Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes

The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simulate the cyclic characteristics of the daily melatonin profile, isolated rat adipocytes were exposed in a circadian-like pattern to melatonin added to the incubating medium for 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day) combined or not with Ins. This intermittent incubation was interrupted when four and a half 24-hr cycles were fulfilled. At the end, either during the induced night (melatonin present) or the induced day (melatonin absent), the rates of lipolysis and D-[U-(14)C]-glucose incorporation into lipids were estimated, in addition to the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and clock gene (Bmal-1b, Clock, Per-1 and Cry-1) mRNA expression. The leptin release was also measured. During the induced night, the following effects were observed: an increase in the mRNA expression of Clock, Per-1 and FAS; a rise in lipogenic response and leptin secretion; and a decrease in the lipolytic activity. The intermittent exposure of adipocytes to melatonin temporally and rhythmically synchronized their metabolic and hormonal function in a circadian fashion, mimicking what is observed in vivo in animals during the daily light-dark cycle. Therefore, this work helps to clarify the physiological relevance of the circadian pattern of melatonin secretion and its interactions with Ins, contributing to a better understanding of the adipocyte biology.

Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demostrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor ROR alpha have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and ROR alpha and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORa were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erb alpha expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erb alpha mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erb alpha mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity. (Author correspondence: cipolla@icb.usp.br)

Patterns of fos activation in rat raphe nuclei during feeding behavior

To analyze the differential recruitment of the raphe nuclei during different phases of feeding behavior, rats were subjected to a food restriction schedule (food for 2 h/day, during 15 days). The animals were submitted to different feeding conditions, constituting the experimental groups: search for food (MFS), food ingestion (MFI), satiety (MFSa) and food restriction control (MFC). A baseline condition (BC) group was included as further control. The MFI and MFC groups, which presented greater autonomic and somatic activation, had more FOS-immunoreactive (FOS-IR) neurons. The MFI group presented more labeled cells in the linear (LRN) and dorsal (DRN) nuclei; the MFC group showed more labeling in the median (MRN), pontine (PRN), magnus (NRM) and obscurus (NRO) nuclei; and the MFSa group had more labeled cells in the pallidus (NRP). The BC exhibited the lowest number of reactive cells. The PRN presented the highest percentage of activation in the raphe while the DRN the lowest. Additional experiments revealed few double-labeled (FOS-IR+ 5-HT-IR) cells within the raphe nuclei in the MFI group, suggesting little serotonergic activation in the raphe during food ingestion. These findings suggest a differential recruitment of raphe nuclei during various phases of feeding behavior. Such findings may reflect changes in behavioral state (e.g., food-induced arousal versus sleep) that lead to greater motor activation, and consequently increased FOS expression. While these data are consistent with the idea that the raphe system acts as gain setter for autonomic and somatic activities, the functional complexity of the raphe is not completely understood. (c) 2008 Elsevier B.V. All rights reserved.