Sandra Andreotti

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

Abstract:  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‐14C]‐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.

High dietary sodium intake increases white adipose tissue mass and plasma leptin in rats.

Objective: Salt restriction has been reported to increase white adipose tissue (WAT) mass in rodents. The objective of this study was to investigate the effect of different sodium content diets on the lipogenic and lipolytic activities of WAT.

Reduced lipolysis and increased lipogenesis in adipose tissue from pinealectomized rats adapted to training

Abstract:  The current study investigated the effects of chronic training and pinealectomy on the lipogenic and lipolytic activity of adipose tissue. Pinealectomized and sham‐operated adult male Wistar rats were distributed in to four subgroups: pinealectomized untrained, pinealectomized trained, control untrained and control trained. At the end of the training period (8 wk) the rats were killed. Blood samples were collected for glucose, insulin and leptin determinations. Peri‐epididymal adipocytes were isolated for measurement of in vitro rates of lipolysis and incorporation of substrates (d‐[U‐14C]‐glucose, l‐[U‐14C]‐lactate, [2‐14C]‐acetate and [1‐14C]‐palmitate) into lipids, and samples of epididymal adipose tissue were homogenized for evaluation of glucose‐6‐phosphate dehydrogenase maximal activity. Pinealectomy resulted in a significantly increased lipolytic capacity in response to isoproterenol and a decrease in circulating leptin levels without affecting the rates of incorporation of different substrates into lipids. However, only in the intact control group did training promote a higher basal and isoproterenol‐stimulated lipolysis, increase the incorporation of palmitate (esterification), decrease the incorporation of acetate (lipogenesis) into lipids and diminish circulating leptin levels. These effects of exercise training were not seen in pinealectomized rats. However, pinealectomized trained animals showed a marked reduction in lipolysis and an increased rate of acetate incorporation. In conclusion, we demonstrated for the first time that the pineal gland plays an important role in the regulation of lipid metabolism in such a way that its absence caused a severe alteration in the balance between lipogenesis and lipolysis, which becomes evident with the adaptation to exercise training.

Metabolic Disorders and Adipose Tissue Insulin Responsiveness in Neonatally STZ-Induced Diabetic Rats Are Improved by Long-Term Melatonin Treatment

Diabetes mellitus is a product of low insulin sensibility and pancreatic β-cell insufficiency. Rats with streptozotocin-induced diabetes during the neonatal period by the fifth day of age develop the classic diabetic picture of hyperglycemia, hypoinsulinemia, polyuria, and polydipsia aggravated by insulin resistance in adulthood. In this study, we investigated whether the effect of long-term treatment with melatonin can improve insulin resistance and other metabolic disorders in these animals. At the fourth week of age, diabetic animals started an 8-wk treatment with melatonin (1 mg/kg body weight) in the drinking water at night. Animals were then killing, and the sc, epididymal (EP), and retroperitoneal (RP) fat pads were excised, weighed, and processed for adipocyte isolation for morphometric analysis as well as for measuring glucose uptake, oxidation, and incorporation of glucose into lipids. Blood samples were collected for biochemical assays. Melatonin treatment reduced hyperglycemia, polydipsia, and polyphagia as well as improved insulin resistance as demonstrated by constant glucose disappearance rate and homeostasis model of assessment-insulin resistance. However, melatonin treatment was unable to recover body weight deficiency, fat mass, and adipocyte size of diabetic animals. Adiponectin and fructosamine levels were completely recovered by melatonin, whereas neither plasma insulin level nor insulin secretion capacity was improved in diabetic animals. Furthermore, melatonin caused a marked delay in the sexual development, leaving genital structures smaller than those of nontreated diabetic animals. Melatonin treatment improved the responsiveness of adipocytes to insulin in diabetic animals measured by tests of glucose uptake (sc, EP, and RP), glucose oxidation, and incorporation of glucose into lipids (EP and RP), an effect that seems partially related to an increased expression of insulin receptor substrate 1, acetyl-coenzyme A carboxylase and fatty acid synthase. In conclusion, melatonin treatment was capable of ameliorating the metabolic abnormalities in this particular diabetes model, including insulin resistance and promoting a better long-term glycemic control.

High Sodium Intake Enhances Insulin‐stimulated Glucose Uptake in Rat Epididymal Adipose Tissue

Objective: This study investigated the effect of different sodium content diets on rat adipose tissue carbohydrate metabolism and insulin sensitivity.

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

Abstract:  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.

Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance.

Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high‐caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high‐caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin‐stimulated glucose uptake in 3T3‐L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin‐induced glucose uptake in 3T3‐L1 adipocytes.

Chronic glucocorticoid treatment enhances lipogenic activity in visceral adipocytes of male Wistar rats

Glucocorticoid (GC) in excess promotes the redistribution of adipose tissue from peripheral to central sites of the body. In this study, we characterized an experimental condition of prolonged GC excess and investigated its effect on the lipogenic metabolism in white adipose tissue.

Pinealectomy interferes with the circadian clock genes expression in white adipose tissue.

Melatonin, the main hormone produced by the pineal gland, is secreted in a circadian manner (24‐hr period), and its oscillation influences several circadian biological rhythms, such as the regulation of clock genes expression (chronobiotic effect) and the modulation of several endocrine functions in peripheral tissues. Assuming that the circadian synchronization of clock genes can play a role in the regulation of energy metabolism and it is influenced by melatonin, our study was designed to assess possible alterations as a consequence of melatonin absence on the circadian expression of clock genes in the epididymal adipose tissue of male Wistar rats and the possible metabolic repercussions to this tissue. Our data show that pinealectomy indeed has impacts on molecular events: it abolishes the daily pattern of the expression of Clock, Per2, and Cry1 clock genes and Pparγ expression, significantly increases the amplitude of daily expression of Rev‐erbα, and affects the pattern of and impairs adipokine production, leading to a decrease in leptin levels. However, regarding some metabolic aspects of adipocyte functions, such as its ability to synthesize triacylglycerols from glucose along 24 hr, was not compromised by pinealectomy, although the daily profile of the lipogenic enzymes expression (ATP‐citrate lyase, malic enzyme, fatty acid synthase, and glucose‐6‐phosphate dehydrogenase) was abolished in pinealectomized animals.

Pinealectomy impairs adipose tissue adaptability to exercise in rats

Abstract:  This study investigated the effects of pinealectomy and exercise training on rat adipose tissue metabolism. Pinealectomized (PINX) and sham‐operated (CONTROL) adult male Wistar rats were subdivided into four subgroups, including PINX untrained, PINX trained, CONTROL untrained and CONTROL trained. At the end of the training period (8 wk), the rats were killed and peri‐epididymal adipocytes were isolated for in vitro insulin‐stimulated glucose uptake, conversion of d‐[U‐14C]‐glucose, l‐[U‐14C]‐lactate, [2‐14C]‐acetate and [1‐14C]‐palmitate into 14CO2, and insulin binding. Pinealectomy resulted in a significantly decreased insulin‐stimulated glucose uptake in adipocytes without affecting insulin‐binding capacity. However, in intact control animals only, training promoted a higher baseline glucose uptake in adipocytes. Training influenced the adipocyte ability to oxidize the different substrates: the rates of glucose and palmitate oxidation increased while the rates of lactate and acetate diminished. Nevertheless, these effects of exercise training were not seen in pinealectomized rats. Additionally, an increase in palmitate oxidation was observed in sedentary pinealectomized animals. In conclusion, these data show that the pineal gland alters the patterns of substrate utilization by the adipocyte, in such a way that its absence disrupts the ability to adapt to the metabolic demands evoked by exercise training in rats.