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.

Intermittent and rhythmic exposure to melatonin in primary cultured adipocytes enhances the insulin and dexamethasone effects on leptin expression.

Considering the cyclic characteristic of production and secretion of pineal melatonin, it is reasonable to assume that this oscillation might be important in determining the variety of its circadian and seasonal effects. To simulate this physiological condition in vitro, isolated adipocytes were exposed to melatonin in a circadian-like pattern by adding the hormone to the incubating medium during 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day). This intermittent procedure was interrupted when three cycles with melatonin were fulfilled (60-hr incubation). Here, we report the effects of melatonin (1 nM) added intermittently or continuously to the incubating medium alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) on leptin release and expression by rat adipocytes. After acute 12-hr incubation neither melatonin nor insulin alone affected leptin expression, but together they increased it by 105%. Dexamethasone increased leptin mRNA content and release (70%) but this effect was not enhanced by melatonin. Nevertheless, after 60 hr under intermittent melatonin, we observed a synergism between melatonin and dexamethasone. This interaction promoted an increment (75% compared with dexamethasone alone) in leptin release and expression. Our results suggest that circadian-like exposure to melatonin potentiates the dexamethasone action and is important to the effects promoted by insulin on leptin expression. Based on an in vitro approach, this work helps to clarify the physiological relevance and the repercussions of the in vivo circadian pattern of melatonin secretion.