Pineal gland (melatonin) affects the parturition time,but not luteal function and fetal growth,in pregnant rats

The purpose of the present study was to investigate the role of pineal gland (melatonin) on parturition time, luteal function, and fetal growth in pregnant rats. Cycling rats were subjected to pinealectomy or sham operation under ether anesthesia; and pinealectomized rats immediately underwent implantation of a melatonin capsule (PINX + Mel group) or a vehicle-containing capsule (PINX group), and sham operated rats also underwent implantation of a vehicle-containing capsule (control group). All rats were maintained under the same photoperiod conditions (14 L:10 D) and were induced pregnancy. Blood samples were obtained on days 7, 12, 15, 17, 19, and 21 of pregnancy to measure serum progesterone concentrations, and parturition times were recorded on days 22 and 23. In the next experiment, pregnant PINX rats received subcutaneous injection of melatonin (10 microg/body) at 08:00 h (PINX + 8 h group) or at 20:00 h (PINX + 20 h group) from day 15 to the end of pregnancy, and parturition times were recorded. Parturition times of rats in the PINX group, the PINX + Mel group or the PINX + 8 h group, but not the PINX + 20 h group, were significantly different compared with those in the control group. Pinealectomy or melatonin implantation did not affect serum progesterone concentrations during pregnancy or the number and weight of fetuses or corpora lutea. The present results indicate that pineal gland (melatonin rhythm) synchronizing with photoperiodic rhythm is likely to be an important determinant of parturition time, but it does not affect progesterone production or fetal growth in pregnant rats.     

Melatonin regulates type 2 deiodinase gene expression in the Syrian hamster

In seasonal species, photoperiod organizes various physiological processes, including reproduction. Recent data indicate that the expression of type 2 iodothyronine deiodinase (Dio2) is modulated by photoperiod in the mediobasal hypothalamus of some seasonal species. Dio2 is believed to control the local synthesis of bioactive T(3) to regulate gonadal response. Here we used in situ hybridization to study Dio2 expression in the hypothalamus of a photoperiodic rodent, the Syrian hamster. Dio2 was highly expressed in reproductively active hamsters in long day, whereas it was dramatically reduced in sexually inhibited hamsters maintained in short day. This contrasted with the laboratory rat, a nonphotoperiodic species, in which no evidence for Dio2 photoperiodic modulation was seen. We also demonstrate that photoperiodic variations of Dio2 expression in hamsters are independent from secondary changes in gonadal steroids. Studies in pinealectomized hamsters showed that the photoperiodic variation of Dio2 expression is melatonin dependent, and injections of long day hamsters with melatonin for only 7 d were sufficient to inhibit Dio2 expression to that of short day levels. Finally, because in some seasonal species thyroid hormones are involved in photorefractoriness, we examined Dio2 expression in short day-refractory hamsters and found that Dio2 mRNA levels remained low despite full reproductive recrudescence. Altogether, these results demonstrate that in the Syrian hamster Dio2 is photoperiodically modulated via a melatonin-dependent process. Furthermore, refractoriness to photoperiod in hamsters appears to occur independently of Dio2. These results raise new perspectives for understanding how thyroid hormones are involved in the control of photoperiodic neuroendocrine processes.     

Melatonin improves metabolic syndrome induced by high fructose intake in rats

Abstract: In this study, we examined whether melatonin improves metabolic syndrome induced by high fructose intake in male Wistar rats. Feeding of a diet containing 60% fructose (HFD) for 4 or 6 wk caused increased serum insulin, triglyceride, total cholesterol, free fatty acids, uric acid, leptin, and lipid peroxide concentrations as well as hepatic triglyceride and cholesterol concentrations, and relative intra‐abdominal fat and liver weights. The 4‐ or 6‐wk HFD feeding reduced serum high‐density lipoprotein cholesterol and adiponectin concentrations. The 6‐wk HFD feeding increased serum tumor necrosis factor‐α concentration and hepatic lipid peroxide concentration and lowered hepatic reduced glutathione concentration. Daily intraperitoneal administration of melatonin (1 or 10 mg/kg body weight), starting at 4‐wk HFD feeding, attenuated these changes at 6‐wk HFD feeding more effectively at its higher dose than at its lower dose. In an oral glucose tolerance test, rats with 4‐ or 6‐wk HFD feeding showed higher serum insulin response curve and normal serum glucose response curve when compared with the corresponding animals that received the control diet. The 4‐ or 6‐wk HFD feeding caused insulin resistance, judging from the scores of HOMR‐IR and QUICKI, which are indices of insulin resistance. The daily administered melatonin (1 or 10 mg/kg body weight) ameliorated the higher serum insulin response curve in the oral glucose tolerance test and insulin resistance at 6‐wk HFD feeding more effectively at its higher dose than at its lower dose. These results indicate that melatonin improves metabolic syndrome induced by high fructose intake in rats.     

Pinealectomy impairs adipose tissue adaptability to exercise in rats

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.     

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.     

Pinealectomy alters adipose tissue adaptability to fasting in rats

This study investigated the effects of pinealectomy and fasting on rat adipose tissue metabolism, as well as on profiles of the hormones directly involved in its regulation (insulin, leptin, and corticosterone). Pinealectomized (PINX) and sham-operated (CONTROL) adult male Wistar rats were killed 6 weeks after surgery, in either fed or fasted (12 and 36 hours) states. Blood samples (for glucose and hormone determinations) and peri-epididymal adipocytes (for in vitro insulin-stimulated glucose uptake, oxidation, and incorporation into lipids) were collected. Pineal ablation decreased insulin-stimulated glucose uptake in adipocytes of both fed and fasted animals without affecting insulin-binding capacity. Pinealectomy attenuated the reduction in the ability to oxidize glucose in both basal and insulin-stimulated states during fasting. This alteration in the ability of adipocytes to oxidize glucose appeared together with a decrease in insulin-induced glucose incorporation into lipids in PINX animals. Additionally, pinealectomized rats showed higher corticosterone levels in both fed and fasted states, and a lower leptinemia with 36 hours of fasting, in comparison to CONTROLs. In conclusion, our data reinforce the hypothesis that the pineal gland has a role in the modulation of adipocyte metabolism, and its absence alters metabolic adaptation to fasting in rats.     

Pinealectomy reduces hepatic and muscular glycogen content and attenuates aerobic power adaptability in trained rats

The current study emphasizes the crucial role of the pineal gland on the effects of chronic training in different tissues focusing on carbohydrate metabolism. We investigated the maximal oxygen uptake (aerobic power), muscle and liver glycogen content, and also the enzymes involved in the carbohydrate metabolism of rat adipose tissue. Pinealectomized and sham-operated adult male Wistar rats were distributed into four groups: pinealectomized (PINX) untrained, pinealectomized trained, control untrained and control trained. The maximal oxygen uptake capability was assayed before and after the training protocol by indirect open circuit calorimetry. The rats were killed after 8 wk of training. Blood samples were collected for glucose and insulin determinations. The glycogen content was assayed in the liver and muscle. Maximal activities of epididymal adipose tissue enzymes (hexokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase and malic enzyme) as well as adipocyte size were determined. The exercise training in control animals promoted an increase in the aerobic power and in liver glycogen content but caused a reduction in the malic enzyme activity in adipose tissue. However, PINX trained animals, in contrast to trained controls, showed a decrease in the aerobic power and in liver and muscle glycogen content, as well as an increase in the activity of the adipocyte enzymes involved in carbohydrate metabolism. In conclusion, these data show that the pineal gland integrity is necessary for the homeostatic control of energy metabolism among adipose, muscle and hepatic tissues. The pinealectomized animals showed alterations in adaptive responses of the maximal oxygen uptake to training. Therefore, the pineal gland must be considered an influential participant in the complex adaptation to exercise and is involved in the improvement of endurance capacity.     

Effects of pinealectomy on the levels and the circadian rhythm of plasma homocysteine in rats

Hyperhomocysteinemia is an independent cardiovascular risk factor. There are several factors including aging that contribute to the development of hyperhomocysteinemia. Nevertheless, the exact mechanisms causing this condition are still debated. We hypothesize that the age-related decrease in melatonin levels may be consequential in hyperhomocysteinemia. Recently, we found that plasma homocysteine (Hcy) levels are increased in pinealectomized (PINX) rats and melatonin reverses this increase. The aim of the present study was to determine if there is a circadian rhythm of plasma Hcy in rats and to examine the effect of pinealectomy on this cycle. Plasma Hcy levels demonstrated a 24-hr rhythm with a peak at 02:00 hr and a nadir at 14:00 hr in both control and PINX rats. Pinealectomy did not change the phase of the rhythm or the nocturnal elevation of plasma Hcy, but it did significantly increase mean plasma Hcy levels compared with those in controls and in rats that were sham pinealectomized (sPINX) (P < 0.05). Melatonin decreases plasma Hcy levels while causing an increase in total glutathione (tGSH). In conclusion, we speculate that decreasing levels of melatonin during aging lead to hyperhomocysteinemia and a decrease in tGSH and the latter may be one of the factors causing hyperhomocysteinemia in the elderly population.     

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.     

Temporal dynamics of type 2 deiodinase expression after melatonin injections in Syrian hamsters

In many species living in temperate zones, reproduction is controlled by the photoperiod. Recent findings have clarified that type 2 iodothyronine deiodinase (Dio2) plays a significant role in the photoperiodic response of gonads in the mediobasal hypothalamus, converting the prohormone T(4) into bioactive T(3). In mammals, Dio2 expression is suppressed by long-term melatonin injections, although the signal transduction pathways that link the melatonin signal to Dio2 expression are unknown. As a first step to approach the problem, we have here investigated the temporal dynamics of the melatonin effect on Dio2 expression using male Syrian hamsters. Dio2 mRNA levels were found to show diurnal rhythms under long-day conditions in an area adjacent to the tuberoinfundibular sulcus and in the ependymal cell layer lining the ventrobasal walls of the third ventricle. Daily sc melatonin injections given in the late afternoon under long-day condition suppressed the Dio2 mRNA levels already at the first day after the onset of the treatment in the ependymal cell layer lining the ventrobasal walls of the third ventricle, and 1 d later in an area adjacent to the tuberoinfundibular sulcus. These suppressive effects were sustained for at least 2 d after a single injection. Furthermore, we examined the temporal changes of the Dio2 expression after the onset of the treatment, showing that the suppression did not occur until midday of the next day. These data suggest that melatonin is involved in the signal transduction mechanisms controlling the photoperiodic response of gonads by acting on Dio2 expression rather rapidly through indirect pathways.     

Melatonin attenuates neuronal apoptosis through up‐regulation of K+–Cl− cotransporter KCC2 expression following traumatic brain injury in rats

Traumatic brain injury (TBI) initiates a complex cascade of neurochemical and signaling changes that leads to neuronal apoptosis, which contributes to poor outcomes for patients with TBI. The neuron‐specific K⁺–Cl^? cotransporter‐2 (KCC2), the principal Cl^? extruder in adult neurons, plays an important role in Cl^? homeostasis and neuronal function. This present study was designed to investigate the expression pattern of KCC2 following TBI and to evaluate whether or not melatonin is able to prevent neuronal apoptosis by modulating KCC2 expression in a Sprague Dawley rat controlled cortical impact model of TBI. The time course study showed decreased mRNA and protein expression of KCC2 in the ipsilateral peri‐core parietal cortex after TBI. Double immunofluorescence staining demonstrated that KCC2 is located in the plasma membrane of neurons. In addition, melatonin (10 mg/kg) was injected intraperitoneally at 5 minutes and repeated at 1, 2, 3, and 4 hours after brain trauma, and brain samples were extracted 24 hours after TBI. Compared to the vehicle group, melatonin treatment altered the down‐regulation of KCC2 expression in both mRNA and protein levels after TBI. Also, melatonin treatment increased the protein levels of brain‐derived neurotrophic factor (BDNF) and phosphorylated extracellular signal‐regulated kinase (p‐ERK). Simultaneously, melatonin administration ameliorated cortical neuronal apoptosis, reduced brain edema, and attenuated neurological deficits after TBI. In conclusion, our findings suggested that melatonin restores KCC2 expression, inhibits neuronal apoptosis and attenuates secondary brain injury after TBI, partially through activation of BDNF/ERK pathway.     

Melatonin increases brown adipose tissue mass and function in Zücker diabetic fatty rats: implications for obesity control

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Previously we demonstrated that melatonin browns subcutaneous fat in Zücker diabetic fatty (ZDF) rats. Other works pointed to melatonin as a signal that increases brown adipose tissue (BAT) mass and function in rodents. However, direct proof of thermogenic properties (uncoupled mitochondria) of the newly recruited BAT in response to melatonin is still lacking. Therefore, in this work, we investigated if melatonin recruits thermogenic BAT in ZDF rats. Zücker lean (ZL) and ZDF animals were subdivided into two groups, control (C) and treated with oral melatonin (M) for 6 weeks. Mitochondrial mass, activity of citrate synthase (CS), and respiratory chain complexes I and IV were lower in C‐ZDF than in C‐ZL animals (P < .001). Melatonin treatment increased BAT weight in ZDF rats (P < .001). Also, it rose mitochondrial mass (P < .01) and activities of CS and complexes I and IV (P < .001) in both, ZDF and ZL rats. Uncoupling protein 1 (UCP1) mRNA and protein were 50% lower in BAT from obese rats. Also, guanosine diphosphate (GDP) binding was lower in ZDF than in lean rats (P < .01). Melatonin treatment of obese rats restored the expression of UCP1 and GDP binding to levels of lean rats and sensitized the thermogenic response to cold exposure. These data demonstrated that melatonin recruits thermogenic BAT in ZDF rats. This may contribute to melatonin's control of body weight and its metabolic benefits.     

Melatonin prevents mitochondrial dysfunction and insulin resistance in rat skeletal muscle

Melatonin has a number of beneficial metabolic actions and reduced levels of melatonin may contribute to type 2 diabetes. The present study investigated the metabolic pathways involved in the effects of melatonin on mitochondrial function and insulin resistance in rat skeletal muscle. The effect of melatonin was tested both in vitro in isolated rats skeletal muscle cells and in vivo using pinealectomized rats (PNX). Insulin resistance was induced in vitro by treating primary rat skeletal muscle cells with palmitic acid for 24 hr. Insulin‐stimulated glucose uptake was reduced by palmitic acid followed by decreased phosphorylation of AKT which was prevented my melatonin. Palmitic acid reduced mitochondrial respiration, genes involved in mitochondrial biogenesis and the levels of tricarboxylic acid cycle intermediates whereas melatonin counteracted all these parameters in insulin‐resistant cells. Melatonin treatment increases CAMKII and p‐CREB but had no effect on p‐AMPK. Silencing of CREB protein by siRNA reduced mitochondrial respiration mimicking the effect of palmitic acid and prevented melatonin‐induced increase in p‐AKT in palmitic acid‐treated cells. PNX rats exhibited mild glucose intolerance, decreased energy expenditure and decreased p‐AKT, mitochondrial respiration, and p‐CREB and PGC‐1 alpha levels in skeletal muscle which were restored by melatonin treatment in PNX rats. In summary, we showed that melatonin could prevent mitochondrial dysfunction and insulin resistance via activation of CREB‐PGC‐1 alpha pathway. Thus, the present work shows that melatonin play an important role in skeletal muscle mitochondrial function which could explain some of the beneficial effects of melatonin in insulin resistance states.     

Rapid modulation of the silent information regulator 1 by melatonin after hypoxia‐ischemia in the neonatal rat brain

Increasing evidence indicates that melatonin possesses protective effects toward different kinds of damage in various organs, including the brain. In a neonatal model of hypoxia‐ischemia (HI), melatonin was neuroprotective and preserved the expression of the silent information regulator 1 (SIRT1) 24 hours after the insult. This study aimed to gain more insight into the role of SIRT1 in the protective effect of melatonin after HI by studying the early (1 hour) modulation of SIRT1 and its downstream targets, and the consequences on necrosis, apoptosis, autophagy, and glial cell activation. We found that melatonin administered 5 minutes after the ischemic insult significantly reduced necrotic cell death assessed 1 hour after its administration. In parallel, we found a reduced activation of the early phases of intrinsic apoptosis, detected by reduced BAX translocation to the mitochondria and preservation of the mitochondrial expression of cytochrome C, indicating a reduced outer mitochondrial membrane permeabilization in the melatonin‐treated ischemic animals. These effects were concomitant to increased expression and activity of SIRT1, reduced expression and acetylation of p53, and increased autophagy activation. Melatonin also reduced HI‐induced glial cells activation. SIRT1 was expressed in neurons after HI and melatonin but not in reactive glial cells expressing GFAP. Colocalization between SIRT1 and GFAP was found in some cells in control conditions. In summary, our results provide more insight into the connection between SIRT1 and melatonin in neuroprotection. The possibility that melatonin‐induced SIRT1 activity might contribute to differentiate neuronal progenitor cells during the neurodegenerative process needs to be further investigated.     

Bipedal ambulation induces experimental scoliosis in C57BL/6J mice with reduced plasma and pineal melatonin levels

In addition to the induction of scoliosis in chickens by pinealectomy (PINX), we previously demonstrated that removal of the pineal gland also produces scoliosis in bipedal rats, which can be inhibited by melatonin treatment. Using C57BL/6J mice with genetically low circulating melatonin levels, the main objective of the present study was to investigate whether bipedal ambulation in C57BL/6J mice has the same effects on spinal deformity as those seen in pinealectomized bipedal rats. The present study consisted of two phases. The aim of the first experiment was to determine whether the C57BL/6J mouse strain actually exhibits depressed plasma concentrations and/or pineal contents of melatonin during both the light and the dark phase of the light:dark cycle. The aims of the second experiment were to evaluate; (i) whether bipedal ambulation alone in the C57BL/6J mouse induces scoliosis, and (ii) whether PINX with bipedal ambulation in another mouse strain, i.e. C3H/HeJ, which normally exhibits diurnal fluctuations in melatonin synthesis and secretion, has effects similar to those of bipedal ambulation alone in C57BL/6J mice. C3H/HeJ mice, serving as controls, showed significant increases in both plasma concentrations and pineal contents of melatonin during the dark phase when compared with the light phase. In contrast, there were no differences in either circulating levels or pineal contents of melatonin between the light and dark phases in C57BL/6J mice. Moreover, plasma melatonin levels were below the detection limit of the assay in both phases and pineal melatonin was < 10% of that in C3H/HeJ mice. Bipedal ambulation for 40 wk in C57BL/6J mice induced scoliosis at a rate of 64.3%, and two of nine scoliotic mice showed two sites of spinal deformity. This type of ambulation in C3H/HeJ mice resulted in scoliosis at a lower rate (25%), and affected animals had only a single scoliotic site. However, PINX combined with bipedal ambulation in C3H/HeJ mice produced scoliosis at a rate (70%) similar to that seen in C57BL/6J mice, and some double deformations were induced. These results confirm our previous observations in rats, and also support our hypothesis that melatonin as well as the bipedal ambulation appear to play a critical pathogenic role in scoliosis in experimental mammals.     

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2^−/− mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.Although reactive oxygen species (ROS) are normal products of cellular metabolism, excessive accumulation of ROS resulting from nutritional imbalance and/or environmental stresses can provoke oxidative damage of diverse cellular macromolecules, such as DNA, RNA, and proteins (1). Accumulation of ROS has been associated with diverse degenerative diseases, such as cancer, neurodegeneration, and obesity-associated metabolic syndrome (2–4). To minimize detrimental consequences of ROS accumulation, cells are equipped with various antioxidant proteins, including superoxide dismutases, catalases, peroxiredoxins, and sestrins (5–7). Several ROS-scavenging chemicals or dietary supplements, such as butylated hydroxyanisole (BHA), N-acetylcysteine (NAC), and antioxidant vitamins, can assist with eliminating excessive amounts of ROS (8–10) and were once considered to be potential inhibitors of degenerative diseases associated with aging and obesity (11–13). However, most animal and human clinical studies failed to demonstrate the benefits of dietary antioxidants in restoring metabolic homeostasis or in promoting health and lifespan (13, 14).Uncoupling protein 1 (Ucp1) is an anion-carrier protein located in the inner membrane of the mitochondria. By dissipating the proton gradient across the mitochondrial inner membrane, Ucp1 uncouples substrate oxidation from ATP synthesis, ultimately reducing ATP production and generating heat (15). Ucp1-mediated mitochondrial uncoupling also suppresses ROS production during respiration (16). Ucp1 expression is induced upon exposure to cold temperature or nutritional overload, and this induction is important for protection of organisms against cold and obesity (17). Despite the significance of Ucp1 in energy metabolism, it is poorly understood how Ucp1 expression is regulated, other than the fact that cAMP and p38 MAPK signaling pathways (18–20) are necessary for Ucp1 induction upon cold stimuli. It also has not yet been explored whether subcellular ROS can regulate Ucp1 expression and subsequent heat generation.Sestrins are a family of stress-inducible proteins that regulate metabolic homeostasis (21). Sestrins have two independent biological activities largely divided into regulating AMP-activated protein kinase (AMPK)-mammalian target of rapamycin complex 1 (mTORC1) signaling and suppressing ROS accumulation (22). Loss of endogenous sestrins can provoke a variety of metabolic pathologies, including insulin resistance, fat accumulation, mitochondrial dysfunction, and oxidative damage (23, 24). Given the known roles of endogenous sestrins in reducing oxidative stress, fat accumulation, and insulin resistance, we thought that overexpression of sestrins may protect animals from developing obesity or obesity-associated metabolic pathologies. Among the three mammalian sestrins (Sestrin1–3), the metabolism-regulating functions of Sestrin2 have been the most rigorously characterized in metabolic organs, such as the liver and adipose tissue (AT) (23, 25). Thus, to examine the effects of Sestrin2 overexpression, we generated tetracyclin-regulated promoter-Sestrin2 (tet-Sesn2) transgenic mice that can express Sestrin2 in a tissue-specific manner when crossed with tissue-specific tetracycline activator (tTA) strains.Using tet-Sesn2 and AT-specific peroxisome proliferator-activated receptor γ (Pparγ)-tTA (Pparγ-tTA) strains, we generated Pparγ-tTA/tet-Sesn2 mice that express Sestrin2 specifically in AT. Given the ROS- and mTORC1-suppressing functions of Sestrin2 (21), we expected that the Pparγ-tTA/tet-Sesn2 (PG-Sn2) strain would exhibit decreased fat accumulation and improved metabolic homeostasis compared with the control strain. However, we found that Sestrin2 overexpression unexpectedly increased fat accumulation, which is associated with dramatic suppression of Ucp1 expression in brown adipose tissue (BAT). The redox-regulating function of Sestrin2, rather than its mTORC1-inhibiting function, was responsible for its Ucp1 regulation. We also discovered that administration of chemical antioxidants, such as BHA or NAC, could inhibit cAMP (in vitro)- or cold (in vivo)-induced Ucp1 expression. These results reveal a critical role of ROS in basal and cold-induced expression of Ucp1 in BAT.     

Physiological and pharmacological effects of melatonin on remote ischemic perconditioning after myocardial ischemia‐reperfusion injury in rats: Role of Cybb,Fas, NfκB,Irisin signaling pathway

It has been found that remote organ/limb temporary ischemia, known as remote ischemic conditioning, can provide protection against the formation of lethal ischemic outcome. Current evidence suggests that aging and age‐releated comorbidities impair the cardioprotective effects of conditionings. In conjuction with aging, decrease in melatonin synthesis from pineal gland can have role in the pathogenesis of aging and age‐related cardiovascular diseases. In this study, we investigated the effects of remote ischemic perconditioning (RIPerC) and physiological and pharmacological concentrations of melatonin on the infarct size, Fas gene, cytochrome b‐245 beta chain (Cybb) gene, nuclear factor‐kappa B (NfκB), and irisin using an in vivo model of myocardial ischemia/reperfusion (I/R) injury. Sprague‐Dawley rats that were divided into two groups first as non‐pinealectomized (Non‐Px) and pinealectomized (Px), and then (a) Control; (b) I/R (30‐minute ischemia, 120‐minute reperfusion caused by left coronary artery ligation); (c) I/R + RIPerC (when myocardial ischemia initiated, three cycles of 5‐minute occlusion followed by 5‐minute reperfusion); (d) I/R + Mel; (e) Px; (f) Px + I/R; (g) Px + I/R + RIPerC; (h) Px + I/R + RIPerC + Mel groups. The infarct size was determined by TTC staining and analyzed by the ImageJ program. Molecular parameters were evaluated by qRT‐PCR and Western blot. Results showed that increased infarct size in Non‐Px groups decreased with RIPerC and melatonin. However, increased infarct size in Px groups was decreased minimally with RIPerC and significantly decreased with RIPerC + Melatonin. Fold change in Fas gene was associated with the infarct size. RIPerC and melatonin reduced expressions of Cybb, NfκB, and irisin genes. The physiological release and pharmacological concentration of melatonin may improve protective effect of RIPerC against I/R‐induced infarct size by modulating Cybb, Fas, NfκB, Irisin signaling pathways.     

Melatonin improves insulin resistance and hepatic steatosis through attenuation of alpha‐2‐HS‐glycoprotein

Melatonin plays an important role in regulating circadian rhythms. It also acts as a potent antioxidant and regulates glucose and lipid metabolism, although the exact action mechanism is not clear. The α2‐HS‐glycoprotein gene (AHSG) and its protein, fetuin‐A (FETUA), are one of the hepatokines and are known to be associated with insulin resistance and type 2 diabetes. The aim of this study was to determine whether melatonin improves hepatic insulin resistance and hepatic steatosis in a FETUA‐dependent manner. In HepG2 cells treated with 300 μmol/L of palmitic acid, phosphorylated AKT expression decreased, and FETUA expression increased, but this effect was inhibited by treatment with 10 μmol/L of melatonin. However, melatonin did not improve insulin resistance in FETUA‐overexpressing cells, indicating that improvement in insulin resistance by melatonin was dependent on downregulation of FETUA. Moreover, melatonin decreased palmitic acid‐induced ER stress markers, CHOP, Bip, ATF‐6, XBP‐1, ATF‐4, and PERK. In addition, in the high‐fat diet (HFD) mice, oral treatment with 100 mg/kg/day melatonin for 10 weeks reduced body weight gain to one‐third of that of the HFD group and hepatic steatosis. Insulin sensitivity and glucose intolerance improved with the upregulation of muscle p‐AKT protein expression. FETUA expression and ER stress markers in the liver and serum of HFD mice were decreased by melatonin treatment. In conclusion, melatonin can improve hepatic insulin resistance and hepatic steatosis through reduction in ER stress and the resultant AHSG expression.