Ana Isabel Esquifino

Melatonin is involved in various functions such as the timing of circadian rhythms, energy metabolism, and body mass gain in experimental animals. However, its effects on adipose tissue lipid metabolism are still unclear. This study analyzes the effects of melatonin on the relative gene expression of lipolytic proteins in rat mesenteric adipose tissue and free fatty acid (FFA) and glycerol plasma levels of male Wistar rats fed a high-fat (HFD) or maintenance diet. Four experimental groups were established: control, obese, and control or obese plus 2.3 mg/kg/day of melatonin in tap water. After 11 weeks, animals were sacrificed at different times throughout a 24 h cycle, and mesenteric adipose tissue and plasma samples were collected and analyzed. Cgi58, Perilipin, and Dgat1 gene expression, as well as FFA and glycerol concentrations, showed rhythm patterns in the control group. HFD disrupted those rhythm patterns and increased FFA and glycerol concentrations during the dark photoperiod. In both melatonin-treated groups, almost all analyzed genes showed circadian patterns. Notably, melatonin significantly prevented the increase in FFA levels during the dark photoperiod in obese rats (obese group: ~1100 mM vs. obese + melatonin group: ~600 μM, similar to control levels). However, the rhythmic pattern observed in control animals was not sustained. According to our results, melatonin could regulate circadian gene transcription of mesenteric adipose tissue lipolysis proteins. The effect of melatonin on preventing elevated FFA plasma levels associated with high-fat diet intake warrants further investigation.

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 SOD^Mn, 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.

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.

The aim of this study was to test two buffer solutions in order to attain a reliable and reproducible analysis of inflammatory cytokines (IL-1β, IL-6, TNF-α, OPG, OPN and OC), in gingival crevicular fluid (GCF) by flow cytometry. 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. 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). The use of PBS buffer with the addition of PI provided reliable measurements of inflammatory biomarkers in GCF samples of healthy volunteers.

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)‐1β, 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‐1β, 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.

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.

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.