Time of day affects MrgD-dependent modulation of cardiomyocyte contractility.

The renin-angiotensin system (RAS) is composed of a series of peptides, receptors, and enzymes that play a pivotal role in maintaining cardiovascular homeostasis. Among the most important players in this system are the angiotensin-II and angiotensin-(1-7) peptides. Our group has recently demonstrated that alamandine (ALA), a peptide with structural and functional similarities to angiotensin-(1-7), interacts with cardiomyocytes, enhancing contractility via the Mas-related G protein-coupled receptor member D (MrgD). It is currently unknown whether this modulation varies along the distinct phases of the day. To address this issue, we assessed the ALA-induced contractility response of cardiomyocytes from mice at four Zeitgeber times (ZTs). At ZT2 (light phase), ALA enhanced cardiomyocyte shortening in an MrgD receptor-dependent manner, which was associated with nitric oxide (NO) production. At ZT14 (dark phase), ALA induced a negative modulation on the cardiomyocyte contraction. ß-Alanine, an MrgD agonist, reproduced the time-of-day effects of ALA on myocyte shortening. NG-nitro-l-arginine methyl ester, an NO synthase inhibitor, blocked the increase in fractional shortening induced by ALA at ZT2. No effect of ALA on myocyte shortening was observed at ZT8 and ZT20. Our results show that ALA/MrgD signaling in cardiomyocytes is subject to temporal modulation. This finding has significant implications for pharmacological approaches that combine chronotherapy for cardiac conditions triggered by disruption of circadian rhythms and hormonal signaling.NEW & NOTEWORTHY Alamandine, a member of the renin-angiotensin system, serves critical roles in cardioprotection, including the modulation of cardiomyocyte contractility. Whether this effect varies along the day is unknown. Our results provide evidence that alamandine via receptor MrgD exerts opposing actions on cardiomyocyte shortening, enhancing, or reducing contraction depending on the time of day. These findings may have significant implications for the development and effectiveness of future cardiac therapies.

Maternal hypothyroidism in mice influences glucose metabolism in adult offspring.

AIMS/HYPOTHESIS: During pregnancy, maternal metabolic disease and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offspring remains poorly understood. METHODS: We investigated this using a mouse model of hypothyroidism, induced by administration of an iodine-deficient diet supplemented with propylthiouracil during gestation. RESULTS: Here, we show that, when fed normal chow, adult mice born to hypothyroid mothers were more glucose-tolerant due to beta cell hyperproliferation (two- to threefold increase in Ki67-positive beta cells) and increased insulin sensitivity. However, following 8 weeks of high-fat feeding, these offspring gained 20% more body weight, became profoundly hyperinsulinaemic (with a 50% increase in fasting insulin concentration), insulin-resistant and glucose-intolerant compared with controls from euthyroid mothers. Furthermore, altered glucose metabolism was maintained in a second generation of animals. CONCLUSIONS/INTERPRETATION: Therefore, gestational hypothyroidism induces long-term alterations in endocrine pancreas function, which may have implications for type 2 diabetes prevention in affected individuals.

Altered myocardial metabolic adaptation to increased fatty acid availability in cardiomyocyte-specific CLOCK mutant mice.

A mismatch between fatty acid availability and utilization leads to cellular/organ dysfunction during cardiometabolic disease states (e.g., obesity, diabetes mellitus). This can precipitate cardiac dysfunction. The heart adapts to increased fatty acid availability at transcriptional, translational, post-translational and metabolic levels, thereby attenuating cardiomyopathy development. We have previously reported that the cardiomyocyte circadian clock regulates transcriptional responsiveness of the heart to acute increases in fatty acid availability (e.g., short-term fasting). The purpose of the present study was to investigate whether the cardiomyocyte circadian clock plays a role in adaptation of the heart to chronic elevations in fatty acid availability. Fatty acid availability was increased in cardiomyocyte-specific CLOCK mutant (CCM) and wild-type (WT) littermate mice for 9weeks in time-of-day-independent (streptozotocin (STZ) induced diabetes) and dependent (high fat diet meal feeding) manners. Indices of myocardial metabolic adaptation (e.g., substrate reliance perturbations) to STZ-induced diabetes and high fat meal feeding were found to be dependent on genotype. Various transcriptional and post-translational mechanisms were investigated, revealing that Cte1 mRNA induction in the heart during STZ-induced diabetes is attenuated in CCM hearts. At the functional level, time-of-day-dependent high fat meal feeding tended to influence cardiac function to a greater extent in WT versus CCM mice. Collectively, these data suggest that CLOCK (a circadian clock component) is important for metabolic adaption of the heart to prolonged elevations in fatty acid availability. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

Biotinylation: a novel posttranslational modification linking cell autonomous circadian clocks with metabolism.

Circadian clocks are critical modulators of metabolism. However, mechanistic links between cell autonomous clocks and metabolic processes remain largely unknown. Here, we report that expression of the biotin transporter slc5a6 gene is decreased in hearts of two distinct genetic mouse models of cardiomyocyte-specific circadian clock disruption [i.e., cardiomyocyte-specific CLOCK mutant (CCM) and cardiomyocyte-specific BMAL1 knockout (CBK) mice]. Biotinylation is an obligate posttranslational modification for five mammalian carboxylases: acetyl-CoA carboxylase α (ACCα), ACCß, pyruvate carboxylase (PC), methylcrotonyl-CoA carboxylase (MCC), and propionyl-CoA carboxylase (PCC). We therefore hypothesized that the cardiomyocyte circadian clock impacts metabolism through biotinylation. Consistent with decreased slc5a6 expression, biotinylation of all carboxylases is significantly decreased (10-46%) in CCM and CBK hearts. In association with decreased biotinylated ACC, oleate oxidation rates are increased in both CCM and CBK hearts. Consistent with decreased biotinylated MCC, leucine oxidation rates are significantly decreased in both CCM and CBK hearts, whereas rates of protein synthesis are increased. Importantly, feeding CBK mice with a biotin-enriched diet for 6 wk normalized myocardial 1) ACC biotinylation and oleate oxidation rates; 2) PCC/MCC biotinylation (and partially restored leucine oxidation rates); and 3) net protein synthesis rates. Furthermore, data suggest that the RRAGD/mTOR/4E-BP1 signaling axis is chronically activated in CBK and CCM hearts. Finally we report that the hepatocyte circadian clock also regulates both slc5a6 expression and protein biotinylation in the liver. Collectively, these findings suggest that biotinylation is a novel mechanism by which cell autonomous circadian clocks influence metabolic pathways.

Melatonin synthesis impairment as a new deleterious outcome of diabetes-derived hyperglycemia.

Melatonin is a neurohormone that works as a nighttime signal for circadian integrity and health maintenance. It is crucial for energy metabolism regulation, and the diabetes effects on its synthesis are unresolved. Using diverse techniques that included pineal microdialysis and ultrahigh-performance liquid chromatography, the present data show a clear acute and sustained melatonin synthesis reduction in diabetic rats as a result of pineal metabolism impairment that is unrelated to cell death. Hyperglycemia is the main cause of several diabetic complications, and its consequences in terms of melatonin production were assessed. Here, we show that local high glucose (HG) concentration is acutely detrimental to pineal melatonin synthesis in rats both in vivo and in vitro. The clinically depressive action of high blood glucose concentration in melatonin levels was also observed in type 1 diabetes patients who presented a negative correlation between hyperglycemia and 6-sulfatoxymelatonin excretion. Additionally, high-mean-glycemia type 1 diabetes patients presented lower 6-sulfatoxymelatonin levels when compared to control subjects. Although further studies are needed to fully clarify the mechanisms, the present results provide evidence that high circulating glucose levels interfere with pineal melatonin production. Given the essential role played by melatonin as a powerful antioxidant and in the control of energy homeostasis, sleep and biological rhythms and knowing that optimal glycemic control is usually an issue for patients with diabetes, melatonin supplementation may be considered as an additional tool to the current treatment.

Adaptations of the aging animal to exercise: role of daily supplementation with melatonin.

The pineal gland, through melatonin, seems to be of fundamental importance in determining the metabolic adaptations of adipose and muscle tissues to physical training. Evidence shows that pinealectomized animals fail to develop adaptive metabolic changes in response to aerobic exercise and therefore do not exhibit the same performance as control-trained animals. The known prominent reduction in melatonin synthesis in aging animals led us to investigate the metabolic adaptations to physical training in aged animals with and without daily melatonin replacement. Male Wistar rats were assigned to four groups: sedentary control (SC), trained control (TC), sedentary treated with melatonin (SM), and trained treated with melatonin (TM). Melatonin supplementation lasted 16 wk, and the animals were subjected to exercise during the last 8 wk of the experiment. After euthanasia, samples of liver, muscle, and adipose tissues were collected for analysis. Trained animals treated with melatonin presented better results in the following parameters: glucose tolerance, physical capacity, citrate synthase activity, hepatic and muscular glycogen content, body weight, protein expression of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and protein kinase activated by adenosine monophosphate (AMPK) in the liver, as well as the protein expression of the glucose transporter type 4 (GLUT4) and AMPK in the muscle. In conclusion, these results demonstrate that melatonin supplementation in aging animals is of great importance for the required metabolic adaptations induced by aerobic exercise. Adequate levels of circulating melatonin are, therefore, necessary to improve energetic metabolism efficiency, reducing body weight and increasing insulin sensitivity.

Melatonin improves insulin sensitivity independently of weight loss in old obese rats.

In aged rats, insulin signaling pathway (ISP) is impaired in tissues that play a pivotal role in glucose homeostasis, such as liver, skeletal muscle, and adipose tissue. Moreover, the aging process is also associated with obesity and reduction in melatonin synthesis from the pineal gland and other organs. The aim of the present work was to evaluate, in male old obese Wistar rats, the effect of melatonin supplementation in the ISP, analyzing the total protein amount and the phosphorylated status (immunoprecipitation and immunoblotting) of the insulin cascade components in the rat hypothalamus, liver, skeletal muscle, and periepididymal adipose tissue. Melatonin was administered in the drinking water for 8- and 12 wk during the night period. Food and water intake and fasting blood glucose remained unchanged. The insulin sensitivity presented a 2.1-fold increase both after 8- and 12 wk of melatonin supplementation. Animals supplemented with melatonin for 12 wk also presented a reduction in body mass. The acute insulin-induced phosphorylation of the analyzed ISP proteins increased 1.3- and 2.3-fold after 8- and 12 wk of melatonin supplementation. The total protein content of the insulin receptor (IR) and the IR substrates (IRS-1, 2) remained unchanged in all investigated tissues, except for the 2-fold increase in the total amount of IRS-1 in the periepididymal adipose tissue. Therefore, the known age-related melatonin synthesis reduction may also be involved in the development of insulin resistance and the adequate supplementation could be an important alternative for the prevention of insulin signaling impairment in aged organisms.

Diurnal, metabolic and thermogenic alterations in a murine model of accelerated aging.

Senescence-Accelerated Mouse-Prone 8 (SAMP8) mice exhibit characteristics of premature aging, including hair loss, cognitive dysfunction, reduced physical activity, impaired metabolic homeostasis, cardiac dysfunction and reduced lifespan. Interestingly, circadian disruption can induce or augment many of these same pathologies. Moreover, previous studies have reported that SAMP8 mice exhibit abnormalities in circadian wheel-running behavior, indicating possible alterations in circadian clock function. These observations led to the hypothesis that 24 h rhythms in behavior and/or circadian clock function are altered in SAMP8 mice and that these alterations may contribute to perturbations in whole-body metabolism. Here, we report that 6-month-old SAMP8 mice exhibit a more prominent biphasic pattern in daily behaviors (food intake and physical activity) and whole-body metabolism (energy expenditure, respiratory exchange ratio), relative to SAMR1 control mice. Consistent with a delayed onset of food intake at the end of the light phase, SAMP8 mice exhibit a phase delay (1.3-1.9 h) in 24 h gene expression rhythms of major circadian clock components (bmal1, rev-erbα, per2, dbp) in peripheral tissues (liver, skeletal muscle, white adipose tissue [WAT], brown adipose tissue [BAT]). Forcing mice to consume food only during the dark period improved alignment of both whole-body metabolism and oscillations in expression of clock genes in peripheral tissues between SAMP8 and SAMR1 mice. Next, interrogation of metabolic genes revealed altered expression of thermogenesis mediators (ucp1, pgc1α, dio2) in WAT and/or BAT in SAMP8 mice. Interestingly, SAMP8 mice exhibit a decreased tolerance to an acute (5 h) cold challenge. Moreover, SAMP8 and SAMR1 mice exhibited differential responses to a chronic (1 week) decrease in ambient temperature; the greatest response in whole-body substrate selection was observed in SAMR1 mice. Collectively, these observations reveal differential behaviors (e.g. 24 h food intake patterns) in SAMP8 mice that are associated with perturbations in peripheral circadian clocks, metabolism and thermogenesis.

Disruption of the Pituitary Circadian Clock Induced by Hypothyroidism and Hyperthyroidism: Consequences on Daily Pituitary Hormone Expression Profiles.

BACKGROUND: The secretion of pituitary hormones oscillates throughout the 24-hour period, indicating that circadian clock-mediated mechanisms regulate this process in the gland. Additionally, pituitary hormone synthesis has been shown to be altered in hypo- and hyperthyroidism. Although thyroid hormones can modulate the other peripheral clocks, the interaction between thyroid hormone levels and circadian clock gene expression in the anterior pituitary has yet to be elucidated. METHODS: Male Wistar rats were divided into three groups: control, hypothyroid, and hyperthyroid. Following the experimental procedures, animals were euthanized every three hours over the course of a 24-hour period. The anterior pituitary glands were excised and processed for mRNA expression analysis by quantitative reverse transcriptase polymerase chain reaction. One- and two-way analysis of variance as well as cosinor analysis were used to evaluate the time-of-day-dependent differential expression for each gene in each experimental group and their interactions. RESULTS: Hyperthyroidism increased the mRNA expression of core clock genes and thyrotrophic embryonic factor (Tef), as well as the mesor and amplitude of brain and muscle Arnt-like protein-1 (Bmal1) and the mesor of nuclear receptor subfamily 1 (Nr1d1) group D member 1, when compared to euthyroid animals. Hypothyroidism disrupted the circadian expression pattern of Bmal1 and period circadian regulator 2 (Per2) and decreased the mesor of Nr1d1 and Tef. Furthermore, it was observed that the pituitary content of Dio2 mRNA was unaltered in hyperthyroidism but substantially elevated in hypothyroidism during the light phase. The upregulated expression was associated with an increased mesor and amplitude, along with an advanced acrophase. The gene expression of all the pituitary hormones was found to be altered in hypo- and hyperthyroidism. Moreover, prolactin (Prl) and luteinizing hormone beta subunit (Lhb) displayed circadian expression patterns in the control group, which were disrupted in both the hypo- and hyperthyroid states. CONCLUSION: Taken together, the data demonstrate that hypo- and hyperthyroidism alter circadian clock gene expression in the anterior pituitary. This suggests that triiodothyronine plays an important role in the regulation of pituitary gland homeostasis, which could ultimately influence the rhythmic synthesis and/or secretion of all the anterior pituitary hormones.

Repercussions of hypo and hyperthyroidism on the heart circadian clock.

Myocardial gene expression and metabolism fluctuate over the course of the day in association with changes in energy supply and demand. Time-of-day-dependent oscillations in myocardial processes have been linked to the intrinsic cardiomyocyte circadian clock. Triiodothyronine (T3) is an important modulator of heart metabolism and function. Recently, our group has reported time-of-day-dependent rhythms in cardiac T3 sensitivity, as well as, T3-mediated acute alterations on core clock components. Hypo and hyperthyroidism are the second most prevalent endocrine disease worldwide. Considering the importance of the cardiomyocyte circadian clock and T3 to cardiac physiology, the aim of this study was to investigate the consequences of chronic hypo and hyperthyroidism on 24-h rhythms of circadian clock genes in the heart. Hypo and hyperthyroidism was induced in rats by thyroidectomy (Tx) and i.p. injections of supraphysiological dose of T3, respectively. Here we report alterations in mRNA levels of the major core clock components (Bmal1, Per2, Nr1d1, and Rora) for both experimental conditions (with the exception of Per2 during hyperthyroid condition). Oscillations in mRNA levels of key glucose and fatty-acid metabolism genes known to be clock controlled (Pdk4, Ucp3, Acot1, and Cd36) were equally affected by the experimental conditions, especially during the hypothyroid state. These findings suggest that chronic alterations in thyroid status significantly impacts 24-h rhythms in circadian clock and metabolic genes in the heart. Whether these perturbations contribute toward the pathogenesis of cardiac dysfunction associated with hypo and hyperthyroidism requires further elucidation.

An overview of the emerging interface between cardiac metabolism, redox biology and the circadian clock.

At various biological levels, mammals must integrate with 24-hr rhythms in their environment. Daily fluctuations in stimuli/stressors of cardiac metabolism and oxidation-reduction (redox) status have been reported over the course of the day. It is therefore not surprising that the heart exhibits dramatic oscillations in various cellular processes over the course of the day, including transcription, translation, ion homeostasis, metabolism, and redox signaling. This temporal partitioning of cardiac processes is governed by a complex interplay between intracellular (e.g., circadian clocks) and extracellular (e.g., neurohumoral factors) influences, thus ensuring appropriate responses to daily stimuli/stresses. The purpose of the current article is to review knowledge regarding control of metabolism and redox biology in the heart over the course of the day, and to highlight whether disruption of these daily rhythms contribute towards cardiac dysfunction observed in various disease states.

Temporal partitioning of adaptive responses of the murine heart to fasting.

Recent studies suggest that the time of day at which food is consumed dramatically influences clinically-relevant cardiometabolic parameters (e.g., adiposity, insulin sensitivity, and cardiac function). Meal feeding benefits may be the result of daily periods of feeding and/or fasting, highlighting the need for improved understanding of the temporal adaptation of cardiometabolic tissues (e.g., heart) to fasting. Such studies may provide mechanistic insight regarding how time-of-day-dependent feeding/fasting cycles influence cardiac function. We hypothesized that fasting during the sleep period elicits beneficial adaptation of the heart at transcriptional, translational, and metabolic levels. To test this hypothesis, temporal adaptation was investigated in wild-type mice fasted for 24-h, or for either the 12-h light/sleep phase or the 12-h dark/awake phase. Fasting maximally induced fatty acid responsive genes (e.g., Pdk4) during the dark/active phase; transcriptional changes were mirrored at translational (e.g., PDK4) and metabolic flux (e.g., glucose/oleate oxidation) levels. Similarly, maximal repression of myocardial p-mTOR and protein synthesis rates occurred during the dark phase; both parameters remained elevated in the heart of fasted mice during the light phase. In contrast, markers of autophagy (e.g., LC3II) exhibited peak responses to fasting during the light phase. Collectively, these data show that responsiveness of the heart to fasting is temporally partitioned. Autophagy peaks during the light/sleep phase, while repression of glucose utilization and protein synthesis is maximized during the dark/active phase. We speculate that sleep phase fasting may benefit cardiac function through augmentation of protein/cellular constituent turnover.

The in vitro maintenance of clock genes expression within the rat pineal gland under standard and norepinephrine-synchronized stimulation.

Although the norepinephrine (NE) synchronization protocol was proved to be an important procedure for further modulating in vitro pineal melatonin synthesis, the maintenance of clock genes under the same conditions remained to be investigated. The aim of this study was to investigate the maintenance of the clock genes expression in pineal gland cultures under standard and NE-synchronized stimulation. The glands were separated into three experimental groups: Control, Standard (acute NE-stimulation), and NE-synchronized. The expression of Bmal1, Per2, Cry2, Rev-erbα, the clock controlled gene Dbp and Arylalkylamine-N-acetyltransferase were investigated, as well as melatonin content. No oscillations were observed in the expression of the investigated genes from the control group. Under Standard NE stimulation, the clock genes did not exhibit a rhythmic pattern of expression. However, in the NE-synchronized condition, a rhythmic expression pattern was observed in all cases. An enhancement in pineal gland responsiveness to NE stimulation, reflected in an advanced synthesis of melatonin was also observed. Our results reinforce our previous hypothesis that NE synchronization of pineal gland culture mimics the natural rhythmic release of NE in the gland, increasing melatonin synthesis and keeping the pineal circadian clock synchronized, ensuring the fine adjustments that are relied in the clockwork machinery.

Lactate activates the somatotropic axis in rats.

Under physical activity a wide variety of cellular metabolic products and hormones are altered in the blood stream, including lactate, a metabolite of pyruvate reduction, and growth hormone (GH). Although a positive correlation between lactate and GH seems to exist during exercise, the role of lactate as a mediator of GH production has never been investigated. Thus, the aim of this study was to investigate whether lactate could activate the somatotropic axis and stimulate GH synthesis/release, contributing to the enhanced somatotropic activity described in exercise conditions. Male adult Wistar rats were acutely treated with sodium lactate [15 or 150µmols, i.p.] at the beginning of the active period (Zeitgeber time 13-14), and euthanized by decapitation 30, 60 and 120min after the injections. Serum GH concentration were determined using ELISA and Gh and Igf-1 mRNA expressions were quantified by qPCR. Serum GH concentration and Gh mRNA expression were increased 30min after lactate injections for both treatments. However, [15µmols] of lactate injection kept GH serum concentration chronically high throughout the experimental period. Igf-1 mRNA expression was increased only 60min after challenge with [15µmols] of lactate, time point which corresponded to 30min after the serum GH peak. The present results led us to conclude that lactate mediates activation of the somatotropic axis, therefore emphasizing its possible role on GH synthesis/release, and further indicating that it could play a part on the increased GH secretion observed in exercise conditions.

Cardiomyocyte-specific BMAL1 plays critical roles in metabolism, signaling, and maintenance of contractile function of the heart.

Circadian clocks are cell autonomous, transcriptionally based, molecular mechanisms that confer the selective advantage of anticipation, enabling cells/organs to respond to environmental factors in a temporally appropriate manner. Critical to circadian clock function are 2 transcription factors, CLOCK and BMAL1. The purpose of the present study was to reveal novel physiologic functions of BMAL1 in the heart, as well as to determine the pathologic consequences of chronic disruption of this circadian clock component. To address this goal, we generated cardiomyocyte-specific Bmal1 knockout (CBK) mice. Following validation of the CBK model, combined microarray and in silico analyses were performed, identifying 19 putative direct BMAL1 target genes, which included a number of metabolic (e.g., ß-hydroxybutyrate dehydrogenase 1 [Bdh1]) and signaling (e.g., the p85α regulatory subunit of phosphatidylinositol 3-kinase [Pik3r1]) genes. Results from subsequent validation studies were consistent with regulation of Bdh1 and Pik3r1 by BMAL1, with predicted impairments in ketone body metabolism and signaling observed in CBK hearts. Furthermore, CBK hearts exhibited depressed glucose utilization, as well as a differential response to a physiologic metabolic stress (i.e., fasting). Consistent with BMAL1 influencing critical functions in the heart, echocardiographic, gravimetric, histologic, and molecular analyses revealed age-onset development of dilated cardiomyopathy in CBK mice, which was associated with a severe reduction in life span. Collectively, our studies reveal that BMAL1 influences metabolism, signaling, and contractile function of the heart.

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 demonstrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor RORα 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 RORα 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 RORα 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-erbα expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erbα mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erbα 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.

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland.

AIMS: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. MAIN METHODS: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3:AANAT complex, respectively. KEY FINDINGS: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced "night". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IRbeta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. SIGNIFICANCE: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland.