates many processes involved in obesity and related metabolic disorders, it could have a therapeutic benefit in the treatment of obesity.

Melatonin and Inflammaging
A role of melatonin in attenuating inflammaging and its progression has been especially discussed with regard to options of treatment under conditions of reduced endogenous melatonin levels. Melatonin is one of the hormones known to decline during aging and, even more in a number of age-related diseases, changes that have been particularly documented in humans [26][27][28] . Interindividual variations observed among elderly persons may be explained, to a certain extent, by differences in the acquisition of melatonin-reducing diseases and disorders. Among these pathological causes of melatonin reduction, neurodegenerative processes have been identified as well as MS-related changes. For instance, decreases in melatonin were observed in coronary heart disease/cardiac syndrome X [28][29][30][31][32][33][34] and in diabetes type 2 [35,36] . In either case, the pathophysiological nexus to inflammation and obesity is well established. Additional evidence from polymorphisms of human melatonin receptor genes indicates that deviations in melatonergic signaling favor the development of prediabetic states, diabetes type 2, elevated cholesterol and coronary heart disease (see [28] ). Moreover, insulin resistance was induced in mice by knocking out the melatonin receptor MT 1 [37] and also by pinealectomy [38,39] .
Counteractions of inflammaging by melatonin seem to occur at different levels. One of them concerns the correction of metabolic dysregulation ( table 1 ), including the prevention of insulin resistance, an inflammation-promoting change and hallmark of MS [40][41][42][43] . Notably, melatonin was effective in suppressing insulin resistance in different models, tissues and methods of induction ( table 1 ).
Although in these studies several regulatory pathways have been found to be modulated by melatonin treatment, the decisive effect at which the relevant routes converge is the reduced serine phosphorylation of IRS-1 (insulin receptor substrate 1), which has sometimes been accompanied by an upregulation of IRS-1 expression. The activated, tyrosine-phosphorylated insulin receptor is known to activate IRS subforms, in particular IRS-1, by tyrosine phosphorylation, a process that is inhibited by serine 307 phosphorylation which causes interruption of insulin signaling [69] . Melatonin and the melatonergic agonist piromelatine have been shown to reverse the blockade of this key step of insulin signal transduction [41,43,52] . Persistent insulin sensitivity has in recent Ref.
In rats fed from weaning with a high-fat diet, melatonin decreased body weight gain, feed efficiency and plasma glucose, leptin and triglyceride levels 44 In middle-aged rats receiving a high-calorie liquid diet, melatonin reduced weight gain and plasma insulin and leptin levels 45 In high-fat diet-fed mice, melatonin improved insulin sensitivity and glucose tolerance 46 In ovariectomized rats, melatonin was effective in reducing obesity 47 -49 In olanzapine-treated rats, melatonin was effective in reducing obesity 50 In gold fish, body weight gain and specific growth rate were reduced by melatonin treatment 51 Melatonin and its analog piromelatonin inhibited weight gain and improved insulin sensitivity in high-fat-fed rats 52 In high-fat-fed rats, melatonin attenuated body weight increase, the increase in plasma glucose, insulin, adiponectin, leptin, triglycerides and cholesterol levels, and counteracted disrupted 24hour patterns 53 Melatonin reduced body weight gain, visceral adiposity, blood triglyceride and insulin levels, and thiobarbituric acid reactive substances under a high-calorie diet in rats 54 In young male Zucker diabetic fatty rats, melatonin treatment reduced mean weight gain without affecting food intake, decreased in a nonsignificant way blood pressure and improved dyslipidemia 55 Melatonin improved MS induced by high-fructose intake in rats without affecting food intake 56 -59 Melatonin and its analog piromelatonin reduced blood pressure in spontaneously hypertensive rats 60 Melatonin prevented the development of MS in male rats exposed to different light/dark regimens 61 Melatonin attenuated high-fat diet-induced fatty liver disease in rats 62 Melatonin, given at the time of reperfusion, prevented ventricular arrhythmias in isolated hearts from fructose-fed rats and spontaneously hypertensive rats 63 Melatonin ameliorated low-grade inflammation and oxidative stress in young Zucker diabetic fatty rats 64 Protective effects of melatonin against metabolic and reproductive disturbances in polycystic ovary syndrome in rats years gained a particular relevance to inflammaging of the brain, because insulin resistance was shown to represent an early sign of low-grade neuroinflammation in dementias, such as Alzheimer's disease, and to aggravate their progression (see [70] ). A further level of action concerns the avoidance of processes that favor or lead to inflammation. This comprises calcium overload, excessive nitric oxide release that results in the formation of peroxynitrite, peroxynitritederived free radicals ( • OH, CO 3 • -, • NO 2 ), and, finally, tyrosine nitration as well as mitochondrial dysfunction with its consequence of oxidative stress (summarized in [17,27] ). All these changes are known to initiate lowgrade inflammation in various organs, which is relevant to aging progression and comprises, in the central nervous system, microglia activation and vicious cycles via overexcitation and damage by oxidants that ultimately cause impaired neuronal and astrocytic functions. In various animal models, melatonin has been shown to counteract these detrimental processes to a substantial extent, by multiple antiexcitatory actions [17,71] , mitochondrial protection [17,27,42,[72][73][74] , reduction of peroxynitriterelated damage [75] and attenuation of microglia activation [27,76,77] . These effects go far beyond the frequently discussed direct antioxidant properties of melatonin based on scavenging of free oxygen radicals. In fact, antioxidative protection by melatonin comprises various mechanisms that reduce the formation of free radicals rather than eliminating those already formed, as outlined in the concept of radical avoidance [78] .
Immunological effects of melatonin represent a third area relevant to inflammaging. In this field, one of the major problems consists of melatonin's multiple roles as an immune modulatory agent, which comprise both proinflammatory and anti-inflammatory actions, which, consequently, also lead to either a pro-oxidant or antioxidant balance [17,27,79] . At first glance, these observations appear to be contradictory, but they may only reflect the conditionality of melatonin's actions. However, the precise reasons for when melatonin behaves in a proor anti-inflammatory way remain to be identified, although the strength of inflammation and the temporal sequence of initiation and healing processes may play a role. Moreover, changes due to immune remodeling in the course of senescence have to be taken into account. With regard to aging and age-associated diseases, proinflammatory/pro-oxidant effects are mainly observed under rheumatic conditions, especially rheumatoid arthritis [80,81] . However, under other conditions concerning senescence, melatonin's anti-inflammatory side seems to prevail. In the liver of aged, ovariectomized female rats, melatonin downregulated proinflammatory cytokines, such as TNF-α, IL-1β and IL-6, and upregulated the antiinflammatory IL-10 [82] . Corresponding findings were obtained in the dentate gyrus, in conjunction with an upregulation of sirtuin 1 [83] , which is assumed to also possess anti-inflammatory properties. Reductions of TNF-α and IL-1β and increased levels of IL-10 were also observed in the liver [84] , pancreas [85] and heart [86] of the senescence-accelerated mouse strain SAMP8. Numerous other reports on anti-inflammatory actions of melatonin that were not obtained under conditions of aging, but in brain trauma, ischemia/reperfusion, hemorrhagic shock and various forms of high-grade inflammation including endotoxinemia and sepsis, have been summarized elsewhere [17] . The applicability of these results to inflammaging and MS remains uncertain, but the data certainly underline melatonin's anti-inflammatory potential.
Melatonin-induced changes in gene expression require detailed analyses beyond the primary signaling pathways transduced by the MT 1 and MT 2 receptors via decreases in cAMP and ERK1/2 activation and modulations by protein-protein interactions [87] . However, from a mechanistic point of view, it is not always possible to discriminate between direct actions and indirect effects via changes in the phase and amplitude of circadian oscillators. While the changes induced in the circadian master clock, the suprachiasmatic nucleus (SCN), are relatively well understood, this is less the case in the numerous peripheral oscillators, which strongly differ in their dependence on the SCN [88] . Since circadian oscillators are cellular machineries, in which the core oscillators are modulated by accessory oscillator components in an often cell type-specific way [89,90] , tissue-dependent differences can be expected. With regard to the cellular generation of circadian rhythms, oscillators may be assumed to be present in any nucleated nonresting cell. Notably, peripheral oscillators exist in cells of particular relevance to MS, such as pancreatic β-cells [91] , hepatocytes, adipocytes, cardiomyocytes [92] and leukocytes [93,94] . Moreover, the effects of melatonin are known in all these cell types, and the factors involved in metabolic sensing are modulated by this hormone, such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), peroxisome proliferator-activated receptor-γ, phosphoinositide 3-kinase, protein kinase B (Akt), including the accessory oscillator components AMP kinase, nicotinamide phosphoribosyl transferase and sirtuin 1 [17,90] . However, the effects of melatonin on all these factors are by far not uniform, but rather often contradictory or, at least, con- Cardinali [17] . Therefore, it is of utmost importance to remain in the context of inflammaging and to discriminate between direct effects and indirect actions via circadian central or peripheral oscillators, demands that frequently have not been considered in respective studies. It would also be important to be aware of the fundamental rules of phase dependency of any action on circadian oscillators, which can lead to either up-or downregulations at different circadian times. Cases of direct effects not mediated by oscillators may be present in the induction of antioxidant enzymes in the rat liver and pancreas under inflammatory conditions, where melatonin promotes the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 that mediates the upregulation of the protective enzymes [95][96][97] . Correspondingly, melatonin reduced proinflammatory factors such as TNF-α, IL-1β and inducible nitric oxide synthase by suppressing the expression of nuclear factor-κB via recruitment of a histone deacetylase to its promoter [95,96] . However, it is important to remain aware of the conditionality of melatonin effects on pro-and anti-inflammatory cytokines, which may be either up-or downregulated by this hormone [79] . Importantly, proinflammatory cytokines such as TNF-α, IL-1β and IL-6 have been shown to be reduced in various models of aging, whereas the anti-inflammatory IL-10 was typically stimulated [27] . Whether this would be also the case under conditions of beginning MS at younger age deserves future attention and thorough analysis, especially as the proinflammatory cytokines are usually upregulated by melato-  [79] . Various other effects of melatonin on gene expression seem to be mediated by the circadian system. In particular, the role of SIRT1 should be considered, which is not only believed to be an aging suppressor; it acts as a protein deacetylase and, moreover, as a component of circadian oscillators that interacts with the BMAL1/CLOCK dimer and is required for high rhythm amplitudes [89,98] . In various models of aging including senescence-accelerated mice, SIRT1 was upregulated by melatonin and caused enhanced deacetylation of various of its substrates, such as PGC-1α, FoxO1, NFκB, and p53 [27,86] . Notably, these effects strongly contrast with opposite effects in epigenetically dysregulated oscillators of cancer cells. Other aspects of epigenetic modulation including possible indirect effects by melatonin via circadian oscillators have recently been summarized [99] .

Evidence of Melatonin's Therapeutic Value in MS: Animal Studies
Treatment with melatonin in rats has the ability to reduce obesity, type 2 diabetes and hepatic steatosis [100,101] . In several animal models of hyperadiposity, melatonin injection was able to normalize most observed alterations and correct the altered biochemical proinflammatory profile ( fig. 1 ; table 1 ).
Moreover, melatonin treatment of streptozotocin-induced type 1 diabetic rats induces the regeneration and proliferation of β-cells in the pancreas leading to a decrease in blood glucose [102] . Loss of melatonin in circulation after pinealectomy of rats results in hyperinsulinemia and accumulation of triglycerides in the liver [103] . The long-term administration of melatonin improves lipid metabolism in type 2 diabetic rats via restoring insulin sensitivity [104] . Melatonin treatment increases glycogen content in the liver of rats [105] and in high fat diet-induced diabetic mice the intraperitoneal injection of 10 mg kg melatonin improved glucose utilization and insulin sensitivity and ameliorated hepatic steatosis [106] . Table 1 summarizes the effect of melatonin in animal models of obesity. Melatonin was usually very effective in reversing hyperadiposity. The reasons for the decrease in body weight after melatonin in the absence of significant differences in food intake are worth exploring. A key piece of evidence in this regard is the observation that melatonin plays a role in seasonal changes in adiposity by increasing the activity of the sympathetic nervous system innervating white fat which leads to lipolysis [107] .
Melatonin not only affects white adipose tissue, but also increases the recruitment of brown adipocytes and increases their metabolic activity in mammals (see [108] ). It has been speculated that the hypertrophic effect and functional activation of brown adipose tissue induced by melatonin can likely be applied to treatment of human obesity. Collectively, the results indicate that the administration of melatonin effectively counteracts some of the disrupting effects seen in diet-induced obesity in animals, in particular insulin resistance, dyslipidemia and obesity. Type 2 diabetic patients have low circulating levels of melatonin [36] with a concurrent and expected upregulation of mRNA expression of melatonin membrane receptor [109] . Furthermore, allelic variants for melatonin receptors were associated with the level of fasting blood glucose and/or increased risk of type 2 diabetes [110][111][112] and with polycystic ovary syndrome [113] . These findings strongly bind melatonin to glucose homeostasis in blood.

Evidence for Melatonin's Therapeutic Value in MS: Clinical Studies
Patients with coronary artery disease show decreased melatonin secretion [31][32][33][34] , and among elderly hypertensive individuals, nocturnal urinary melatonin excretion was inversely associated with the nondipper pattern [114] . In turn, administered melatonin proved capable of reducing nocturnal blood pressure in hypertensives [117][118][119][120] and attenuated age-dependent disturbances of cardiovascular rhythms [121] . A meta-analysis of randomized controlled trials suggests that melatonin-controlled release is effective and safe in improving nocturnal hypertension [136] . As a pleiotropic molecule, melatonin may exert its antihypertensive and anti-remodeling effects through its antioxidant and scavenging properties, preserving the availability of nitric oxide and having sympathoplegic effects that provide cardiovascular protection in MS.
As well as in animal models, clinical studies have shown that melatonin improves lipid profiles in MS patients. Melatonin treatment (1 mg/kg for 30 days) increased the levels of high-density lipoprotein cholesterol Cardinali   in the morning. The daily systolic and diastolic BP rhythms were bimodal. In reference to previously reported data of younger subjects, mean BP was elevated, particularly the nocturnal fall was less pronounced. Also, the overall systolic BP variability was higher as was the percentage of the 12-hour component. Both values and also the systolic and diastolic BP were reduced during melatonin treatment. The hypotensive effect of melatonin was most pronounced between 03.00 and 08.   in peri-and postmenopausal women [137] . Several mechanisms may explain the hypolipidemic effects of melatonin, such as reduced intestinal absorption of cholesterol [138] or inhibiting cholesterol biosynthesis [139] . Catecholamine-induced hypercoagulability in acute stress that contributes to the growth of thrombus after rupture of coronary plaque was prevented by the administration of melatonin [122] . This was probably mediated by the reported inhibitory effects of melatonin on platelet aggregation [123][124][125] . In light of these results, melatonin may have a protective effect in reducing atherothrombotic risk in MS.
Several studies support the beneficial role of melatonin in patients with MS. Melatonin treatment ameliorated MS in obese patients [126,127] as well as in bipolar and schizophrenic patients after treatment with second generation antipsychotics [128][129][130] . Melatonin administration normalized MS in elderly hypertensive patients [140] and improved the enzymatic profile in patients with alcoholic liver steatosis [131,132] . The combination of melatonin and zinc acetate, when used alone or in combination with metformin improved glycemic control in type 2 diabetic patients [133] , and an inverse relationship between urinary 6-sulfatoxy melatonin excretion and insulin levels and insulin resistance was reported in healthy women in the Nurses' Health Study cohort [141] . However, a recent placebo-controlled single-blind study including 21 healthy women reported that melatonin (5 mg) decreased glucose tolerance [135] . Further studies are needed to clarify this controversy.
Overall, the results discussed above suggest that melatonin therapy may be beneficial for patients with MS. Undoubtedly, more studies are needed to evaluate an appropriate time/duration of treatment/dose relationship in the administration of melatonin in patients with MS.
As with many diseases, particularly those related to MS, hypertension, cardiovascular disease, obesity, diabetes, etc., the evidence supports the hypothesis that metabolic rhythm attenuation and/or disruption contribute to the etiology of the disease. Diabetes mellitus, a significant risk factor for developing heart disease and/or MS in humans, is associated with a phase change in the cardiac circadian clock [142] . Actually, metformin, a diabetes medication commonly used under Clinical Commissioning Group guidelines, increases the circadian amplitude of the metabolic sensor AMP kinase and modulates liver casein kinase 1α (CK1α) and muscle CK1ε, two regulators of the respective circadian core oscillators, effects that influence the expression and temporal patterns of several clock components and key genes of energy metabolism [143] .

Conclusions
Melatonin can provide an innovative strategy in MS by combining its effects on the circadian rhythm with its cytoprotective properties. Melatonin protects against several MS comorbidities, such as diabetes and concomitant oxyradical-mediated damage, inflammation, microvascular disease and atherothrombotic risk. At an early stage of the treatment of MS, a nondrug approach such as changing the lifestyle, low-fat diet and exercise is commonly recommended. Patients who are refractory to these changes are treated with antihypertensive drugs (antidiabetic, lipid-lowering drugs) that can have significant side effects.
Melatonin may thus be beneficial from the initial phases of MS treatment. It has a high safety profile and shows a reduced toxicity, thus differing from many pharmaceutical agents used in MS patients. Moreover, melatonin is usually remarkably well tolerated at very high doses [144] . As melatonin is a short-lived molecule that has a limited duration of action (half-life from 0.54 to 0.67 h, analogs with a high affinity for melatonin receptors and a longer duration of action have been synthesized to treat circadian disorders [145] . To what extent the new melatonergic agents approved by the US Food and Drug Administration or the European Medicines Agency (ramelteon, agomelatine, tasimelteon) share the protective activity of melatonin in MS remains to be defined. There is evidence that ramelteon given daily in drinking water (8 mg/kg) for 8 weeks to spontaneously hypertensive male Wistar-Kyoto rats significantly attenuated systolic blood pressure and body weight gain associated with age [146] . In addition, an investigational melatonergic agonist, piromelatine (NEU-P11), has been reported to be similarly effective or even superior to melatonin in improving some MS-associated parameters [52,60,147] .
Given higher binding affinities, longer half-life and high relative potencies of the various melatonin agonists, studies using 2 or 3 mg/day of melatonin are probably inadequate to provide an adequate comparison with the effects of the natural compound. Doses that considerably exceed those usually applied have been found to be safe, e.g. in the treatment of ALS patients who received either 60 mg/day orally for up to 13 months [148] or 300 mg/ day enterally for up to 2 years [144] . In a phase I dose escalation study in healthy volunteers to assess the tolerability and pharmacokinetics of 20-, 30-, 50-, and 100-mg oral doses of melatonin, no adverse effects after oral melatonin other than mild transient drowsiness, with no effects on sleeping patterns, were seen [149] . Therefore, further clinical trials using dosages of melatonin in the range of 50-100 mg/day appear to be reasonable and are warranted. The priorities for populations, outcomes, and durations of these studies must be defined.