Effect of Melatonin on Redox Enzymes Daily Gene Expression in Perirenal and Subcutaneous Adipose Tissue of a Diet Induced Obesity Model.

Increased adiposity is related to oxidative stress, inflammation and metabolic disorders. Our group has shown that melatonin totally or partially prevents the alterations that obesity causes in some neuroendocrine and inflammatory parameters indicative of oxidative stress. This study analyzes the effects of HFD on the relative gene expression of several redox balance enzymes on adult male Wistar rats subcutaneous (SAT) and perirenal adipose tissue (PRAT) and the possible preventive role of melatonin. Three experimental groups were established: control, high fat diet (HFD) and HFD plus 25 µg/mL melatonin in tap water. After 11 weeks, animals were sacrificed at 09:00 a.m. and 01:00 a.m. and PRAT and SAT were collected for selected redox enzymes qRT-PCR. Differential expression of redox enzyme genes, except for SODMn, GPx and catalase, was observed in the control group as a function of fat depot. HFD causes the disappearance of the temporal changes in the expression of the genes studied in the two fat depots analyzed. PRAT seems to be more sensitive than SAT to increased oxidative stress induced by obesity. Melatonin combined with a HFD intake, partially prevents the effects of the HFD on the gene expression of the redox enzymes. According to our results, melatonin selectively prevents changes in the relative gene expression of redox enzymes in PRAT and SAT of animals fed an HFD.

Early Appearance of Epicardial Adipose Tissue through Human Development.

Background: Epicardial adipose tissue (EAT) is a visceral fat depot with unique anatomic, biomolecular and genetic features. Due to its proximity to the coronary arteries and myocardium, dysfunctional EAT may contribute to the development and progression of cardiovascular and metabolic-related adiposity-based chronic diseases. The aim of this work was to describe, by morphological techniques, the early origin of EAT. Methods: EAT adipogenesis was studied in 41 embryos from 32 gestational days (GD) to 8 gestational weeks (GW) and in 23 fetuses until full term (from 9 to 36 GW). Results: This process comprises five stages. Stage 1 appears as mesenchyme at 33-35 GD. Stage 2 is characterized by angiogenesis at 42-45 GD. Stage 3 covers up to 34 GW with the appearance of small fibers in the extracellular matrix. Stage 4 is visible around the coronary arteries, as multilocular adipocytes in primitive fat lobules, and Stage 5 is present with unilocular adipocytes in the definitive fat lobules. EAT precursor tissue appears as early as the end of the first gestational month in the atrioventricular grooves. Unilocular adipocytes appear at the eighth gestational month. Conclusions: Due to its early origin, plasticity and clinical implications, factors such as maternal health and nutrition might influence EAT early development in consequence.

Melatonin as adjunctive therapy in the treatment of periodontitis associated with obesity.

AIMS: To study the effect of adjunctive systemic administration of melatonin to standard mechanical periodontal therapy in obese rats with experimental periodontitis. MATERIALS AND METHODS: In 42 Wistar rats with an initial body weight of 180 g., half (n = 21) were fed with a high-fat diet to induce obesity. In both obese and normal-weight groups, experimental periodontitis was subsequently induced through oral gavages with a combination of Porphyromonas gingivalis and Fusobacterium nucleatum. Both groups were randomly allocated to either no treatment or periodontal treatment consisting on standard mechanical debridement, with either adjunctive chlorhexidine or melatonin. Outcomes were evaluated by the changes in clinical parameters (probing depth modified gingival index, plaque dental index and bleeding on probing [BOP]), in bone resorption and in the levels of biomarkers in plasma and in gingival tissue (inflammatory cytokines, insulin, leptin, osteocalcin, osteopontin, plasminogen activator inhibitor-1, intercellular adhesion molecule 1, E-selectin and lipids). RESULTS: In the obese-periodontitis group, adjunctive melatonin administration resulted in reduced gingival inflammation and BOP, with significant reductions in probing depth and enhanced bone repair demonstrated by micro-CT (15% reduction in alveolar bone destruction) when compared with the same group treated with adjunctive CHX or the normal-weight rats with either melatonin or CHX. In this melatonin-treated obese-periodontitis group, a significant impact on biochemical biomarkers was also demonstrated in both gingival and plasma samples, when compared with the other groups, with significant reductions in pro-inflammatory cytokines. CONCLUSIONS: Adjunctive melatonin therapy significantly reduced alveolar bone loss and exerted a protective anti-inflammatory effect mainly in those experimental animals affected by the co-morbidity of periodontitis and obesity.

Obesity and periodontitis: An experimental study to evaluate periodontal and systemic effects of comorbidity.

BACKGROUND: Obesity and overweight have been associated with periodontitis. This study aims to evaluate periodontal and systemic effects of this association in a validated experimental model. METHODS: Twenty-eight male Wistar rats were randomly divided into four groups: 1) control group (Con) (fed with standard diet); 2) high-fat diet group (HFD) (fed with a diet containing 35.2% fat); 3) control group with induced periodontitis (Con-Perio); and 4) HFD group with induced periodontitis (HFD-Perio). To induce periodontitis, oral gavages with Porphyromonas gingivalis ATCC W83K1 and Fusobacterium nucleatum DMSZ 20482 were used. Periodontal outcomes were evaluated by inflammatory parameters, periodontal probing depth (PD), and modified gingival index (MGI). Systemic effects were evaluated by measuring levels of inflammatory cytokines, insulin, adiponectin, and leptin using multiplex immunoassays and levels of visfatin, resistin, lipid profiles, transaminases, and plasma endotoxin using colorimetric tests and the glucose tolerance test. RESULTS: Clinical parameters (PD and MGI) were significantly increased (P < 0.05) in the induced periodontitis groups compared with controls. The HFD-Perio group demonstrated significantly higher PD compared with Con-Perio group. Lipid profiles, cytokines, and adipocytokines showed significantly elevated levels in the HFD-Perio group compared with the other groups. Similarly, glucose levels in the HFD-Perio group were significantly higher (P < 0.05) than in the HFD group, and hepatic damage parameters demonstrated a tendency toward higher levels in the HFD-Perio group. CONCLUSION: Obesity and periodontitis demonstrated a comorbidity effect on both systemic inflammatory and metabolic dysregulation biomarkers, with increased glucose, dyslipidemia and hepatic damage.

Citrus essential oils inhalation by mice: Behavioral testing, GCMS plasma analysis, corticosterone, and melatonin levels evaluation.

The use of orange essential oils (EOs) as a complementary treatment is very common in Brazilian popular culture. The levels of melatonin (MEL) and corticosterone (CORT) hormones were investigated simultaneously, by the Luminex™ immunoassay system in mice plasma, after Citrus aurantium and Citrus sinensis EOs inhalation for 30 min. The plasma was analyzed by headspace through gas chromatography coupled to mass spectrometry for investigation of the EO components. Mice were submitted to behavioral testing to research anxiolytic-like, sedative, and antidepressant-like effects. The inhalation of atmosphere obtained from vaporization of 10% solution of this Citrus EO separately did not affect MEL or CORT plasma levels; that is, the MEL and CORT levels did not present variation in function of the EO in the schedule used. On the other hand, the imipramine positive control used altered the level of MEL as expected. The EO constituents were detected in plasma at different ratios that is present in inhaled EO. Behavioral tests showed that the inhalation of 10% C. sinensis EO presents an anxiolytic-like and sedative effect. Thus, C. sinensis EO can be a valuable tool for treatment of the anxiety disturbs, apparently without interference with MEL and CORT physiological levels.

Melatonin counteracts changes in hypothalamic gene expression of signals regulating feeding behavior in high-fat fed rats.

BACKGROUND: Previous studies indicate that the administration of melatonin caused body weight and abdominal visceral fat reductions in rodent models of hyperadiposity. The objective of the present study performed in high-fat fed rats was to evaluate the activity of melatonin on gene expression of some medial basal hypothalamus (MBH) signals involved in feeding behavior regulation, including neuropeptide Y (NPY), proopiomelanocortin (POMC), prolactin-releasing peptide (PrRP), leptin- and insulin-receptors (R) and insulin-R substrate (IRS)-1 and -2. Blood levels of leptin and adiponectin were also measured. METHODS: Adult Wistar male rats were divided into four groups (n=16 per group): (i) control diet (3% fat); (ii) high-fat (35%) diet; (iii) high-fat diet+melatonin; (iv) control diet+melatonin. Rats had free access to high-fat or control chow and one of the following drinking solutions: (a) tap water; (b) 25 µg/mL of melatonin. RESULTS: After 10 weeks, the high-fat fed rats showed augmented MBH mRNA levels of NPY, leptin-R, PrRP, insulin-R, IRS-1 and IRS-2. The concomitant administration of melatonin counteracted this increase. Feeding of rats with a high-fat diet augmented expression of the MBH POMC gene through an effect insensitive to melatonin treatment. The augmented levels of circulating leptin and adiponectin seen in high-fat fed rats were counteracted by melatonin as was the augmented body weight: melatonin significantly attenuated a body weight increase in high-fat fed rats without affecting chow or water consumption. Melatonin augmented plasma leptin and adiponectin in control rats. CONCLUSIONS: The results indicate that an effect on gene expression of feeding behavior signals at the central nervous system (CNS) may complement a peripheral rise of the energy expenditure produced by melatonin to decrease body weight in high-fat fed rats.

Optimization of buffer solutions to analyze inflammatory cytokines in gingival crevicular fluid by multiplex flow cytometry.

OBJECTIVE: the aim of this study was to test two buffer solutions in order to attain a reliable and reproducible analysis of inflammatory cytokines (IL-1ß, IL-6, TNF-α, OPG, OPN and OC), in gingival crevicular fluid (GCF) by flow cytometry. MATERIAL AND METHODS: GCF samples from healthy volunteers were collected with perio-paper strips and diluted either in phosphate buffered saline (PBS) or Tris-HCl buffer, with and without protease inhibitors (PI). Cytokine immunoassays were carried out by flow cytometry (Luminex Xmap 200) generating standard curves. RESULTS: standards curves generated with the use of phosphate-buffered saline (PBS) demonstrated best adjustment for cytokines IL-1ß, IL-6 and TNF- α levels, when using Tris-HCl (p<0.05). CONCLUSIONS: The use of PBS buffer with the addition of PI provided reliable measurements of inflammatory biomarkers in GCF samples of healthy volunteers.

Melatonin normalizes clinical and biochemical parameters of mild inflammation in diet-induced metabolic syndrome in rats.

The objective of this study was to evaluate the efficacy of melatonin to affect mild inflammation in the metabolic syndrome (MS) induced by a high-fat diet in rats. Adult Wistar male rats were divided into four groups (n = 16/group): (i) control diet (3% fat); (ii) high-fat (35%) diet; (iii) high-fat diet + melatonin; and (iv) melatonin. Rats had free access to high-fat or control chow and one of the following drinking solutions for 10 wk: (a) tap water; (b) 25 µg/mL of melatonin. Plasma interleukin (IL)-1ß, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and C-reactive protein (CRP) were measured at two time intervals, that is, the middle of daylight period and the middle of the scotophase. In addition, a number of somatic and metabolic components employed clinically to monitor the MS were measured. Melatonin decreased the augmented circulating levels of IL-1ß, IL-6, TNF-α, IFN-γ, and CRP seen in obese rats and restored the depressed levels of IL-4 and IL-10. Rats fed with the high-fat diet showed significantly higher body weights and augmented systolic blood pressure from the third and fourth week onwards, respectively, melatonin effectively preventing these changes. In high-fat-fed rats, circulating low-density lipoprotein-cholesterol, total cholesterol, and triglyceride concentration augmented significantly, melatonin being effective to counteract these changes. Melatonin-treated rats showed a decreased insulin resistance, the highest values of plasma high-density lipoprotein-cholesterol, and the lowest values of plasma uric acid. The results indicate that melatonin is able to normalize the altered biochemical pro-inflammatory profile seen in rats fed with a high-fat diet.

Cadmium as an endocrine disruptor: correlation with anterior pituitary redox and circadian clock mechanisms and prevention by melatonin.

To examine the effect of a low dose of cadmium (Cd) as an endocrine disruptor, male Wistar rats received CdCl2 (5ppm Cd) in drinking water or drinking water alone. After 1 month, the rats were euthanized at one of six time intervals around the clock and the 24-h pattern of adenohypophysial prolactin (PRL) synthesis and release, lipid peroxidation, and redox enzyme and metallothionein (MT) gene expression was examined. Cd suppressed 24-h rhythmicity in expression of the PRL gene and in circulating PRL by increasing them at early photophase only, in correlation with an augmented pituitary lipid peroxidation and redox enzyme expression. CdCl2 treatment effectively disrupted the 24-h variation in expression of every pituitary parameter tested except for MT-3. In a second experiment the effect of melatonin (3µg/ml in drinking water) was assessed at early photophase, the time of maximal endocrine-disrupting effect of Cd. Melatonin treatment blunted the effect of Cd on PRL synthesis and release, decreased Cd-induced lipid peroxidation, and counteracted the effect of Cd on expression of most redox enzymes. A third experiment was performed to examine whether melatonin could counteract Cd-induced changes in the 24-h pattern of pituitary circadian clock gene expression and plasma PRL, luteinizing hormone (LH), thyrotropin (TSH), and corticosterone levels. Rats receiving CdCl2 exhibited a suppressed daily rhythm of Clock expression and a significant disruption in daily rhythms of pituitary Bmal1, Per1, Per2, Cry1, and Cry2. The coadministration of melatonin restored rhythmicity in Clock and Bmal1 expression but shifted the maxima in pituitary Per1, Cry1, and Cry2 expression to the scotophase. Melatonin also counteracted the effect of Cd on 24-h rhythmicity of circulating PRL, LH, TSH, and corticosterone. The results highlight the occurrence of a significant endocrine disruptor effect of a low dose of Cd. Generally melatonin counteracted the effects of Cd and ameliorated partially the circadian disruption caused by the pollutant.

Melatonin supplementation decreases prolactin synthesis and release in rat adenohypophysis: correlation with anterior pituitary redox state and circadian clock mechanisms.

In the laboratory rat, a number of physiological parameters display seasonal changes even under constant conditions of temperature, lighting, and food availability. Since there is evidence that prolactin (PRL) is, among the endocrine signals, a major mediator of seasonal adaptations, the authors aimed to examine whether melatonin administration in drinking water resembling in length the exposure to a winter photoperiod could affect accordingly the 24-h pattern of PRL synthesis and release and some of their anterior pituitary redox state and circadian clock modulatory mechanisms. Melatonin (3 µg/mL drinking water) or vehicle was given for 1 mo, and rats were euthanized at six time intervals during a 24-h cycle. High concentrations of melatonin (>2000 pg/mL) were detected in melatonin-treated rats from beginning of scotophase (at 21:00 h) to early photophase (at 09:00 h) as compared with a considerably narrower high-melatonin phase observed in controls. By cosinor analysis, melatonin-treated rats had significantly decreased MESOR (24-h time-series average) values of anterior pituitary PRL gene expression and circulating PRL, with acrophases (peak time) located in the middle of the scotophase, as in the control group. Melatonin treatment disrupted the 24-h pattern of anterior pituitary gene expression of nitric oxide synthase (NOS)-1 and -2, heme oxygenase-1 and -2, glutathione peroxidase, glutathione reductase, Cu/Zn- and Mn-superoxide dismutase, and catalase by shifting their acrophases to early/middle scotophase or amplifying the maxima. Only the inhibitory effect of melatonin on pituitary NOS-2 gene expression correlated temporally with inhibition of PRL production. Gene expression of metallothionein-1 and -3 showed maxima at early/middle photophase after melatonin treatment. The 24-h pattern of anterior pituitary lipid peroxidation did not vary after treatment. In vehicle-treated rats, Clock and Bmal1 expression peaked in the anterior pituitary at middle scotophase, whereas that of Per1 and Per2 and of Cry1 and Cry2 peaked at the middle and late photophase, respectively. Treatment with melatonin raised mean expression of anterior pituitary Per2, Cry1, and Cry2. In the case of Per1, decreased MESOR was observed, although the single significant difference found between the experimental groups when analyzed at individual time intervals was increase at early scotophase in the anterior pituitary of melatonin-treated rats. Melatonin significantly phase-delayed expression of Per1, Per2, and Cry1, also phase-delayed the plasma corticosterone circadian rhythm, and increased the amplitude of plasma corticosterone and thyrotropin rhythms. The results indicate that under prolonged duration of a daily melatonin signal, rat anterior pituitary PRL synthesis and release are depressed, together with significant changes in the redox and circadian mechanisms controlling them.

Discontinuous versus continuous drinking of ethanol in peripubertal rats: effect on 24-hour pattern of hypophyseal-gonadal axis activity and anterior pituitary oxidative stress.

AIMS: Discontinuous (weekend) consumption of alcohol is common in adolescents and young adults. This study therefore assesses, in peripubertal male rats, the effect of discontinuous as compared to chronic feeding of ethanol or control liquid diet. METHODS: Animals received an ethanol liquid diet (6.2 % w/v) starting on day 35 of life. Every week for 5 weeks, the discontinuous ethanol group received the ethanol diet for 3 consecutive days and the control liquid diet for 4 days. At the 5th week, 24 h after the last ethanol administration to the discontinuously ethanol-treated animals, rats were killed at 4-hour intervals beginning at 09.00 h. Chronically administered rats received the ethanol diet until immediately before study. RESULTS: Disrupted 24-hour rhythmicity together with a significant nocturnal increase in plasma luteinizing hormone (LH), testosterone and prolactin (PRL) occurred in the discontinuous ethanol group. Plasma ethanol levels were undetectable at 24 h after the last ethanol treatment. In contrast, after chronic ethanol administration, plasma PRL was increased late in scotophase while LH and testosterone decreased; blood ethanol levels were 2-fold greater than those in discontinuously ethanol-administered rats killed immediately after ethanol withdrawal. Circulating testosterone positively correlated with LH levels in control rats only. Chronic administration of ethanol significantly augmented mean expression of pituitary nitric oxide synthase (NOS)-2, heme oxygenase (HO)-1, Per1 and Per2 genes and disrupted their diurnal rhythmicity. Decreased NOS-1 and NOS-2 expression during scotophase, together with suppression of the rhythm in Per1 and Per2 expression, were found in the discontinuous ethanol group. CONCLUSIONS: Abstinence after discontinuous drinking of alcohol in rats, as compared to chronic administration of ethanol, is accompanied by increases of plasma LH and testosterone, a greater PRL response and a less pronounced oxidative damage of the anterior pituitary.

Cadmium-Induced Disruption in 24-h Expression of Clock and Redox Enzyme Genes in Rat Medial Basal Hypothalamus: Prevention by Melatonin.

In a previous study we reported that a low daily p.o. dose of cadmium (Cd) disrupted the circadian expression of clock and redox enzyme genes in rat medial basal hypothalamus (MBH). To assess whether melatonin could counteract Cd activity, male Wistar rats (45 days of age) received CdCl(2) (5 ppm) and melatonin (3 µg/mL) or vehicle (0.015% ethanol) in drinking water. Groups of animals receiving melatonin or vehicle alone were also included. After 1 month, MBH mRNA levels were measured by real-time PCR analysis at six time intervals in a 24-h cycle. In control MBH Bmal1 expression peaked at early scotophase, Per1 expression at late afternoon, and Per2 and Cry2 expression at mid-scotophase, whereas neither Clock nor Cry1 expression showed significant 24-h variations. This pattern was significantly disrupted (Clock, Bmal1) or changed in phase (Per1, Per2, Cry2) by CdCl(2) while melatonin counteracted the changes brought about by Cd on Per1 expression only. In animals receiving melatonin alone the 24-h pattern of MBH Per2 and Cry2 expression was disrupted. CdCl(2) disrupted the 24-h rhythmicity of Cu/Zn- and Mn-superoxide dismutase (SOD), nitric oxide synthase (NOS)-1, NOS-2, heme oxygenase (HO)-1, and HO-2 gene expression, most of the effects being counteracted by melatonin. In particular, the co-administration of melatonin and CdCl(2) increased Cu/Zn-SOD gene expression and decreased that of glutathione peroxidase (GPx), glutathione reductase (GSR), and HO-2. In animals receiving melatonin alone, significant increases in mean Cu/Zn and Mn-SOD gene expression, and decreases in that of GPx, GSR, NOS-1, NOS-2, HO-1, and HO-2, were found. The results indicate that the interfering effect of melatonin on the activity of a low dose of CdCl(2) on MBH clock and redox enzyme genes is mainly exerted at the level of redox enzyme gene expression.

Melatonin and the metabolic syndrome: physiopathologic and therapeutical implications.

Metabolic syndrome (MS) patients exhibit sleep/wake disturbances and other circadian abnormalities, and these may be associated with more rapid weight increase and development of diabetes and atherosclerotic disease. On this basis, the successful management of MS may require an ideal drug that besides antagonizing the trigger factors of MS could also correct the disturbed sleep-wake rhythm. Melatonin is an effective chronobiotic agent able to change the phase and amplitude of circadian rhythms. Melatonin has also significant cytoprotective properties preventing a number of MS sequelae in animal models of diabetes and obesity. A small number of controlled trials indicate that melatonin is useful to treat the metabolic and cardiovascular comorbidities of MS. Whether the recently introduced melatonergic agents (ramelteon, agomelatine, tasimelteon) have the potential for treating sleep disorders in MS patients and, more generally, for arresting the progression of disease, merits further investigation.

Continuous versus discontinuous drinking of an ethanol liquid diet in peripubertal rats: effect on 24-h variation of lymph node and splenic mitogenic responses and lymphocyte subset populations.

Excessive alcohol consumption continues to be a major public health problem, particularly in the adolescent and young adult populations. Generally, such a behavior tends to be confined to the weekends, to attain frequently binge drinking. This study in peripubertal male rats compares the effect of the discontinuous feeding of a liquid diet containing a moderate amount of ethanol (6.2% wt/vol) to that of continuous ethanol administration or a control diet, taking as end points the 24-h variations of plasma prolactin levels and mitogenic responses and lymphocyte subset populations in submaxillary lymph nodes and spleen. Animals received the ethanol liquid diet starting on day 35 of life, the diet being similar to that given to controls except for that maltose was isocalorically replaced by ethanol. Ethanol provided 36% of the total caloric content. Every week, the discontinuous ethanol group received the ethanol diet for 3 days and the control liquid diet for the remaining 4 days. After 4 weeks, rats were killed at six time intervals, beginning at 0900 h. A significant decrease of splenic cells' response to concanavalin A, and of lymph node and splenic cells' response to lipopolysaccharide was found in rats under the discontinuous ethanol regime, when compared with control- or ethanol-chronic rats. Under discontinuous ethanol feeding, mean values of lymph node and splenic CD8(+) and CD4(+)-CD8(+) cells decreased, whereas those of lymph node and splenic T cells, and splenic B cells, augmented. In rats chronically fed with ethanol, splenic mean levels of CD8(+) and CD4(+)-CD8(+) cells augmented. Both modalities of ethanol administration disrupted the 24 h variation in immune function seen in controls. Mean plasma prolactin levels increased by 3.6-fold and 8.5-fold in rats chronically or discontinuously fed with alcohol, respectively. The immune parameters examined in an additional group of rats fed regular chow and water ad libitum did not differ significantly from control liquid diet. The results support the view that the discontinuous drinking of a moderate amount of ethanol can be more harmful for the immune system than a continuous ethanol intake, presumably by inducing a greater stress as indicated by the augmented plasma prolactin levels observed.

Melatonin effect on plasma adiponectin, leptin, insulin, glucose, triglycerides and cholesterol in normal and high fat-fed rats.

Melatonin effect on body weight progression, mean levels and 24-hr pattern of circulating adiponectin, leptin, insulin, glucose, triglycerides and cholesterol were examined in rats fed a normal or a high-fat diet. In experiment 1, rats fed a normal diet were divided into two groups: receiving melatonin (25 µg/mL drinking water) or vehicle for 9 wk. In experiment 2, animals were divided into three groups: two fed with a high-fat diet (35% fat) and melatonin (25 µg/mL) or vehicle in drinking water for 11 wk, while a third group was given a normal diet (4% fat). At the end of experiments, groups of eight rats were killed at six different time intervals throughout a 24-hr period. Melatonin administration for 9 wk decreased body weight gain from the 3rd wk on without affecting food intake. A significant reduction in circulating insulin, glucose and triglyceride mean levels and disrupted daily patterns of plasma adiponectin, leptin and insulin were observed after melatonin. In high fat-fed rats, melatonin attenuated body weight increase, hyperglycemia and hyperinsulinemia, as well as the increase in mean plasma adiponectin, leptin, triglycerides and cholesterol levels. The high-fat diet disrupted normal 24-hr patterns of circulating adiponectin, insulin and cholesterol, the effects on insulin and cholesterol being counteracted by melatonin. Nocturnal plasma melatonin concentration in control and obese rats receiving melatonin for 11 wk attained values 21-24-fold greater than controls. The results indicate that melatonin counteracts some of the disrupting effects of diet-induced obesity in rats.

Cadmium chronotoxicity at pituitary level: effects on plasma ACTH, GH, and TSH daily pattern.

Cadmium is an endocrine disruptor that has been shown to induce chronotoxic effects. The present study was designed to evaluate the possible cadmium effects on the daily secretory pattern of adrenocorticotropin hormone (ACTH), growth hormone (GH), and thyroid-stimulating hormone (TSH) in adult male Sprague-Dawley rats. For this purpose, animals were treated with cadmium at two different doses [25 and 50 mg/l cadmium chloride (CdCl(2))] in the drinking water for 30 days. Control age-matched rats received cadmium-free water. After the treatment, rats were killed at six different time intervals throughout a 24-h cycle. Cadmium exposure modified the 24-h pattern of plasma ACTH and GH levels, as the peak of ACTH content between 12:00 and 16:00 h in controls appeared at 12:00 h in the group treated with the lowest dose used, while it appeared between 16:00 and 20:00 h in rats exposed to 50 mg/l CdCl(2). In addition, the peak of GH content found at 04:00 h in controls moved to 16:00 h in rats exposed to 25 mg/l CdCl(2), and the highest dose used abolished 24-h changes of GH secretion. The metal treatment did not modify ACTH secretory pattern. Exposure to cadmium also increased ACTH and TSH medium levels around the clock with both doses used. These results suggest that cadmium modifies ACTH and TSH medium levels around the clock, as well as disrupted ACTH and GH secretory pattern, thus confirming the metal chronotoxicity at pituitary level.

Effect of cadmium on 24-hour pattern in expression of redox enzyme and clock genes in rat medial basal hypothalamus.

The effect of cadmium (Cd) in the brain has been attributed to an increase in reactive oxygen species in cells, particularly when high amounts of the metal are given. In this study we examined the effect of a low dose of Cd (7.5 microg/day) on 24-h changes in expression of redox pathway enzyme and circadian genes in rat medial basal hypothalamus (MBH). Rats receiving CdCl(2) (5 ppm in drinking water) or tap water for 1 month were killed at six different time intervals throughout a 24 h cycle. MBH mRNA levels were measured by real-time PCR analysis. In CdCl(2) treated rats a disruption of 24-h pattern of hypothalamic gene expression of nitric oxide synthase (NOS)-1 and -2, heme oxygenase (HO)-1 and -2, Mn- superoxide dismutase (SOD), catalase, glutathione peroxidase and glutathione reductase was detectable. Mean levels of MBH mRNA for HO-2, Mn-SOD and catalase augmented after Cd intake, whereas those of NOS-2 decreased. After CdCl(2) intake rats the 24-h pattern of clock gene expression in MBH seen in controls was significantly suppressed (Bmal1) or changed in phase (Per1, Per2, Cry2) while in the case of Clock significant 24-h variations were induced. The results are compatible with the view that a low amount of Cd given in tap water brought about significant changes in circadian expression of redox enzyme and clock genes in rat MBH.

24-Hour variation in gene expression of redox pathway enzymes in rat hypothalamus: effect of melatonin treatment.

The 24-h changes in medial basal hypothalamic (MBH) gene expression of redox pathway enzymes nitric oxide synthase (NOS)-1 and NOS-2, heme oxygenase (HO)-1 and HO-2, Cu/Zn- and Mn-superoxide dismutases (SOD) and catalase were examined in adult male Wistar rats kept under an alternating regimen of light/dark. Half of the animals received melatonin (approximately 60 microg/day) in the drinking water. After 1 month, rats were killed at six different time intervals, throughout a 24-h cycle. MBH mRNA levels were measured by real-time PCR analysis. In controls, gene expression of NOS-2 and HO-2 peaked at the early light phase while that of HO-1 showed a maximum at the middle of the dark phase. None of MBH mRNAs encoding NOS-1, Cu/Zn-SOD, Mn-SOD and catalase exhibited significant 24-h variations in control rats. Melatonin administration decreased significantly mRNAs for NOS-1, NOS-2, HO-1 and HO-2 as well as changed their 24-h profile. Melatonin augmented gene expression of the antioxidant enzymes Cu/Zn-SOD, Mn-SOD or catalase at certain time intervals only. The results are compatible with the view that the principal indirect (i.e. gene expression of redox pathway enzymes) effect of melatonin on redox pathway in the hypothalamus is mainly exerted via down-regulation of pro-oxidant enzyme mRNAs.

Effect of a high-fat diet on 24-hour pattern of circulating adipocytokines in rats.

We have shown a significant disruption of 24-h pattern of plasma pituitary, adrenal, and gonadal hormones in high-fat-fed rats. Our objective was to assess the effect of a high-fat diet (35% fat) on mean levels and 24-h pattern of several adipocytokines in rats. A normal diet-fed rats (4% fat) were used as controls. When body weight of high-fat-fed rats attained values about 25% higher than controls (after 66 days of treatment), the animals were killed at six different time intervals throughout a 24-h cycle. Plasma concentrations of insulin, adiponectin, interleukin (IL)-1, leptin, ghrelin, plasminogen activator inhibitor-1 (PAI-1), and monocyte chemoattractant protein-1 (MCP-1) were measured in a multianalyte profiling by using the Luminex-100 system. Tumor necrosis factor alpha (TNFalpha) and IL-6 were measured by enzyme-linked immunosorbent assay. A significant hyperglycemia developed in high-fat-fed rats, together with a significant increase in plasma insulin. Mean levels of plasma adiponectin, IL-1, IL-6, TNFalpha, and leptin augmented, and ghrelin decreased, in high-fat-fed rats. The normal daily pattern of plasma insulin, adiponectin, IL-1, IL-6, TNFalpha, leptin, ghrelin, and MCP-1 became disrupted in high-fat-fed rats. The results indicate that a high-fat diet may bring about signs of insulin resistance and mild inflammation in rats, together with the disruption in daily variations of circulating insulin and ghrelin, and of several adipocytokines including leptin, adiponectin, IL-1, IL-6, TNFalpha, and MCP-1.

Melatonin and the immune system in aging.

Aging is associated with a decline in immune function (immunosenescence), a condition known to correlate with increased incidence of cancer as well as infectious and degenerative diseases. Innate, cellular and humoral immunity all exhibit increased deterioration with age. Circulating melatonin decreases with age, and in recent years much interest has been focused on its immunomodulatory effect. Melatonin stimulates the production of progenitor cells for granulocytes and macrophages. It also stimulates the production of natural killer cells and CD4+ cells and inhibits CD8+ cells. The production and release of various cytokines from natural killer cells and T helper lymphocytes are enhanced by melatonin. Melatonin has the potential therapeutic value to enhance immune function in aged individuals.