Novel protective role of the circadian nuclear receptor retinoic acid‐related orphan receptor‐α in diabetic cardiomyopathy

Diabetic cardiomyopathy is a major complication that significantly contributes to morbidity and mortality in diabetics with few therapies. Moreover, antidiabetic drugs reported inconsistent or even adverse cardiovascular effects, suggesting that it is important to exploit novel therapeutic targets against diabetic cardiomyopathy. Here, we observed that the nuclear melatonin receptor, the retinoic acid‐related orphan receptor‐α (RORα), was downregulated in diabetic hearts. By utilizing a mouse line with RORα disruption, we demonstrated that RORα deficiency led to significantly augmented diastolic dysfunction and cardiac remodeling induced by diabetes. Microscopic and molecular analyses further indicated that the detrimental effects of RORα deficiency were associated with aggravated myocardial apoptosis, autophagy dysfunction, and oxidative stress by disrupting antioxidant gene expression. By contrast, restoration of cardiac RORα levels in transgenic mice significantly improved cardiac functional and structural parameters at 8 weeks after diabetes induction. Consistent with genetic manipulation, pharmacological activation of RORα by melatonin and SR1078 (a synthetic agonist) showed beneficial effects against diabetic cardiomyopathy, while the RORα inhibitor SR3335 significantly exacerbated cardiac impairments in diabetic mice. Collectively, these findings suggest that cardiac‐targeted manipulation of nuclear melatonin receptor RORα may hold promise for delaying diabetic cardiomyopathy development.     

Melatonin differentially regulates pathological and physiological cardiac hypertrophy: Crucial role of circadian nuclear receptor RORα signaling

Exercise‐induced physiological hypertrophy provides protection against cardiovascular disease, whereas disease‐induced pathological hypertrophy leads to heart failure. Emerging evidence suggests pleiotropic roles of melatonin in cardiac disease; however, the effects of melatonin on physiological vs pathological cardiac hypertrophy remain unknown. Using swimming‐induced physiological hypertrophy and pressure overload‐induced pathological hypertrophy models, we found that melatonin treatment significantly improved pathological hypertrophic responses accompanied by alleviated oxidative stress in myocardium but did not affect physiological cardiac hypertrophy and oxidative stress levels. As an important mediator of melatonin, the retinoid‐related orphan nuclear receptor‐α (RORα) was significantly decreased in human and murine pathological hypertrophic cardiomyocytes, but not in swimming‐induced physiological hypertrophic murine hearts. In vivo and in vitro loss‐of‐function experiments indicated that RORα deficiency significantly aggravated pathological cardiac hypertrophy, and notably weakened the anti‐hypertrophic effects of melatonin. Mechanistically, RORα mediated the cardioprotection of melatonin in pathological hypertrophy mainly by transactivation of manganese‐dependent superoxide dismutase (MnSOD) via binding to the RORα response element located in the promoter region of the MnSOD gene. Furthermore, MnSOD overexpression reversed the pro‐hypertrophic effects of RORα deficiency, while MnSOD silencing abolished the anti‐hypertrophic effects of RORα overexpression in pathological cardiac hypertrophy. Collectively, our findings provide the first evidence that melatonin exerts an anti‐hypertrophic effect on pathological but not physiological cardiac hypertrophy via alleviating oxidative stress through transactivation of the antioxidant enzyme MnSOD in a RORα‐dependent manner.     

The nuclear melatonin receptor RORα is a novel endogenous defender against myocardial ischemia/reperfusion injury

Circadian rhythm disruption or decrease in levels of circadian hormones such as melatonin increases ischemic heart disease risk. The nuclear melatonin receptors RORs are pivotally involved in circadian rhythm regulation and melatonin effects mediation. However, the functional roles of RORs in the heart have never been investigated and were therefore the subject of this study on myocardial ischemia/reperfusion (MI/R) injury pathogenesis. RORα and RORγ subtypes were detected in the adult mouse heart, and RORα but not RORγ was downregulated after MI/R. To determine the pathological consequence of MI/R‐induced reduction of RORα, we subjected RORα‐deficient staggerer mice and wild‐type (WT) littermates to MI/R injury, resulting in significantly increased myocardial infarct size, myocardial apoptosis and exacerbated contractile dysfunction in the former. Mechanistically, RORα deficiency promoted MI/R‐induced endoplasmic reticulum stress, mitochondrial impairments, and autophagy dysfunction. Moreover, RORα deficiency augmented MI/R‐induced oxidative/nitrative stress. Given the emerging evidence of RORα as an essential melatonin effects mediator, we further investigated the RORα roles in melatonin‐exerted cardioprotection, in particular against MI/R injury, which was significantly attenuated in RORα‐deficient mice, but negligibly affected by cardiac‐specific silencing of RORγ. Finally, to determine cell type‐specific effects of RORα, we generated mice with cardiomyocyte‐specific RORα overexpression and they were less vulnerable to MI/R injury. In summary, our study provides the first direct evidence that the nuclear melatonin receptor RORα is a novel endogenous protective receptor against MI/R injury and an important mediator of melatonin‐exerted cardioprotection; melatonin‐RORα axis signaling thus appears important in protection against ischemic heart injury.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

Melatonin safeguards against fatty liver by antagonizing TRAFs‐mediated ASK1 deubiquitination and stabilization in a β‐arrestin‐1 dependent manner

Melatonin has been previously shown to prevent nonalcoholic fatty liver disease (NAFLD), yet the underlying mechanisms are poorly understood. Here, we identified a previously unknown regulatory action of melatonin on apoptosis signal‐regulating kinase 1 (ASK1) signaling pathway in the pathogenesis and development of NAFLD. Although melatonin administration did not alter food intake, it significantly alleviated fatty liver phenotypes, including the body weight gain, insulin resistance, hepatic lipid accumulation, steatohepatitis, and fibrosis in a high‐fat diet (HFD)‐induced NAFLD mouse model (in vivo). The protection of melatonin against NAFLD was not affected by inactivation of Kupffer cell in this model. In NAFLD mice liver, ASK1 signal cascade was substantially activated, evidence by the enhancement of total ASK1, phospho‐ASK1, phospho‐MKK3/6, phospho‐p38, phospho‐MKK4/7, and phospho‐JNK. Melatonin treatment significantly suppressed the ASK1 upregulation and the phosphorylation of ASK1, MKK3/6, MKK4/7, p38, and JNK. Mechanistically, we found that lipid stress triggered the interaction between ASK1 and TNF receptor‐associated factors (TRAFs), including TRAF1, TRAF2, and TRAF6, which resulted in ASK1 deubiquitination and thereby increased ASK1 protein stability. Melatonin did not alter ASK1 mRNA level; however, it activated a scaffold protein β‐arrestin‐1 and enabled it to bind to ASK1, which antagonized the TRAFs‐mediated ASK1 deubiquitination, and thus reduced ASK1 protein stability. Consistent with these findings, knockout of β‐arrestin‐1 in mice partly abolished the protection of melatonin against NAFLD. Taken together, our results for the first time demonstrate that melatonin safeguards against NAFLD by eliminating ASK1 activation via inhibiting TRAFs‐mediated ASK1 deubiquitination and stabilization in a β‐arrestin‐1 dependent manner.     

Melatonin modulates TLR4‐mediated inflammatory genes through MyD88‐ and TRIF‐dependent signaling pathways in lipopolysaccharide‐stimulated RAW264.7 cells

Abstract: Increasing evidence demonstrates that melatonin has an anti‐inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll‐like receptor 4 (TLR4)‐mediated molecule myeloid differentiation factor 88 (MyD88)‐dependent and TRIF‐dependent signaling pathways in lipopolysaccharide (LPS)‐stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 μg/mL) in the absence or presence of melatonin (10, 100, 1000 μm ). As expected, melatonin inhibited TLR4‐mediated tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, IL‐6, IL‐8, and IL‐10 in LPS‐stimulated macrophages. In addition, melatonin significantly attenuated LPS‐induced upregulation of cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS‐stimulated macrophages. Although it had no effect on TLR4‐mediated phosphorylation of c‐Jun N‐terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF‐κB) in LPS‐stimulated macrophages. In addition, melatonin inhibited TLR4‐mediated Akt phosphorylation in LPS‐stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of interferon (IFN)‐regulated factor‐3 (IRF3), which was involved in TLR4‐mediated TRIF‐dependent signaling pathway, in LPS‐stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS‐induced IFN‐β in macrophages. In conclusion, melatonin modulates TLR4‐mediated inflammatory genes through MyD88‐dependent and TRIF‐dependent signaling pathways.     

Melatonin alleviates adipose inflammation through elevating α‐ketoglutarate and diverting adipose‐derived exosomes to macrophages in mice

Obesity is associated with macrophage infiltration and metabolic inflammation, both of which promote metabolic disease progression. Melatonin is reported to possess anti‐inflammatory properties by inhibiting inflammatory response of adipocytes and macrophages activation. However, the effects of melatonin on the communication between adipocytes and macrophages during adipose inflammation remain elusive. Here, we demonstrated melatonin alleviated inflammation and elevated α‐ketoglutarate (αKG) level in adipose tissue of obese mice. Mitochondrial isocitrate dehydrogenase 2 (Idh2) mRNA level was also elevated by melatonin in adipocytes leading to increase αKG level. Further analysis revealed αKG was the target for melatonin inhibition of adipose inflammation. Moreover, sirtuin 1 (Sirt1) physically interacted with IDH2 and formed a complex to increase the circadian amplitude of Idh2 and αKG content in melatonin‐inhibited adipose inflammation. Notably, melatonin promoted exosomes secretion from adipocyte and increased adipose‐derived exosomal αKG level. Our results also confirmed that melatonin alleviated adipocyte inflammation and increased ratio of M2 to M1 macrophages by transporting of exosomal αKG to macrophages and promoting TET‐mediated DNA demethylation. Furthermore, exosomal αKG attenuated signal transducers and activators of transduction‐3 (STAT3)/NF‐κB signal by its receptor oxoglutarate receptor 1 (OXGR1) in adipocytes. Melatonin also attenuated adipose inflammation and deceased macrophage number in chronic jet‐lag mice. In summary, our results demonstrate melatonin alleviates metabolic inflammation by increasing cellular and exosomal αKG level in adipose tissue. Our data reveal a novel function of melatonin on adipocytes and macrophages communication, suggesting a new potential therapy for melatonin to prevent and treat obesity caused systemic inflammatory disease.     

Melatonin prevents the dynamin‐related protein 1‐dependent mitochondrial fission and oxidative insult in the cortical neurons after 1‐methyl‐4‐phenylpyridinium treatment

Mitochondrial dysfunction and oxidative stress are involved in the pathogenesis of Parkinson's disease (PD). Mitochondrial morphology is dynamic and precisely regulated by the mitochondrial fission and fusion machinery. Aberrant mitochondrial fragmentation controlled by the mitochondrial fission protein, dynamin‐related protein 1 (Drp1), may result in cell death. Our previous results showed that melatonin protected neurons by inhibiting oxidative stress in a 1‐methyl‐4‐phenylpyridinium (MPP⁺)‐induced PD model. However, the effect of melatonin on mitochondrial dynamics remains uncharacterized. Herein, we investigated the effect of melatonin and the role of Drp1 on MPP⁺‐induced mitochondrial fission in rat primary cortical neurons. We found that MPP⁺ induced a rapid increase in the ratio of GSSG:total glutathione (a marker of oxidative stress) and mitochondrial fragmentation, Drp1 upregulation within 4 hours, and finally resulted in neuron loss 48 hours after the treatment. Neurons overexpressing wild‐type Drp1 promoted mitochondrial and nuclear fragmentation; however, neurons overexpressing dominant‐negative Drp1^K38A or cotreated with melatonin exhibited significantly reduced MPP⁺‐induced mitochondrial fragmentation and neuron death. Moreover, melatonin cotreatment prevented an MPP⁺‐induced high ratio of GSSG and mitochondrial Drp1 upregulation. The prevention of mitochondrial fission by melatonin was not found in neurons transfected with wild‐type Drp1. These results provide a new insight that the neuroprotective effect of melatonin against MPP⁺ toxicity is mediated by inhibiting the oxidative stress and Drp1‐mediated mitochondrial fragmentation.     

Melatonin antagonizes hypoxia‐mediated glioblastoma cell migration and invasion via inhibition of HIF‐1α

Hypoxia is a crucial factor in tumor aggressiveness and resistance to therapy, especially in glioblastoma. Our previous results have shown that melatonin exerts antimigratory and anti‐invasive action in glioblastoma cells under normoxia. However, the effect of melatonin on migration and invasion of glioblastoma cells under hypoxic condition remains poorly understood. Here, we show that melatonin strongly reduced hypoxia‐mediated invasion and migration of U251 and U87 glioblastoma cells. In addition, we found that melatonin significantly blocked HIF‐1α protein expression and suppressed the expression of downstream target genes, matrix metalloproteinase 2 (MMP‐2) and vascular endothelial growth factor (VEGF). Furthermore, melatonin destabilized hypoxia‐induced HIF‐1α protein via its antioxidant activity against ROS produced by glioblastoma cells in response to hypoxia. Along with this, HIF‐1α silencing by small interfering RNA markedly inhibited glioblastoma cell migration and invasion, and this appeared to be associated with MMP‐2 and VEGF under hypoxia. Taken together, our findings suggest that melatonin suppresses hypoxia‐induced glioblastoma cell migration and invasion via inhibition of HIF‐1α. Considering the fact that overexpression of the HIF‐1α protein is often detected in glioblastoma multiforme, melatonin may prove to be a potent therapeutic agent for this tumor.     

Melatonin prevents cell death and mitochondrial dysfunction via a SIRT1‐dependent mechanism during ischemic‐stroke in mice

Silent information regulator 1 (SIRT1), a type of histone deacetylase, is a highly effective therapeutic target for protection against ischemia reperfusion (IR) injury (IRI). Previous studies showed that melatonin preserves SIRT1 expression in neuronal cells of newborn rats after hypoxia–ischemia. However, the definite role of SIRT1 in the protective effect of melatonin against cerebral IRI in adult has not been explored. In this study, the brain of adult mice was subjected to IRI. Prior to this procedure, the mice were given intraperitoneal with or without the SIRT1 inhibitor, EX527. Melatonin conferred a cerebral‐protective effect, as shown by reduced infarct volume, lowered brain edema, and increased neurological scores. The melatonin‐induced upregulation of SIRT1 was also associated with an increase in the anti‐apoptotic factor, Bcl2, and a reduction in the pro‐apoptotic factor Bax. Moreover, melatonin resulted in a well‐preserved mitochondrial membrane potential, mitochondrial Complex I activity, and mitochondrial cytochrome c level while it reduced cytosolic cytochrome c level. However, the melatonin‐elevated mitochondrial function was reversed by EX527 treatment. In summary, our results demonstrate that melatonin treatment attenuates cerebral IRI by reducing IR‐induced mitochondrial dysfunction through the activation of SIRT1 signaling.     

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing

Melatonin, a circadian rhythm–promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)‐MSCs pretreated with melatonin enhanced wound closure, granulation, and re‐epithelialization at mouse skin wound sites, where relatively more UCB‐MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB‐MSCs. Melatonin (1 μm ) significantly increased the motility of UCB‐MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT₂). We found that Gαq coupled with MT₂ and that the binding of Gαq to MT₂ uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization‐related proteins such as profilin‐1, cofilin‐1, and F‐actin in UCB‐MSCs. Finally, a lack of MT₂ expression in UCB‐MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT₂ triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB‐MSCs motility.     

Melatonin influences somatostatin secretion from human pancreatic δ‐cells via MT1 and MT2 receptors

Melatonin is an effector of the diurnal clock on pancreatic islets. The membrane receptor‐transmitted inhibitory influence of melatonin on insulin secretion is well established and contrasts with the reported stimulation of glucagon release from α‐cells. Virtually, nothing is known concerning the melatonin‐mediated effects on islet δ‐cells. Analysis of a human pancreatic δ‐cell model, the cell line QGP‐1, and the use of a somatostatin‐specific radioimmunoassay showed that melatonin primarily has an inhibitory effect on somatostatin secretion in the physiological concentration range. In the pharmacological range, melatonin elicited slightly increased somatostatin release from δ‐cells. Cyclic adenosine monophosphate (cAMP) is the major second messenger dose‐dependently stimulating somatostatin secretion, in experiments employing the membrane‐permeable 8‐Br‐cAMP. 8‐Br‐cyclic guanosine monophosphate proved to be of only minor relevance to somatostatin release. As the inhibitory effect of 1 nm melatonin was reversed after incubation of QGP‐1 cells with the nonselective melatonin receptor antagonist luzindole, but not with the MT2‐selective antagonist 4‐P‐PDOT (4‐phenyl‐2‐propionamidotetraline), an involvement of the MT1 receptor can be assumed. Somatostatin release from the δ‐cells at low glucose concentrations was significantly inhibited during co‐incubation with 1 nm melatonin, an effect which was less pronounced at higher glucose levels. Transient expression experiments, overexpressing MT1, MT2, or a deletion variant as a control, indicated that the MT1 and not the MT2 receptor was the major transmitter of the inhibitory melatonin effect. These data point to a significant influence of melatonin on pancreatic δ‐cells and on somatostatin release.     

Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor‐κB activation

Constitutive activation and gemcitabine induction of nuclear factor‐κB (NF‐κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF‐κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF‐κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF‐κB by suppressing IκBα phosphorylation and decreased the expression of NF‐κB response genes in MiaPaCa‐2, AsPc‐1, Panc‐28 cells and gemcitabine resistance MiaPaCa‐2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor‐independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug‐treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF‐κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF‐κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.     

Melatonin enhances antioxidant action of α‐tocopherol and ascorbate against NADPH‐ and iron‐dependent lipid peroxidation in human placental mitochondria

Abstract: Human placental mitochondria might be a significant source of NADPH‐ and iron‐dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH‐ and iron‐dependent lipid peroxidation of human placental mitochondria in a concentration‐dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal‐chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and α‐tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 μm ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 μm ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro‐oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and α‐tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.     

Melatonin prevents Drp1‐mediated mitochondrial fission in diabetic hearts through SIRT1‐PGC1α pathway

Myocardial contractile dysfunction is associated with an increase in mitochondrial fission in patients with diabetes. However, whether mitochondrial fission directly promotes diabetes‐induced cardiac dysfunction is still unknown. Melatonin exerts a substantial influence on the regulation of mitochondrial fission/fusion. This study investigated whether melatonin protects against diabetes‐induced cardiac dysfunction via regulation of mitochondrial fission/fusion and explored its underlying mechanisms. Here, we show that melatonin prevented diabetes‐induced cardiac dysfunction by inhibiting dynamin‐related protein 1 (Drp1)‐mediated mitochondrial fission. Melatonin treatment decreased Drp1 expression, inhibited mitochondrial fragmentation, suppressed oxidative stress, reduced cardiomyocyte apoptosis, improved mitochondrial function and cardiac function in streptozotocin (STZ )‐induced diabetic mice, but not in SIRT 1^?/? diabetic mice. In high glucose‐exposed H9c2 cells, melatonin treatment increased the expression of SIRT 1 and PGC ‐1α and inhibited Drp1‐mediated mitochondrial fission and mitochondria‐derived superoxide production. In contrast, SIRT 1 or PGC ‐1α siRNA knockdown blunted the inhibitory effects of melatonin on Drp1 expression and mitochondrial fission. These data indicated that melatonin exerted its cardioprotective effects by reducing Drp1‐mediated mitochondrial fission in a SIRT 1/PGC ‐1α‐dependent manner. Moreover, chromatin immunoprecipitation analysis revealed that PGC ‐1α directly regulated the expression of Drp1 by binding to its promoter. Inhibition of mitochondrial fission with Drp1 inhibitor mdivi‐1 suppressed oxidative stress, alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. These findings show that melatonin attenuates the development of diabetes‐induced cardiac dysfunction by preventing mitochondrial fission through SIRT 1‐PGC 1α pathway, which negatively regulates the expression of Drp1 directly. Inhibition of mitochondrial fission may be a potential target for delaying cardiac complications in patients with diabetes.     

Melatonin ameliorates Aβ42‐induced alteration of βAPP‐processing secretases via the melatonin receptor through the Pin1/GSK3β/NF‐κB pathway in SH‐SY5Y cells

Melatonin is involved in the physiological regulation of the β‐amyloid precursor protein (βAPP)‐cleaving secretases which are responsible for generation of the neurotoxic amyloid beta (Aβ) peptide, one of the hallmarks of Alzheimer's disease (AD) pathology. In this study, we aimed to determine the underlying mechanisms of this regulation under pathological conditions. We establish that melatonin prevents Aβ₄₂‐induced downregulation of a disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) as well as upregulation of β‐site APP‐cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) in SH‐SY5Y cell cultures. We also demonstrate that the intrinsic mechanisms of the observed effects occurred via regulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and glycogen synthase kinase (GSK)‐3β as melatonin reversed Aβ₄₂‐induced upregulation and nuclear translocation of NF‐κBp65 as well as activation of GSK3β via its receptor activation. Furthermore, specific blocking of the NF‐κB and GSK3β pathways partially abrogated the Aβ₄₂‐induced reduction in the BACE1 and PS1 levels. In addition, GSK3β blockage affected α‐secretase cleavage and modulated nuclear translocation of NF‐κB. Importantly, our study for the first time shows that peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1) is a crucial target of melatonin. The compromised levels and/or genetic variation of Pin1 are associated with age‐dependent tau and Aβ pathologies and neuronal degeneration. Interestingly, melatonin alleviated the Aβ₄₂‐induced reduction of nuclear Pin1 levels and preserved the functional integrity of this isomerase. Our findings illustrate that melatonin attenuates Aβ₄₂‐induced alterations of βAPP‐cleaving secretases possibly via the Pin1/GSK3β/NF‐κB pathway.