Melatonin improves mitochondrial function in inguinal white adipose tissue of Zücker diabetic fatty rats

Mitochondrial dysfunction in adipose tissue may contribute to obesity‐related metabolic derangements such as type 2 diabetes mellitus (T2DM). Because mitochondria are a target for melatonin action, the goal of this study was to investigate the effects of melatonin on mitochondrial function in white (WAT) and beige inguinal adipose tissue of Zücker diabetic fatty (ZDF) rats, a model of obesity‐related T2DM. In this experimental model, melatonin reduces obesity and improves the metabolic profile. At 6 wk of age, ZDF rats and lean littermates (ZL) were subdivided into two groups, each composed of four rats: control (C‐ZDF and C‐ZL) and treated with oral melatonin in the drinking water (10 mg/kg/day) for 6 wk (M‐ZDF and M‐ZL). After the treatment period, animals were sacrificed, tissues dissected, and mitochondrial function assessed in isolated organelles. Melatonin increased the respiratory control ratio (RCR) in mitochondria from white fat of both lean (by 26.5%, P < 0.01) and obese (by 34.5%, P < 0.01) rats mainly through a reduction of proton leaking component of respiration (state 4) (28% decrease in ZL, P < 0.01 and 35% in ZDF, P < 0.01). However, melatonin treatment lowered the RCR in beige mitochondria of both lean (by 7%, P < 0.05) and obese (by 13%, P < 0.05) rats by maintaining high rates of uncoupled respiration. Melatonin also lowered mitochondrial oxidative status by reducing nitrite levels and by increasing superoxide dismutase activity. Moreover, melatonin treatment also caused a profound inhibition of Ca‐induced opening of mPTP in isolated mitochondria from both types of fat, white and beige, in both lean and obese rats. These results demonstrate that chronic oral melatonin improves mitochondrial respiration and reduces the oxidative status and susceptibility to apoptosis in white and beige adipocytes. These melatonin effects help to prevent mitochondrial dysfunction and thereby to improve obesity‐related metabolic disorders such as diabetes and dyslipidemia of ZDF rats.     

Melatonin increases brown adipose tissue mass and function in Zücker diabetic fatty rats: implications for obesity control

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Previously we demonstrated that melatonin browns subcutaneous fat in Zücker diabetic fatty (ZDF) rats. Other works pointed to melatonin as a signal that increases brown adipose tissue (BAT) mass and function in rodents. However, direct proof of thermogenic properties (uncoupled mitochondria) of the newly recruited BAT in response to melatonin is still lacking. Therefore, in this work, we investigated if melatonin recruits thermogenic BAT in ZDF rats. Zücker lean (ZL) and ZDF animals were subdivided into two groups, control (C) and treated with oral melatonin (M) for 6 weeks. Mitochondrial mass, activity of citrate synthase (CS), and respiratory chain complexes I and IV were lower in C‐ZDF than in C‐ZL animals (P < .001). Melatonin treatment increased BAT weight in ZDF rats (P < .001). Also, it rose mitochondrial mass (P < .01) and activities of CS and complexes I and IV (P < .001) in both, ZDF and ZL rats. Uncoupling protein 1 (UCP1) mRNA and protein were 50% lower in BAT from obese rats. Also, guanosine diphosphate (GDP) binding was lower in ZDF than in lean rats (P < .01). Melatonin treatment of obese rats restored the expression of UCP1 and GDP binding to levels of lean rats and sensitized the thermogenic response to cold exposure. These data demonstrated that melatonin recruits thermogenic BAT in ZDF rats. This may contribute to melatonin's control of body weight and its metabolic benefits.     

Melatonin induces browning of inguinal white adipose tissue in Zucker diabetic fatty rats

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Identification of brown fat (beige/brite) in white adipose tissue (WAT) prompted us to investigate whether melatonin is a brown‐fat inducer. We used Zücker diabetic fatty (ZDF) rats, a model of obesity‐related type 2 diabetes and a strain in which melatonin reduces obesity and improves their metabolic profiles. At 5 wk of age, ZDF rats and lean littermates (ZL) were subdivided into two groups, each composed of four rats: control and those treated with oral melatonin in the drinking water (10 mg/kg/day) for 6 wk. Melatonin induced browning of inguinal WAT in both ZDF and ZL rats. Hematoxylin–eosin staining showed patches of brown‐like adipocytes in inguinal WAT in ZDF rats and also increased the amounts in ZL animals. Inguinal skin temperature was similar in untreated lean and obese rats. Melatonin increased inguinal temperature by 1.36 ± 0.02°C in ZL and by 0.55 ± 0.04°C in ZDF rats and sensitized the thermogenic effect of acute cold exposure in both groups. Melatonin increased the amounts of thermogenic proteins, uncoupling protein 1 (UCP1) (by ~2‐fold, P < 0.01) and PGC‐1α (by 25%, P < 0.05) in extracts from beige inguinal areas in ZL rats. Melatonin also induced measurable amounts of UCP1 and stimulated by ~2‐fold the levels of PGC‐1α in ZDF animals. Locomotor activity and circulating irisin levels were not affected by melatonin. These results demonstrate that chronic oral melatonin drives WAT into a brown‐fat‐like function in ZDF rats. This may contribute to melatonin′s control of body weight and its metabolic benefits.     

Melatonin ameliorates low‐grade inflammation and oxidative stress in young Zucker diabetic fatty rats

The aim of this study was to investigate the effects of melatonin on low‐grade inflammation and oxidative stress in young male Zucker diabetic fatty (ZDF) rats, an experimental model of metabolic syndrome and type 2 diabetes mellitus (T2DM). ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups, each composed of 10 rats: naive (N), vehicle treated (V), and melatonin treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Pro‐inflammatory state was evaluated by plasma levels of interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), and C‐reactive protein (CRP). Also, oxidative stress was assessed by plasma lipid peroxidation (LPO), both basal and after Fe²⁺/H₂O₂ inducement. ZDF rats exhibited higher levels of IL‐6 (112.4 ± 1.5 pg/mL), TNF‐α (11.0 ± 0.1 pg/mL) and CRP (828 ± 16.0 µg/mL) compared with lean rats (IL‐6, 89.9 ± 1.0, P < 0.01; TNF‐α, 9.7 ± 0.4, P < 0.01; CRP, 508 ± 21.5, P < 0.001). Melatonin lowered IL‐6 (10%, P < 0.05), TNF‐α (10%, P < 0.05), and CRP (21%, P < 0.01). Basal and Fe²⁺/H₂O₂‐induced LPO, expressed as malondialdehyde equivalents (µmol/L), were higher in ZDF rats (basal, 3.2 ± 0.1 versus 2.5 ± 0.1 in ZL, P < 0.01; Fe²⁺/H₂O₂‐induced, 8.7 ± 0.2 versus 5.5 ± 0.3 in ZL; P < 0.001). Melatonin improved basal LPO (15%, P < 0.05) in ZDF rats, and Fe²⁺/H₂O₂‐ induced LPO in both ZL (15.2%, P < 0.01) and ZDF rats (39%, P < 0.001). These results demonstrated that oral melatonin administration ameliorates the pro‐inflammatory state and oxidative stress, which underlie the development of insulin resistance and their consequences, metabolic syndrome, diabetes, and cardiovascular disease.     

Beneficial effects of melatonin on obesity and lipid profile in young Zucker diabetic fatty rats

Abstract: The study objective was to investigate the effects of melatonin on obesity and obesity‐associated systolic hypertension and dyslipidemia in young male Zucker diabetic fatty (ZDF) rats, an experimental model of the metabolic syndrome. ZDF rats (n = 30) and lean littermates (ZL) (n = 30) were used. At 6 wk of age, both lean and fatty animals were subdivided into three groups (n = 10): naive (N), vehicle‐treated (V), and melatonin‐treated (M) (10 mg/kg/day) for 6 wk. Vehicle and melatonin were added to the drinking water. Melatonin reduced mean weight gain (51 ± 2/100 g BW) versus N‐ZDF group (58 ± 3, P < 0.05) without food intake differences. M‐ZDF rats showed an apparent reduction in systolic hypertension that proved not to be statistically significant, and a significant improvement in dyslipidemia, with a reduction in hypertriglyceridemia from 580 ± 40 to 420.6 ± 40.9 mg/dL (P < 0.01). Melatonin raised high‐density‐lipoprotein (HDL) cholesterol in ZDF (from 81.6 ± 4.9 to 103.1 ± 4.5 mg/dL, P < 0.01) and ZL rats (from 62.8 ± 4.8 to 73.5 ± 4.8 mg/dL, P < 0.05) and significantly reduced low‐density‐lipoprotein (LDL) cholesterol in ZDF rats from 5.20 ± 0.4 to 4.14 ± 0.3 mg/dL (P < 0.05) but had no effect on total cholesterol levels. To our knowledge, this is the first evidence of a positive effect of melatonin on overweight and lipid pattern of obese Zucker diabetic rats, supporting the proposition that melatonin administration may ameliorate overweight and lipid metabolism in humans. Because these benefits occurred in youth, before advanced metabolic and vascular complications, melatonin might help to prevent cardiovascular disease associated with obesity and dyslipidemia.     

Systemic combined melatonin–mitochondria treatment improves acute respiratory distress syndrome in the rat

Despite high in‐hospital mortality associated with acute respiratory distress syndrome (ARDS), there is no effective therapeutic strategy. We tested the hypothesis that combined melatonin–mitochondria treatment ameliorates 100% oxygen‐induced ARDS in rats. Adult male Sprague‐Dawley rats (n = 40) were equally categorized into normal controls, ARDS, ARDS‐melatonin, ARDS with intravenous liver‐derived mitochondria (1500 μg per rat 6 hr after ARDS induction), and ARDS receiving combined melatonin–mitochondria. The results showed that 22 hr after ARDS induction, oxygen saturation (saO₂) was lowest in the ARDS group and highest in normal controls, significantly lower in ARDS‐melatonin and ARDS‐mitochondria than in combined melatonin–mitochondria group, and significantly lower in ARDS‐mitochondria than in ARDS‐melatonin group. Conversely, right ventricular systolic blood pressure and lung weight showed an opposite pattern compared with saO₂ among all groups (all P < 0.001). Histological integrity of alveolar sacs showed a pattern identical to saO₂, whereas lung crowding score exhibited an opposite pattern (all P < 0.001). Albumin level and inflammatory cells (MPO+, CD40+, CD11b/c+) from bronchoalveolar lavage fluid showed a pattern opposite to saO₂ (all P < 0.001). Protein expression of indices of inflammation (MMP‐9, TNF‐α, NF‐κB), oxidative stress (oxidized protein, NO‐1, NOX‐2, NOX‐4), apoptosis (mitochondrial Bax, cleaved caspase‐3, and PARP), fibrosis (Smad3, TGF‐β), mitochondrial damage (cytochrome C), and DNA damage (γ‐H2AX+) exhibited an opposite pattern compared to saO₂ in all groups, whereas protein (HO‐1, NQO‐1, GR, GPx) and cellular (HO‐1+) expressions of antioxidants exhibited a progressively increased pattern from normal controls to ARDS combined melatonin–mitochondria group (all P < 0.001). In conclusion, combined melatonin–mitochondrial was superior to either treatment alone in attenuating ARDS in this rat model.     

Melatonin pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia–reperfusion injury in rats through suppression of mitochondrial permeability transition

We tested the hypothesis that melatonin (Mel) enhances exogenous mitochondria (Mito) treatment against rodent hepatic ischemia–reperfusion (IR) injury. In vitro study utilized three groups of hepatocytes (i.e. nontreatment, menadione, and menadione–melatonin treatment, 4.0 × 10⁵ each), while in vivo study used adult male Sprague Dawley rats (n = 40) equally divided into sham‐control (SC), IR (60‐min left‐lobe ischemia + 72‐hr reperfusion), IR‐Mel (melatonin at 30 min/6/8 hr after reperfusion), IR‐Mito (mitochondria 15,000 μg/rat 30 min after reperfusion), and IR‐Mel‐Mito. Following menadione treatment in vitro, oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (cleaved caspase‐3/PARP), DNA damage (γ‐H2AX/CD90/XRCC1), mitochondria damage (cytosolic cytochrome c) biomarkers, and mitochondrial permeability transition were found to be lower, whereas mitochondrial cytochrome c were found to be higher in hepatocytes with melatonin treatment compared to those without (all P < 0.001). In vivo study demonstrated highest liver injury score and serum AST in IR group, but lowest in SC group and higher in IR‐Mito group than that in groups IR‐Mel and IR‐Mel‐Mito, and higher in IR‐Mel group than that in IR‐Mel‐Mito group after 72‐hr reperfusion (all P < 0.003). Protein expressions of inflammatory (TNF‐α/NF‐κB/IL‐1β/MMP‐9), oxidative stress (NOX‐1/NOX‐2/oxidized protein), apoptotic (caspase‐3/PARP/Bax), and mitochondria damage (cytosolic cytochrome c) biomarkers displayed an identical pattern, whereas mitochondria integrity marker (mitochondrial cytochrome c) showed an opposite pattern compared to that of liver injury score (all P < 0.001) among five groups. Microscopically, expressions of apoptotic nuclei, inflammatory (MPO ⁺ /CD68 ⁺ /CD14 ⁺ cells), and DNA damage (γ‐H2AX ⁺ cells) biomarkers exhibited an identical pattern compared to that of liver injury score (all P < 0.001) among five groups. Melatonin‐supported mitochondria treatment offered an additional benefit of alleviating hepatic IR injury.     

Melatonin inhibits cholangiocarcinoma and reduces liver injury in Opisthorchis viverrini‐infected and N‐nitrosodimethylamine‐treated hamsters

The human liver fluke Opisthorchis viverrini infection and N‐nitrosodimethylamine (NDMA) administration induce cholangiocarcinoma (CCA) and liver injury in hamsters. Melatonin protects against liver injury and reduces the alteration of mitochondrial structure, mitochondrial membrane potential, and mitochondrial pro‐ and anti‐apoptotic pathways in various cancer types. To investigate the chemopreventive effect of melatonin on CCA genesis and liver injury, hamsters were treated with a combination of O. viverrini infection and NDMA concurrently administered with melatonin (10 mg/kg and 50 mg/kg) for 120 days. Melatonin treatment at 50 mg/kg caused a significant reduction in liver/body weight ratios and decreased tumor volumes leading to an increase in the survival of animals. In the tumorous tissues, the high‐dose melatonin reduced DNA fragmentation and mitochondrial apoptosis by inducing anti‐apoptotic protein (Bcl‐2) in the mitochondrial fraction and down‐regulating cytochrome c, pro‐apoptotic protein (Bax), and caspase‐3 in tumor cytosol. Moreover, a high‐dose melatonin treatment significantly increased mitochondrial antioxidant enzymes and prevented mitochondrial ultrastructure changes in the tumor. Overall, melatonin has potent chemopreventive effects in inhibiting CCA genesis and also reduces liver injury in hamster CCA, which, in part, might involve in the suppression of CCA by reducing tumor mitochondria alteration.     

Melatonin treatment improves adipose‐derived mesenchymal stem cell therapy for acute lung ischemia–reperfusion injury

This study investigated whether melatonin‐treated adipose‐derived mesenchymal stem cells (ADMSC) offered superior protection against acute lung ischemia–reperfusion (IR) injury. Adult male Sprague‐Dawley rats (n = 30) were randomized equally into five groups: sham controls, lung IR–saline, lung IR–melatonin, lung IR–melatonin–normal ADMSC, and lung IR–melatonin–apoptotic ADMSC. Arterial oxygen saturation was lowest in lung IR–saline; lower in lung IR–melatonin than sham controls, lung IR–melatonin–normal ADMSC, and lung IR–melatonin–apoptotic ADMSC; lower in lung IR–melatonin–normal ADMSC than sham controls and lung IR–melatonin–apoptotic ADMSC; lower in lung IR–melatonin–apoptotic ADMSC than sham controls (P < 0.0001 in each case). Right ventricular systolic blood pressure (RVSBP) showed a reversed pattern among all groups (all P < 0.0001). Changes in histological scoring of lung parenchymal damage and CD68+ cells showed a similar pattern compared with RVSBP in all groups (all P < 0.001). Changes in inflammatory protein expressions such as VCAM‐1, ICAM‐1, oxidative stress, TNF‐α, NF‐κB, PDGF, and angiotensin II receptor, and changes in apoptotic protein expressions of cleaved caspase 3 and PARP, and mitochondrial Bax, displayed identical patterns compared with RVSBP in all groups (all P < 0.001). Numbers of antioxidant (GR+, GPx+, NQO‐1+) and endothelial cell biomarkers (CD31+ and vWF+) were lower in sham controls, lung IR–saline, and lung IR–melatonin than lung IR–melatonin–normal ADMSC and lung IR–melatonin–apoptotic ADMSC, and lower in lung IR–melatonin–normal ADMSC than lung IR–melatonin–apoptotic ADMSC (P < 0.001 in each case). In conclusion, when the animals were treated with melatonin, the apoptotic ADMSC were superior to normal ADMSC for protection of lung from acute IR injury.     

Melatonin improves glucose homeostasis in young Zucker diabetic fatty rats

The aim of this study was to investigate the effects of melatonin on glucose homeostasis in young male Zucker diabetic fatty (ZDF) rats, an experimental model of metabolic syndrome and type 2 diabetes mellitus (T2DM). ZDF rats (n=30) and lean littermates (ZL) (n=30) were used. At 6wk of age, both lean and fatty animals were subdivided into three groups, each composed of ten rats: naive (N), vehicle treated (V), and melatonin treated (M) (10mg/kg/day) for 6wk. Vehicle and melatonin were added to the drinking water. ZDF rats developed DM (fasting hyperglycemia, 460±39.8mg/dL; HbA(1) c 8.3±0.5%) with both insulin resistance (HOMA-IR 9.28±0.9 versus 1.2±0.1 in ZL) and decreased β-cell function (HOMA1-%B) by 75%, compared with ZL rats. Melatonin reduced fasting hyperglycemia by 18.6% (P<0.05) and HbA(1) c by 11% (P<0.05) in ZDF rats. Also, melatonin lowered insulinemia by 15.9% (P<0.05) and HOMA-IR by 31% (P<0.01) and increased HOMA1-%B by 14.4% (P<0.05). In addition, melatonin decreased hyperleptinemia by 34% (P<0.001) and raised hypoadiponectinemia by 40% (P<0.001) in ZDF rats. Moreover, melatonin reduced serum free fatty acid levels by 13.5% (P<0.05). These data demonstrate that oral melatonin administration ameliorates glucose homeostasis in young ZDF rats by improving both insulin action and β-cell function. These observations have implications on melatonin's possible use as a new pharmacologic therapy for improving glucose homeostasis and of obesity-related T2DM, in young subjects.     

Tissue-specific alterations of methyl group metabolism with DNA hypermethylation in the Zucker (type 2) diabetic fatty rat

Background Altered methyl group and homocysteine metabolism were tissue‐specific, persistent, and preceded hepatic DNA hypomethylation in type 1 diabetic rats. Similar metabolic perturbations have been shown in the Zucker (type 2) diabetic fatty (ZDF) rat in the pre‐diabetic and early diabetic stages, but tissue specificity and potential impact on epigenetic marks are unknown, particularly during pathogenesis. Methods ZDF (fa/fa) and lean (+/?) control rats were killed at 12 and 21 weeks of age, representing early and advanced diabetic conditions. Blood and tissues were analysed with respect to methyl group and homocysteine metabolism, including DNA methylation. Results At 12 weeks, hepatic glycine N‐methyltransferase (GNMT), methionine synthase, and cystathionine β‐synthase (CBS) activity and/or abundance were increased in ZDF rats. At 21 weeks, GNMT activity was increased in liver and kidney; however, only hepatic CBS protein abundance (12 weeks) and betaine‐homocysteine S‐methyltransferase mRNA expression (21 weeks) were significantly elevated (78 and 100%, respectively). Hepatic phosphatidylethanolamine N‐methyltransferase expression was also elevated in the ZDF rat. Homocysteine concentrations were decreased in plasma and kidney, but not in liver, at 12 and 21 weeks. In contrast to hepatic DNA hypomethylation in the type 1 diabetic rat, genomic DNA was hypermethylated at 12 and 21 weeks in the liver of ZDF rats, concomitant with an increase in DNA methyltransferase 1 expression at 21 weeks. Conclusions The pathogenesis of type 2 diabetes in the ZDF rat was associated with tissue and disease stage‐specific aberrations of methyl group and homocysteine metabolism, with persistent hepatic global DNA hypermethylation. Copyright © 2011 John Wiley & Sons, Ltd.     

Dynamin‐related protein 1‐mediated mitochondrial fission contributes to post‐traumatic cardiac dysfunction in rats and the protective effect of melatonin

Mechanical trauma (MT ) causes myocardial injury and cardiac dysfunction. However, the underlying mechanism remains largely unclear. This study investigated the role of mitochondrial dynamics in post‐traumatic cardiac dysfunction and the protective effects of melatonin. Adult male Sprague Dawley rats were subjected to 5‐minute rotations (200 revolutions at a rate of 40 rpm) to induce MT model. Melatonin was administrated intraperitoneally 5 minute after MT . Mitochondrial morphology, myocardial injury, and cardiac function were determined in vivo. There was smaller size of mitochondria and increased number of mitochondria per μm² in the hearts after MT when the secondary myocardial injury was induced. Melatonin treatment at the dose of 30 mg/kg reduced serine 616 phosphorylation of Drp1 and inhibited mitochondrial Drp1 translocation and mitochondrial fission in the hearts of rats subjected to MT , which contributed to the reduction of myocardial injury and the improvement of cardiac function. In vitro, H9c2 cells cultured in 20% traumatic plasma (TP ) for 12 hour showed enhanced mitochondrial fission, mitochondrial membrane potential (?Ψm) loss, mitochondrial cytochrome c release, and decreased mitochondrial complex I‐IV activities. Pretreatment with melatonin (100 μmol/L) efficiently inhibited TP ‐induced mitochondrial fission, ?Ψm loss, cytochrome c release, and improved mitochondrial function. Melatonin's protective effects were attributed to its role in suppressing plasma TNF ‐α overproduction, which was responsible for Drp1‐mediated mitochondrial fission. Taken together, our results demonstrate for the first time that abnormal mitochondrial dynamics is involved in post‐traumatic cardiac dysfunction. Melatonin has significant pharmacological potential in protecting against MT ‐induced cardiac dysfunction by preventing excessive mitochondrial fission.     

Melatonin‐mediated downregulation of ZNF746 suppresses bladder tumorigenesis mainly through inhibiting the AKT‐MMP‐9 signaling pathway

There still lacking effective treatment for bladder cancer. This study investigated whether melatonin (Mel) can suppress the growth and invasion of bladder cancer cells. Male C57B/L6 mice were categorized into control group (ie, subcutaneous injection of HT1197 bladder cancer cell line at the back] and treatment group [subcutaneous HT1197 cells + intraperitoneal Mel (100 mg/kg/d) from day 8 to day 21 after tumor cell injection]. In vitro Mel suppressed cell growth of four bladder cancer cell lines (ie, T24, RT4, HT1197, HT1376), cell migration in HT1197/HT1376, mitochondrial membrane potential (MMP) in T24 and colony formation in RT4 cells as well as arrested the cell cycle at G0 phase and inhibited the mitotic phase of T24 cells (all P < 0.0001). Protein expression of ZNF746 in RT4/T24 cells and protein expression phosphorylated (p)‐AKT/MMP‐2/MMP‐9 in HT1197/HT1376 cells were reduced following Mel treatment (all P < 0.001). Transfection of T24 cells with plasmid‐based shRNA (ie, ZNF746‐silencing) downregulated the protein expression of MMP‐9, cell growth, and invasion and attachment to endothelial cells but upregulated the colony formation (all P < 0.001). Mel suppressed oxidative stress and MMP but upregulated mitochondria mass in ZNF746‐silenced T24 cells, whereas these parameters exhibited a similar patter to Mel treatment in ZNF746‐silenced T24 cells (all P < 0.0001). In vivo study demonstrated that Mel treatment significantly suppressed cellular expressions of MMP‐9/MMP‐2, protein expressions of ZNF746/p‐AKT, and tumor size (all P < 0.001). Mel treatment suppressed the growth, migration, and invasion of bladder carcinoma cells through downregulating ZNF746‐regulated MMP‐9/MMP‐2 signaling.     

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,given at the time of reperfusion,prevents ventricular arrhythmias in isolated hearts from fructose‐fed rats and spontaneously hypertensive rats

Melatonin reduces reperfusion arrhythmias when administered before coronary occlusion, but in the clinical context of acute coronary syndromes, most of the therapies are administered at the time of reperfusion. Patients frequently have physiological modifications that can reduce the response to therapeutic interventions. This work determined whether acute melatonin administration starting at the moment of reperfusion protects against ventricular arrhythmias in Langendorff‐perfused hearts isolated from fructose‐fed rats (FFR), a dietary model of metabolic syndrome, and from spontaneous hypertensive rats (SHR). In both experimental models, we confirmed metabolic alterations, a reduction in myocardial total antioxidant capacity and an increase in arterial pressure and NADPH oxidase activity, and in FFR, we also found a decrease in eNOS activity. Melatonin (50 μm ) initiated at reperfusion after 15‐min regional ischemia reduced the incidence of ventricular fibrillation from 83% to 33% for the WKY strain, from 92% to 25% in FFR, and from 100% to 33% in SHR (P = 0.0361, P = 0.0028, P = 0.0013, respectively, by Fisher's exact test, n = 12 each). Although, ventricular tachycardia incidence was high at the beginning of reperfusion, the severity of the arrhythmias progressively declined in melatonin‐treated hearts. Melatonin induced a shortening of the action potential duration at the beginning of reperfusion and in the SHR group also a faster recovery of action potential amplitude. We conclude that melatonin protects against ventricular fibrillation when administered at reperfusion, and these effects are maintained in hearts from rats exposed to major cardiovascular risk factors. These results further support the ongoing translation to clinical trials of this agent.     

Daily melatonin protects the endothelial lineage and functional integrity against the aging process,oxidative stress,and toxic environment and restores blood flow in critical limb ischemia area in mice

We tested the hypothesis that daily melatonin treatment protects endothelial lineage and functional integrity against the aging process, oxidative stress/endothelial denudation (ED), and toxic environment and restored blood flow in murine critical limb ischemia (CLI). In vitro study using HUVECs, in vivo models (ie, CLI through left femoral artery ligation and ED through carotid artery wire injury), and model of lipopolysaccharide‐induced aortic injury in young (3 months old) and aged (8 months old) mice were used to elucidate effects of melatonin treatment on vascular endothelial integrity. In vitro study showed that menadione‐induced oxidative stress (NOX‐1/NOX‐2), inflammation (TNF‐α/NF‐kB), apoptosis (cleaved caspase‐3/PARP), and mitochondrial damage (cytosolic cytochrome c) in HUVECs were suppressed by melatonin but reversed by SIRT3‐siRNA (all P < .001). In vivo, reduced numbers of circulating endothelial progenitor cells (EPCs) (C‐kit/CD31+/Sca‐1/KDR+/CXCR4/CD34+), and angiogenesis (Matrigel assay of bone marrow‐derived EPC and ex vivo aortic ring cultures) in older (compared with younger) mice were significantly reversed through daily melatonin administration (20 mg/kg/d, ip) (all P < .001). Aortic vasorelaxation and nitric oxide release were impaired in older mice and reversed in age‐match mice receiving melatonin (all P < .01). ED‐induced intimal/medial hyperplasia, reduced blood flow to ischemic limb, and angiogenesis (reduced CD31+/vWF+ cells/small vessel number) were improved after daily melatonin treatment (all P < .0001). Lipopolysaccharide‐induced aortic endothelial cell detachment, which was more severe in aged mice, was also alleviated after daily melatonin treatment (P < .0001). Daily melatonin treatment protected both structural and functional integrity of vascular endothelium against aging‐, oxidative stress‐, lipopolysaccharide‐, and ischemia‐induced damage probably through upregulating the SIRT signaling pathway.     

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl₄)‐induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid‐Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl₄ followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS‐stained glycogen‐laden cells. In addition, melatonin increased bone morphogenic protein (BMP)‐2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal‐regulated kinase (ERK), and nuclear factor‐κB (NF‐κB) in hDPSCs. Melatonin‐induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF‐κB. Compared to treatment of CCl₄‐injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF‐κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.     

Melatonin improves neuroplasticity by upregulating the growth‐associated protein‐43 (GAP‐43) and NMDAR postsynaptic density‐95 (PSD‐95) proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to transient focal cerebral ischemia even during a long‐term recovery period

Recent evidence shows that the NMDAR postsynaptic density‐95 (PSD‐95), growth‐associated protein‐43 (GAP‐43), and matrix metalloproteinase‐9 (MMP‐9) protein enhance neuroplasticity at the subacute stage of stroke. Here, we evaluated whether melatonin would modulate the PSD‐95, GAP‐43, and MMP‐9 proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to experimental stroke. Adult male Sprague–Dawley rats were treated with melatonin (5 mg/kg) or vehicle at reperfusion onset after transient occlusion of the right middle cerebral artery (tMCAO) for 90 min. Animals were euthanized for Western immunoblot analyses for the PSD‐95 and GAP‐43 proteins and gelatin zymography for the MMP‐9 activity at 7 days postinsult. Another set of animals was sacrificed for histologic and Golgi–Cox‐impregnated sections at 28 days postinsult. In cultured neurons exposed to glutamate excitotoxicity, melatonin significantly upregulated the GAP‐43 and PSD‐95 expressions and improved dendritic aborizations (P < 0.05, respectively). Relative to controls, melatonin‐treated stroke animals caused a significant improvement in GAP‐43 and PSD‐95 expressions as well as the MMP‐9 activity in the ischemic brain (P < 0.05). Consequently, melatonin also significantly promoted the dendritic spine density and reduced infarction in the ischemic brain, and improved neurobehaviors as well at 28 days postinsult (P < 0.05, respectively). Together, melatonin upregulates GAP‐43, PSD‐95, and MMP‐9 proteins, which likely accounts for its actions to improve neuroplasticity in cultured neurons exposed to glutamate excitotoxicity and to enhance long‐term neuroprotection, neuroplasticity, and brain remodeling in stroke rats.     

Hepatic steatosis and metabolic risk factors among patients with chronic hepatitis B: The multicentre,prospective CAP-Asia study

We aimed to compare the severity of liver disease, metabolic profile and cardiovascular disease (CVD) risk of chronic hepatitis B (CHB) patients with and without hepatic steatosis and patients with non-alcoholic fatty liver disease (NAFLD). Patients with NAFLD and CHB were prospectively enrolled from 10 Asian centres. Fibroscan was performed for all patients and hepatic steatosis was defined based on controlled attenuation parameter >248 dB/m. CVD risk was assessed using the Framingham risk score. The data for 1080 patients were analysed (67% NAFLD, 33% CHB). A high proportion (59%) of CHB patients had hepatic steatosis. There was a significant stepwise increase in alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transpeptidase, controlled attenuation parameter and liver stiffness measurement, from CHB patients without hepatic steatosis to CHB patients with hepatic steatosis to NAFLD patients (p < 0.001 for all comparisons). There was a significant stepwise increase in the proportion of patients with metabolic syndrome and in CVD risk, with very high or extreme CVD risk seen in 20%, 48% and 61%, across the groups (p < 0.001 between CHB patients with and without hepatic steatosis and p < 0.05 between CHB patients with hepatic steatosis and NAFLD patients). In conclusion, there was a high proportion of CHB patients with hepatic steatosis, which should be diagnosed, as they may have more severe liver disease, so that this and their metabolic risk factors can be assessed and managed accordingly for a better long-term outcome