help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zalatan, F.
Right arrow Articles by Blask, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zalatan, F.
Right arrow Articles by Blask, D. E.
Endocrinology Vol. 142, No. 9 3783-3790
Copyright © 2001 by The Endocrine Society

ARTICLES

Inhibition of Isoproterenol-Induced Lipolysis in Rat Inguinal Adipocytes in Vitro by Physiological Melatonin via a Receptor-Mediated Mechanism

Fred Zalatan, Jean A. Krause and David E. Blask

Laboratory of Experimental Neuroendocrinology/Oncology, Bassett Research Institute, Cooperstown, New York 13326

Address all correspondence and requests for reprints to: Dr. David E. Blask, Bassett Research Institute, Cooperstown, New York 13326. E-mail: dblask{at}usa.net


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because the pineal hormone melatonin has been implicated in affecting adiposity in rats and fatty acid transport in certain rat tumor models, we tested whether melatonin regulates lipolysis in a normal cell system in vitro. Adipocytes were isolated from the inguinal fat pads (i.e. sc fat) of Sprague Dawley male rats during mid-light phase. Lipolysis was stimulated with isoproterenol (3 µM), and cells were incubated for 4 h in the presence or absence of a physiological circulating concentration of melatonin (1 nM). Lipolysis was measured by determining the amount of glycerol present in the incubation buffer, expressed as nmol glycerol/mg cellular fatty acid. We observed a 20- to 30-fold stimulation of basal lipolysis by isoproterenol, and this stimulation was inhibited 50–70% by melatonin. Melatonin exhibited this effect over a wide range of concentrations tested (100 pM–1 µM) with an IC50 of approximately 500 pM. The effect by melatonin (1 nM) was completely blocked by pertussis toxin (50 ng/ml), by 8-bromo-cAMP (10 nM), and by the melatonin receptor antagonist S-20928 (1 nM). These results suggest that the antilipolytic effect occurs through one of the Gi protein-coupled melatonin receptors because we have shown that both the mt1 (Mel 1a) and MT2 (Mel 1b) melatonin receptors are expressed in inguinal adipocytes. Melatonin inhibition of lipolysis was not observed in adipocytes isolated from rat epididymal fat pads (i.e. visceral fat), even though these cells also express both the mt1 and MT2 receptors. The results indicate that physiological circulating concentrations of melatonin inhibit isoproterenol-induced lipolysis in rat adipocytes via a G protein-coupled melatonin receptor-mediated signal transduction pathway in a site-specific manner.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXTENSIVE STUDIES OF mammalian adipocyte physiology in vitro have resulted in a broader understanding of the hormonal regulation of fatty acid release at the cellular level. These studies revealed that a variety of hormones stimulate lipolysis through the activation of hormone-sensitive lipase, which catalyzes the hydrolysis of triglycerides into fatty acids and glycerol. These products are then released through the cell membrane and, in vivo, are used as energy sources to meet the metabolic demands of the organism (1, 2, 3).

Lipolytic activating agents include catecholamines (such as epinephrine and isoproterenol), adrenocorticotropic hormone, and glucagon. All of these substances indirectly activate hormone-sensitive lipase by increasing the level of cAMP. In the case of the catecholamines, the signal pathway begins with binding to Gs-protein linked ß-adrenergic receptors, thereby increasing cAMP and activating protein kinase A, leading to the phosphorylation and activation of the lipase (1, 2, 3, 4). Recent studies suggest that phosphorylation of hormone-sensitive lipase is not sufficient for complete induction of lipolysis and that the lipase needs to be transported from the cytosol to the lipid droplet, where the enzyme has access to triglycerides (1, 2).

Other studies investigated inhibitory lipolytic agents, which include adenosine, nicotinic acid, and the E-series PGs (1, 2, 3). These agents lower the cAMP concentration through specific receptors linked to a Gi protein, resulting in the inactivation of the hormone-sensitive lipase (1, 2, 5). Insulin also inhibits lipolysis, but multiple pathways appear to be involved that are distinct from the Gi pathway (6). One mechanism by which insulin inhibits lipolysis is by activating cGMP-inhibited cAMP phosphodiesterase, thereby decreasing the level of cAMP (7). Subsequent studies showed a pathway involving phosphoinositide 3-kinase as playing a central role in regulating insulin’s antilipolytic action (8, 9).

Melatonin, a circadian hormone produced primarily at night by the pineal gland (10), has been implicated in the oncostatic inhibition of cancer cell growth both in vitro (11) and in vivo (12). Some of melatonin’s actions in the regulation of circadian and seasonal physiology are thought to be mediated through high affinity, Gi protein-linked melatonin receptors, notably mt1 and MT2 (also known as Mel 1a and Mel 1b, respectively) (13, 14, 15). Other actions of melatonin such as in the protection against oxidative damage (16) and regulation of gene expression (17) may not operate through high affinity receptors. For example, the direct antioxidant effects of melatonin are due to the free radical scavenging properties inherent in its chemical structure (16), whereas melatonin’s indirect antioxidant actions result from its capacity to stimulate several antioxidative enzymes (18). Because melatonin can freely diffuse through cell membranes, these effects are unlikely to be receptor-mediated. However, a melatonin receptor-based mechanism cannot be completely ruled out in the case of melatonin’s indirect antioxidant actions. The mechanisms involved in the modulation of gene expression by melatonin (17) are unclear.

In a rat hepatoma tumor model in vivo, melatonin has also been shown to inhibit growth by reducing tumor uptake of the cancer-promoting agent, linoleic acid (12). This process is thought to be receptor-mediated because it is blocked by pertussis toxin, which ADP-ribosylates some Gi proteins, thereby uncoupling them from their associated receptors and inhibiting the signaling pathway (19). Given the finding that melatonin blocks fatty acid uptake in a tumor model as well as suppresses adiposity in rats (20, 21), we investigated whether melatonin is involved in fatty acid release in a normal cell system. Because previous studies of melatonin’s involvement in lipolysis were inconclusive (22), we specifically tested whether melatonin inhibited lipolysis in rat adipocytes in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of adipocytes
Preparation of adipocytes from Sprague Dawley rats (Harlan Sprague Dawley, Inc.) was based on the method of Rodbell (23), slightly modified by Honnor et al. (24). Animals were provided with laboratory chow RMH 1000 (Agway, Inc., Syracuse, NY) and tap water ad libitum. Animals were approved for use in these studies by the Institutional Animal Care and Use Committee of the Bassett Research Institute and were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Approximately 4–6 g of the inguinal fat pad (i.e. sc fat) or epididymal fat pad (i.e. visceral fat) were surgically removed from a CO2 anesthetized 200–250 g male rat (rats were kept on an alternating 12-h light, 12-h dark cycle with lights on from 0600–1800 h). Fat pads were minced and suspended in 50 ml of Krebs-Ringer solution (0.1 M NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.25 mM MgSO4, 1.25 mM NaH2PO4) containing 25 mM HEPES, 5 mM glucose, 1% BSA (1) (BSA fraction V; Sigma, St. Louis, MO), and 50 mg collagenase type II (Sigma); the buffer was adjusted to pH 7.4 after the addition of BSA. The suspension, contained in a 125-ml flask, was incubated at 37 C for 1 h at 120 rpm. Cells were washed by transferring the suspension to a 50 ml conical tube and spinning at 200 x g for 3 min; a Pasteur pipet was then used to dip through the floating adipocyte layer and remove the buffer. Cells were then washed three times in the same buffer without collagenase (25 ml/wash). After the final wash, most of the buffer was removed, and the cell layer (which included undigested material) was filtered through one strip of gauze into a 14 ml culture tube. Any remaining buffer below the cell layer was then removed (the volume of the cell layer was typically 1–3 ml).

Adipocyte incubations
Incubations were set up in 12 x 75 mm borosilicate tubes in a final volume of 0.8 ml. The buffer used was the same used for adipocyte preparation except that collagenase was absent, and 4% BSA (essentially fatty acid free; Sigma) was used instead of 1% BSA (fraction V). A 50 µl aliquot of the prepared cell layer (roughly 100,000–200,000 cells) was added followed by the addition, where indicated, of 3 µM isoproterenol, 1 nM melatonin (or varying concentrations), 50 ng/ml of pertussis toxin (Sigma; 19, 25), 10 nM 8-bromo-cAMP (Calbiochem), or 1 nM melatonin receptor antagonist S-20928 (Servier). Samples were incubated at 37 C (usually for 4 h; a zero time point control group was included in each experiment) at 100 rpm. Incubations were stopped by placing vials on ice for 10 min, then transferring the buffer to a 1.5 ml tube, avoiding the top cell layer. Tubes were spun at low speed for 5 sec, and the buffer was transferred to a fresh tube, again avoiding any residual cell layer as well as the bottom pellet. This step was repeated two more times, followed by incubation of the aliquots at 60 C for 20 min to inactivate any residual enzymatic activity. Aliquots were stored at -20 C until glycerol measurements were performed (see below).

Total fatty acid extractions
Aliquots (50 µl) of adipocytes were set aside from the experimental samples and frozen at -80 C. At a later time, they were extracted for total fatty acid using the method of Folch et al. (26) modified by McDonald-Gibson (27). Heptadecanoic acid (300 µg) was added initially as an internal standard. The methyl esters of prepared fatty acid samples were analyzed by gas chromatography using a Hewlett-Packard Co. Model 5890A chromatograph equipped with a flame-ionization detector (220 C), an integrator (model 3396A), and an autoinjector (model 7673S). Separations were performed using a 0.25 mm x 30 m fused-silica capillary column (model 2330, Supelco) at 190 C with helium as the carrier gas (20 cm/sec). The average total mg of fatty acid (typically of 2–4 aliquots) was used in the unit calculation for glycerol released. The amount of total fatty acid in a 50 µl aliquot was usually between 12–18 mg, with little variation between samples from a single experiment.

Previously published work has used cell number, DNA content, packed cell volume, or the wet weight of the isolated fat pad in the unit calculation for glycerol released during lipolysis (protein content, which is normally used in biochemical analysis, cannot be used with adipocytes because, in most preparations, a high amount of BSA is added). Each of these methods, however, has disadvantages. Cell number is difficult to calculate because adipocytes float in aqueous buffers, making it difficult to prepare a representative aliquot to count cells. Another problem with cell number is that adipocytes vary greatly in size, which gives less meaning to the absolute number of cells present. DNA content may be a more accurate standard than cell number, but achieving a good yield of nucleic acid from fat cells can be cumbersome. Packed cell volume poses problems due to the fragility of adipocytes during centrifugation, as cell lysis will result in an underestimation of packed volume. The wet weight of the fat pad is inaccurate because not all of the adipocytes are subsequently recovered from the isolated tissue, especially when using the inguinal fat pad. We believe we have improved the unit calculation in our present study by expressing the glycerol released as nmol/mg cellular fatty acid. We have observed consistent results with this method, which, like protein content in many biochemical assays, accurately represents the amount of tissue present and allows for easy comparisons of measurements from different experiments.

Glycerol assays
Glycerol assays were done using the Triglyceride (GPO-Trinder) Reagent A (Sigma). Sample volume was 25–80 µl, diluted up to 100 µl with water. The reconstituted Reagent A (900 µl) was then added; absorbance was read at OD540 after 15 min with an Ultrospec 4000 UV/Visible Spectrophotometer (Amersham Pharmacia Biotech). Measurements of glycerol released were calculated as nmol/mg cellular fatty acid. Mean glycerol released (± SE) was compared among treatment groups either by one-way ANOVA followed by Student’s-Newman-Kewls post hoc test or by t test. Differences were considered significant at P < 0.05.

PCR analysis
Total RNA was isolated from rat inguinal and epididymal adipocytes using the SV Total RNA Isolation System (Promega Corp.). The mRNA (2–4 µg) was reversed transcribed with MMLV-Reverse Transcriptase (Life Technologies, Inc.) and the resulting cDNA was amplified using the PCR for either a mt1 or MT2 melatonin receptor product with Taq Polymerase (Promega Corp.) at 1.5 mM MgCl2 and a 58 C annealing temperature for 36 cycles using a RoboCycler (Stratagene, La Jolla, CA). For detection of the mt1 product, the 5' primer used was 5'-TGCTACATTTGCCACAGTCTC corresponding to a start of position 1 of the known sequence in rat1. The 3' primer used was 5'-GACCTATGAAGTTGAGTGGGG, amplifying a 397 bp product. For detection of the MT2 product, the 5' primer used was 5'-CTGGTGCATCTGTCACAGTG corresponding to a start of position 3 of the known sequence in rat2. The 3' primer used was 5'-CACAAACACTGCGAACATG, amplifying a 358-bp product. Because neither primer set used spanned an intron, PCR was done with RNA alone to ensure that the product observed did not arise from residual genomic DNA. Products were analyzed on 1.2% agarose gels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A physiological melatonin concentration partially inhibits isoproterenol-stimulated lipolysis in inguinal adipocytes
Because melatonin has been shown to modulate fatty acid uptake (12), we tested whether it affects fatty acid release. Rat adipocytes were isolated from the inguinal fat pad and incubated for 4 h in the presence or absence of isoproterenol (3 µM), a ß-adrenergic agonist that stimulates lipolysis (4). Cells were also treated in the presence or absence of a physiological blood concentration of melatonin (1 nM), which is in the range of the peak levels found in the circulation (10). It is important to note, however, that in some cells the intracellular concentrations of melatonin (e.g. brain and bone marrow cells) and body fluids (e.g. bile and cerebrospinal fluid) may exceed those in the blood by several orders of magnitude (i.e. micrograms). This would make local physiological concentrations of melatonin in tissues and cells much higher than in blood (28).

Table 1Go shows a representative set of experiments in which basal lipolysis increased almost 3-fold over the time 0 control value after 4 h of incubation. Melatonin did not significantly effect this low level of basal lipolysis. Isoproterenol induced a 31-fold increase in glycerol release over the 4 h control. Melatonin caused a 65% inhibition of isoproterenol-stimulated glycerol release. Exogenous glycerol (1 µmol) was added to cell samples with or without 1 nM melatonin to test whether melatonin was decreasing glycerol levels in the medium not by inhibiting lipolysis, but by stimulating cellular uptake and/or metabolism of glycerol. Glycerol levels remained constant throughout a 4 h incubation, and no decrease in glycerol was observed in the presence of melatonin (Zalatan, F., unpublished results).


View this table:
[in this window]
[in a new window]
  		Table 1. Melatonin inhibition of isoproterenol-induced lipolysis

 
A time course study depicted in Fig. 1Go shows that the level of lipolysis in the isoproterenol-stimulated cells was 35-fold higher than basal lipolysis after 2 h. There was a steady increase in glycerol release due to isoproterenol, with the melatonin inhibition becoming apparent after 2 h. The level of lipolysis in cells treated with isoproterenol and melatonin was 26-fold greater than basal lipolysis and 25% less than the isoproterenol group at 2 h. The effect of melatonin was magnified after 4 h as isoproterenol continued to stimulate lipolysis whereas glycerol release in the melatonin/isoproterenol samples leveled off, and a 61% inhibition by melatonin was observed. The pH of the medium remained constant throughout the incubation. There was only a slight increase in lactate dehydrogenase activity during the incubation, indicating that cell damage was minimal, and there was no difference in activity between the isoproterenol and melatonin/isoproterenol groups (Zalatan, F., unpublished results).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 1. Time course of melatonin inhibition of lipolysis. Adipocytes from the inguinal fat pad were isolated from a male Sprague Dawley rat. Cells were distributed to sample vials and incubated at 37 C for 1, 2, or 4 h with either vehicle (•), 3 µM isoproterenol ({blacksquare}), or isoproterenol + 1 nM melatonin ({circ}). One set of samples was not incubated, and this set was used for the time 0 point. Glycerol release was measured and calculated as the mean of duplicate samples ± SE. Total fatty acid was extracted from representative samples of cell aliquots to estimate the amount of cellular fatty acid present in the experimental samples, and this estimate was used in the unit calculation of glycerol released. The data were combined from four independent experiments (n = 4/time point). a, P < 0.05 vs. Time 0 control and vs. control at 1 h, 2 h, and 4 h. b, P < 0.05 vs. Isoproterenol 1 h. c, P < 0.05 vs. isoproterenol 4 h.

 
Dose-response effects of melatonin on isoproterenol- induced lipolysis
Several concentrations of melatonin, ranging from 1 pM–1 µM and encompassing physiological levels found in blood during darkness and local physiological concentrations found in both tissues and cells (27), were tested for their antilipolytic effect (Fig. 2Go). Melatonin inhibited isoproterenol-induced lipolysis in a dose-dependent manner with an IC50 of approximately 500 pM. The antilipolytic effect of melatonin appeared to saturate at 1 nM. The lowest concentration of melatonin that began to affect lipolysis was 100 pM (25% inhibition).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Dose response effect of melatonin on lipolysis. Adipocytes were prepared and incubated as in Fig. 1Go. Cells were treated with either vehicle (control), three different concentrations of melatonin alone (1 µM, 1 nM, and 1 pM), isoproterenol (3 µM), or isoproterenol + seven different concentrations of melatonin (1 µM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM). Glycerol release was then measured and calculated as the mean of duplicate or triplicate samples ± SE. The data were combined from three independent experiments (n = 3/treatment group). a, P < 0.05 vs. Isoproterenol. b, P < 0.05 vs. Melatonin (1 pM) + Isoproterenol and vs. Melatonin (10 pM) + Isoproterenol.

 
The melatonin effect on lipolysis is blocked by pertussis toxin, by 8-bromo-cAMP, and by melatonin receptor antagonist S-20928
To determine whether the inhibition of lipolysis by melatonin is a receptor mediated event, we tested whether pertussis toxin, which uncouples the signaling of some Gi/o proteins from their associated receptors, would block the antilipolytic effect of a physiological concentration of melatonin (1 nM). Table 2Go shows that melatonin inhibited isoproterenol-induced lipolysis by 57%, and that pertussis toxin (50 ng/ml) completely blocked this effect of melatonin. Pertussis toxin alone was capable of stimulating lipolysis, consistent with previously published work (25). The concentration we used stimulated lipolysis approximately 8-fold, making the amount of glycerol released by this group much lower than that seen with the melatonin/isoproterenol group. Therefore, one would not expect that the stimulation by pertussis toxin would mask its ability to block melatonin action. Pertussis toxin did not add to the effect of isoproterenol, which stimulated lipolysis 21-fold.


View this table:
[in this window]
[in a new window]
  		Table 2. Pertussis toxin inhibition of the melatonin effect on lipolysis

 
To test whether melatonin mediates its antilipolytic effect by lowering cAMP levels, we used 8-bromo-cAMP, a membrane-permeable analog of cAMP, to detect whether it could prevent melatonin action. At a concentration of 10 nM, 8-bromo-cAMP alone did not affect basal or isoproterenol-stimulated lipolysis (Table 3Go). Cells incubated with melatonin (1 nM) and isoproterenol (3 µM) showed a 56% decrease in lipolysis compared with isoproterenol alone. Cells incubated with a combination of melatonin, isoproterenol, and 8-bromo-cAMP, however, showed the same level of lipolysis as cells incubated with isoproterenol alone. Cells treated with a combination of melatonin and 8-bromo-cAMP had the same amount of lipolysis as the control group.


View this table:
[in this window]
[in a new window]
  		Table 3. 8-Bromo-cAMP inhibition of the melatonin effect on lipolysis

 
To demonstrate that the melatonin effect on lipolysis was mediated by a melatonin receptor, we used the melatonin receptor antagonist S-20928 (29). Treatment of cells with a combination of melatonin (1 nM), isoproterenol (3 µM), and S-20928 (1 nM) showed no decrease in lipolysis compared with cells treated with isoproterenol alone (Table 4Go). Cells treated with S-20928 in combination with melatonin also showed no decrease in basal lipolysis. By itself, S-20928 had no effect on either isoproterenol-stimulated or basal lipolysis.


View this table:
[in this window]
[in a new window]
  		Table 4. Melatonin receptor antagonist inhibition of the melatonin effect on lipolysis

 
Melatonin inhibition of lipolysis is not observed in epididymal adipocytes
Because epididymal adipocytes are studied in the majority of published work on lipolysis in vitro, we decided to test whether these cells show a similar response to melatonin as the inguinal adipocytes. Epididymal and inguinal adipocytes were isolated from the same set of rats and compared in their ability to respond to the antilipolytic effect of 1 nM melatonin. Both types of adipocytes responded to isoproterenol stimulation in a similar manner (Table 5Go). However, neither basal nor induced lipolysis in epididymal adipocytes was inhibited by melatonin, whereas inguinal adipocytes responded as in previous experiments. Epididymal adipocytes showed no response even in the presence of 1 µM melatonin (Zalatan, F., unpublished results).


View this table:
[in this window]
[in a new window]
  		Table 5. Melatonin effect compared between epididymal and inguinal adipocytes

 
Melatonin receptors are expressed in inguinal and epididymal adipocytes
RT-PCR was used to test whether inguinal and epididymal adipocytes express the mRNA coding for melatonin receptors mt1 (Mel 1a) and MT2 (Mel 1b) (Fig. 3Go). Primers specific for mt1 were used to successfully amplify a 397 bp PCR product from inguinal adipocyte cDNA. No product was observed from a reaction with RNA alone. Expression of MT2, using primers amplifying a 358 bp product, was also detected from inguinal adipocyte cDNA. In reactions with epididymal adipocyte cDNA, both mt1 and MT2 expression was also detected. No product was observed from RNA alone in either case.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. Expression of the mt1 and MT2 receptors in rat inguinal and epididymal adipocytes. Lane A, reaction with inguinal adipocyte cDNA using primers specific for mt1, yielding the expected 397 bp product; Lane B, negative control reaction with the mt1 primers using an equivalent amount of inguinal adipocyte total RNA; Lane C, reaction with inguinal adipocyte cDNA using primers specific for MT2, yielding the expected 358 bp product; Lane D, negative control reaction with the MT2 primers using an equivalent amount of inguinal adipocyte total RNA. Lane E, reaction with epididymal adipocyte cDNA using primers specific for mt1, yielding the 397 bp product; Lane F, negative control reaction with the mt1 primers using an equivalent amount of epididymal adipocyte total RNA; Lane G, reaction with epididymal adipocyte cDNA using primers specific for MT2, yielding the 358 bp product; Lane H, negative control reaction with the MT2 primers using an equivalent amount of epididymal adipocyte total RNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Very little is known about melatonin’s role in fatty acid transport/glycerol release across the cell membrane. Although other studies implicated an effect of melatonin in the regulation of lipolysis, these reports were either very preliminary or used hormone concentrations that greatly exceeded those normally found in the blood during darkness (22). An early study showed that pharmacological melatonin (1 mM) actually increased glycerol release from rat epididymal fat pads in vitro in response to epinephrine (30). The same study showed that pharmacological melatonin (1 mM) moderately inhibited ACTH-induced glycerol release in rabbit retroperitoneal fat but not in rat epididymal fat. In another report, pharmacological melatonin (0.1–1 mM) moderately inhibited both basal and hormone-induced lipolysis in rat epididymal adipocytes (31). In the current study, it is significant that a physiological concentration of melatonin (1 nM) exerted its effect in the presence of a saturating amount of isoproterenol in rat inguinal adipocytes but, in agreement with some of the earlier studies mentioned above, not in rat epididymal adipocytes.

It is likely that melatonin is exerting its effect through a Gi-associated receptor because this effect is blocked by pertussis toxin; this is consistent with the fact that the known melatonin receptors are primarily Gi-linked (32, 33). Although the isoproterenol stimulation of lipolysis acts through ß-adrenergic receptors (4), which can be Gi-linked in certain systems (34), it is less likely that melatonin is acting through these receptors because the pathway of isoproterenol-stimulated lipolysis is Gs-linked and should not be affected by pertussis toxin. Our results with 8-bromo-cAMP, which prevented melatonin’s antilipolytic effect, support the idea that melatonin inhibits lipolysis by lowering cAMP levels, presumably through a Gi pathway. We also observed an inhibitory, albeit inconsistent, effect of melatonin on basal lipolysis in inguinal adipocytes. The amount of basal lipolysis that we observed was generally quite low, making it difficult in some experiments to detect a significant difference below the basal level. In addition, the expression of the mt1 and MT2 melatonin receptors was detected in inguinal adipocytes, and melatonin’s antilipolytic effect was blocked by the melatonin receptor antagonist S-20928. The IC50 of approximately 500 pM in our dose-response study is consistent with an effect mediated via physiological nocturnal circulating melatonin levels found in rats (i.e. 300–700 pM) (35, 36) and the high affinity of the mt1 and MT2 receptors (13, 14, 15). Taken together, these results provide strong support for a melatonin receptor-based mechanism mediating the antilipolytic effect of melatonin.

Melatonin may be acting similarly to adenosine, a well-studied lipolysis inhibitor (1, 2, 3). Adenosine’s action is mediated through a Gi-linked adenosine receptor, resulting in an inhibition of adenylate cyclase. This lowers the concentration of cAMP and subsequently inactivates hormone-sensitive lipase plus a variety of other enzymes (1, 2, 3, 4, 5). It is known that rat epididymal adipocytes express Gi1, Gi2, and Gi3 (37), and we expect that the mt1 and MT2 receptors are linked to at least one of these G-proteins in inguinal adipocytes, most likely Gi2, and Gi3 (32).

The kinetics of melatonin inhibition of lipolysis differ markedly from adenosine. Our time course showed that a 2 h incubation is generally needed to observe a partial effect by melatonin, compared with a complete block by adenosine within minutes, which has been observed in previous studies (1, 2, 3, 5). It is possible that the melatonin receptor involved is more loosely coupled to a Gi protein than the adenosine receptor, thereby resulting in a slower cellular response to melatonin, although this would be inconsistent with the tight coupling recently reported in cell culture studies (38). It is also possible that because melatonin is very lipophilic and easily diffuses into the cell, it may quickly become less available for plasma membrane receptor binding, thus resulting in a delayed response. Support for this postulate is derived from studies showing that melatonin is rapidly taken-up and retained in high concentrations by tissues and cells (28, 39), and more specifically that endogenous melatonin achieves concentrations in sc adipose tissue from human mammary glands that are several orders of magnitude greater than circulating levels (40). Such high cellular and tissue concentrations of melatonin may be maintained by specific intracellular melatonin binding proteins (28, 39).

The slow melatonin response may also indicate regulation of the expression of an unknown gene product that is involved in melatonin inhibition of glycerol release. This would not, however, be consistent with recent observations from our laboratory showing that both the release (i.e. fasted state) and uptake (i.e. fed state) of fatty acids in rat inguinal fat pads were totally blocked within 2–10 min of perfusion in situ with melatonin (100 pM), distinguishing melatonin from other known lipolytic inhibitors (41). Therefore, this rapid suppression of fatty acid transport by melatonin in vivo makes it highly unlikely that regulation of gene expression is involved. In addition, melatonin has the ability to block fatty acid uptake and subsequent growth in a perfused rat hepatoma (12), which also distinguishes melatonin from other antilipolytic factors. These studies support the idea that melatonin may play a role in cancer cachexia (42), which is characterized by lipid mobilization and weight loss (43). The results point to a potentially positive dual effect of melatonin during cachexia; melatonin inhibits loss of stored fatty acids while also inhibiting tumor utilization of any circulating fatty acids (12, 41).

Because melatonin’s role in both uptake and release of fatty acids appears to be mediated through melatonin receptors, it will be of considerable interest to determine how these receptors can regulate both processes. Our laboratory has previously speculated that the melatonin signaling pathway ultimately regulates the activity of at least one of the known fatty acid transport proteins (12, 44). These transport proteins, already known for their ability to facilitate the uptake of fatty acids, would have to be capable of facilitating release as well, something that has not as yet been shown (45). In fact, it is still a matter of debate whether fatty acids enter the cell exclusively by diffusion, by a transport process, or both (46). The rapidity with which melatonin suppresses both fatty acid uptake in the hepatoma model and uptake/release in the inguinal fat pad model in vivo supports the idea that the melatonin receptor pathway is functionally linked to a regulated transport mechanism (12, 41).

What is not addressed by these observations, however, is melatonin’s inhibition of glycerol release. If melatonin is indeed acting indirectly through a fatty acid transport protein, it would of necessity block any facilitated transport mechanism involved in the release of glycerol. This would be very interesting because recent studies have revealed new evidence for a facilitated mechanism regulating glycerol release involving aquaporin (47). Alternatively, melatonin inhibition of fatty acid/glycerol release could have a mode of action different from its mechanism of inhibition of fatty acid uptake, such as through suppression of lipase activity. Even more intriguing is the idea that, because at least two melatonin receptors are expressed in inguinal adipocytes, one receptor might regulate fatty acid uptake, whereas the other might regulate fatty acid and glycerol release. A recent report has indeed demonstrated differences in the signaling pathways of the mt1 and MT2 receptors (48).

Another unique aspect of our lipolysis studies was the use of the rat inguinal fat pad. Most published work involving adipocytes deals with the epididymal fat pad, which is much easier to prepare due to the decreased amount of associated connective tissue. We found that melatonin failed to inhibit lipolysis in epididymal cells, a result supported by preliminary studies of epididymal fat pads perfused in situ (Dauchy, R., L. Sauer, and D. Blask, unpublished results). This is further indication that melatonin is a unique antilipolytic factor, because factors such as adenosine inhibit lipolysis in both inguinal and epididymal cells (3, 25 ; Zalatan, F., unpublished results). Surprisingly, both mt1 and MT2 receptor expression was detected in epididymal adipocytes using RT-PCR, suggesting that these cells either lack an intracellular component needed to transduce the melatonin signal for inhibition of lipolysis, or they contain an inhibitory component that blocks the signal transduction. Alternatively, the density of melatonin receptors actually expressed in epididymal adipocytes may be too low to transduce a melatonin signal.

Site-related differences in the lipolytic response to stimulatory and inhibitory agents have been documented between sc and visceral adipocytes. For example in humans and in rodent species, ß-adrenergic stimulation of lipolysis is greater in visceral adipocytes from epididymal fat tissue than in sc adipocytes (49, 50, 51). Epididymal adipocytes are presumably more sensitive to norepinephrine-stimulated lipolysis due to greater hormone-sensitive lipase activity and different lipid droplet characteristics in these cells (50). Other evidence also suggests that the relative balance between {alpha}- and ß-adrenergic receptors may contribute to the mechanisms of site-specific lipolytic responses (51). Conversely, sc adipocytes have been reported to be more sensitive than epididymal adipocytes to the antilipolytic action of adenosine (52). Therefore, it is not altogether surprising that a similar differential sensitivity to the antilipolytic activity of melatonin exists in adipocytes of different origin.

The physiological significance of melatonin’s ability to inhibit lipolysis and block fatty acid uptake in a site-specific manner in laboratory rats, which are a nonphotoperiodic, nonseasonally breeding animals (53), is unclear. In Siberian hamsters, which are a photoperiodically sensitive, seasonally breeding species, long duration melatonin infusions in pinealectomized animals were done to mimic short day winter melatonin profiles experienced by these animals under natural photoperiodic conditions. These infusions induced the expected short-day winter response characterized by reproductive atrophy and decreased body and epididymal white fat weight. The decrease in epididymal fat weight was accompanied by a reduction in epididymal fat lipoprotein lipase activity and carcass lipid content indicating that melatonin reduces body weight in this species by blocking fatty acid uptake by adipocytes (54, 55, 56). During the winter part of the annual breeding cycle, a melatonin-induced reduction in epididymal fat stores may compromise the normal function of the epididymus in the maturation and motility of spermatozoa because epididymal fat may maintain optimal heat insulation for this important reproductive structure and function. Interestingly, sc adipocytes were not as sensitive to this effect as were epididymal adipocytes, suggesting that this is an adaptive phenomenon so that animals maintain some sc fat for insulation during the winter (56).

In contrast, the lack of an effect of melatonin on either epididymal lipolysis or fatty acid uptake in nonphotoperiodic rats, which can breed throughout the year, may reflect less dependence of this species on melatonin for regulation of epididymal fat metabolism, and thus epididymal function, as part of its normal physiology. On the other hand, the ability of melatonin to inhibit fatty acid release and uptake by sc adipocytes may reflect an important physiological circadian regulation of this fat depot in maintaining optimal energy balance and heat insulation. In fact, it has recently been shown that the diminution of the circadian amplitude of the endogenous melatonin signal in aging rats results in increased body weight, visceral adiposity, and associated adverse metabolic consequences (20). Restoration of the nocturnal melatonin signal to a more youthful level in aging animals decreased body weight, intraabdominal adiposity, and plasma insulin and leptin levels without altering food intake or total adiposity (21).

In summary, we have shown that a physiological concentration of melatonin inhibits lipolysis in rat inguinal but not epididymal adipocytes in vitro. This finding gives melatonin the unique quality of regulating the release of fatty acids in rat adipocytes in a site-specific manner. Based on other work involving melatonin’s inhibition of fatty acid uptake, it is very likely that further studies will reveal that melatonin is involved in a tightly controlled on/off switch regulating the amount of circulating fatty acids in the rat, which may be important in the chronobiology of intermediary lipid metabolism.


   Acknowledgments
 
We thank Robert Dauchy for help with preliminary experiments and gas chromatography, Darin Lynch and Randall Zuckerman for technical assistance, Theodore Peters, Jr., Allan Green, and Eugene Holowachuk for helpful advice, and Leonard Sauer for his initial ideas in developing this project. We also thank the Institut de Recherches Internationales Servier (Courbevoie Cedex, France) for its gift of the melatonin receptor antagonist S-20928.


   Footnotes
 
This work was supported by NIH Grant CA-76197 (to D.E.B.) and the Stephen C. Clark Research Fund.

Abbreviation: Mel, Melatonin.

Received February 9, 2001.

Accepted for publication May 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Carey GB 1998 Mechanisms regulating adipocyte lipolysis. Adv Exp Med Biol 441:157–170[Medline]
  2. Londos C, Brasaemle DL, Schultz CJ, Adler-Wailes DC, Levin DM, Kimmel AR, Rondinone CM 1999 On the control of lipolysis in adipocytes. Ann NY Acad Sci 892:155–168[CrossRef][Medline]
  3. Kather H, Bieger W, Michel G, Aktories K, Jakobs KH 1985 Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms. J Clin Invest 76:1559–1565
  4. Lafontan M, Berlan M 1995 Fat cell {alpha}2-adrenoceptors: the regulation of fat cell function and lipolysis. Endocr Rev 16:716–738[CrossRef][Medline]
  5. Gasic S, Tian B, Green A 1999 Tumor necrosis factor {alpha} stimulates lipolysis in adipocytes by decreasing Gi protein concentrations. J Biol Chem 274:6770–6775[Abstract/Free Full Text]
  6. Engfeldt P, Hellmer J, Wahrenberg H, Arner P 1988 Effects of insulin on adrenoceptor binding and the rate of catecholamine-induced lipolysis in isolated human fat cells. J Biol Chem 263:15553–15560[Abstract/Free Full Text]
  7. Smith CJ, Vasta V, Degerman E, Belfrage P, Manganiello VC 1991 Hormone-sensitive cyclic GMP-inhibited cyclic AMP phosphodiesterase in rat adipocytes. J Biol Chem 266:13385–13390[Abstract/Free Full Text]
  8. Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M 1994 Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. J Biol Chem 269:3568–3573[Abstract/Free Full Text]
  9. Shepherd PR, Withers DJ, Siddle K 1998 Phosphoinositide 3-kinase: the key switch mechanism in insulin signaling. Biochem J 333:471–490
  10. Reiter RJ 1991 Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 12:151–180[CrossRef][Medline]
  11. Blask DE, Wilson ST, Zalatan F 1997 Physiological melatonin inhibition of human breast cancer cell growth in vitro: evidence for a glutathione-mediated pathway. Cancer Res 57:1909–1914[Abstract/Free Full Text]
  12. Blask DE, Sauer LA, Dauchy RT, Holowachuk EW, Ruhoff MS, Kopff HS 1999 Melatonin inhibition of cancer growth in vivo involves suppression of tumor fatty acid metabolism via melatonin receptor-mediated signal transduction events. Cancer Res 59:4693–4701[Abstract/Free Full Text]
  13. Reppert SM, Weaver DR, Ebisawa T 1994 Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian responses. Neuron 13:1177–1185[CrossRef][Medline]
  14. Godson C, Reppert SM 1997 The Mel 1a melatonin receptor is coupled to parallel signal transduction pathways. Endocrinology 138:397–404[Abstract/Free Full Text]
  15. Reppert SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt SA, Gusella JF 1995 Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel 1b melatonin receptor. Proc Natl Acad Sci USA 92:8734–8738[Abstract/Free Full Text]
  16. Reiter RJ, Tan D, Poeggeler B, Li-dun C, Menendez-Pelaez A 1994 Melatonin, free radicals and cancer initiation. In: Maestroni GJM, Conti A, Reiter RJ, eds. Advances in Pineal Research: 7. John Libbey & Company, London and Paris, 211–228
  17. Molis TM., Spriggs LL, Jupiter Y, Hill SM 1995 Melatonin modulation of estrogen-receptor proteins, growth factors, and proto-oncogenes in human breast cancer. J Pineal Res 18:93–103[Medline]
  18. Reiter RJ, Tan D, Osuna C, Gitto E 2000 Actions of melatonin in the reduction of oxidative stress. J Biomed Sci 7:444–458[Medline]
  19. Moreno FJ, Mills I, Garcia-Sainz JA, Fain, J 1983 Effects of pertussis toxin treatment on the metabolism of rat adipocytes. J Biol Chem 258:10938–10943[Abstract/Free Full Text]
  20. Rasmussen DD, Boldt BM, Wilkinson CW, Yellon SM, Matsumoto AM 1999 Daily melatonin administration at middle age suppresses male rat visceral fat, plasma leptin, and plasma insulin to youthful levels. Endocrinology 140:1009–1012[Abstract/Free Full Text]
  21. Wolden-Hanson T, Mitton DR, McCants RL, Yellon SM, Wilkinson CW, Matsumoto AM, Rasmussen DD 2000 Daily melatonin administration to middle-aged male rats suppresses body weight, intraabdominal adiposity, and plasma leptin and insulin independent of food intake and total body fat. Endocrinology 141:487–497[Abstract/Free Full Text]
  22. Vaughan MK, Vaughan, GM 1993 Metabolic and thyroidal consequences of melatonin administration in mammals. In: Yu HS, Reiter RJ, eds. Melatonin: biosynthesis, physiological effects, and clinical applications. Boca Raton, FL: CRC Press; 311–347
  23. Rodbell M 1964 Metabolism of isolated fat cells. J Biol Chem 239:375–380[Free Full Text]
  24. Honnor RC, Dhillon GS, Londos C 1985 cAMP-dependent protein kinase and lipolysis in rat adipocytes. J Biol Chem 260:15122–15129[Abstract/Free Full Text]
  25. Olansky L, Myers GA, Pohl SL, Hewlett EL 1983 Promotion of lipolysis in rat adipocytes by pertussis toxin: reversal of endogenous inhibition. Proc Natl Acad Sci USA 80:6547–6551[Abstract/Free Full Text]
  26. Folch J, Lees M, Sloane-Stanley GH 1957 A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–507[Free Full Text]
  27. McDonald-Gibson RG 1987 Quantitative measurements of arachidonic acid in tissues or fluids. In: Benedetto C, McDonald-Gibson RG, Nigam S, Slater TF, eds. Prostaglandins and related substances. Washington, D.C: IRL Press; 259–268
  28. Reiter RJ 1998 Oxidative damage in the central nervous system: protection by melatonin. Prog Neurobiol 56:359–384[CrossRef][Medline]
  29. Guardiola-Lemaitre B, Delagrange P 1997 Melatonin agonists and antagonists: pharmacological tools or therapeutic agents? In: Webb SM, Puig-Domingo M, Moller M, Pevet P, eds. Pineal update. Westbury, NY: PJD Publications; 301–319
  30. Vaughan M, Barchas J 1966 Effects of melatonin and related compounds on the release of glycerol from rat adipose tissue in vitro. J Pharmacol Exp Ther 152:298–303[Abstract/Free Full Text]
  31. Ng TB, Wong CM 1986 Effects of pineal indoles and arginine vasotocin on lipolysis and lipogenesis in isolated adipocytes. J Pineal Res 3:55–66[Medline]
  32. Brydon L, Roka F, Petit L, de Coppet P, Tissot M, Barrett P, Morgan PJ, Nanoff C, Strosberg AD, Jockers R 1999 Dual signaling of human Mel 1a melatonin receptors via Gi2, Gi3, and Gq/11 proteins. Mol Endocrinol 13:2025–2038[Abstract/Free Full Text]
  33. Barrett P, Morris M, Choi WS, Ross A, Morgan PJ 1999 Melatonin receptors and signal transduction mechanisms. Biol Signals Recept 8:6–14[CrossRef][Medline]
  34. Soeder KJ, Snedden SK, Cao W, et al. 1999 The ß-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism. J Biol Chem 274:12017–12022[Abstract/Free Full Text]
  35. Binkley SA 1981 Pineal biochemistry: comparative aspects and circadian rhythms. In: Reiter RJ, ed. The pineal gland vol. 1–anatomy and biochemistry. Boca Raton, FL: CRC Press; 155–172
  36. Dauchy RT, Blask DE, Sauer LA, Brainard GC, Krause JA 1999 Dim light during darkness stimulates tumor progression by enhancing tumor fatty acid uptake and metabolism. Cancer Lett 144:131–136[CrossRef][Medline]
  37. Mitchell FM, Griffiths SL, Saggerson ED, Housley MD, Knowler JT, Milligan G 1989 Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Biochem J 262:403–408[Medline]
  38. Roka F, Brydon L, Waldhoer M, et al. 1999 Tight association of the human Mel 1a melatonin receptor and Gi: precoupling and constitutive activity. Mol Pharmacol 56:1014–1024[Abstract/Free Full Text]
  39. Messner M, Hardeland R, Rodenbeck A, Huether G 1998 Tissue retention and subcellular distribution of continuously infused melatonin in rats under near physiological conditions. J Pineal Res 25:251–259[Medline]
  40. Maestroni GJM, Conti A 1996 Melatonin in human breast cancer tissue: association with nuclear grade and estrogen receptor status. Lab Invest 75:557–561[Medline]
  41. Sauer LA, Dauchy RT, Blask DE 2001 Melatonin inhibits fatty acid transport in inguinal fat pads of hepatoma 7288CTC-bearing and normal Buffalo rats via receptor-mediated signal transduction. Life Sci 68:2835–2844[CrossRef][Medline]
  42. Lissoni P, Paolorossi F, Tancini G, et al. 1996 Is there a role for melatonin in the treatment of neoplastic cachexia? Eur J Cancer 32A:1340–1343
  43. Argiles JP, Lopez-Soriano J, Busquets S, Lopez-Soriano FJ 1997 Journey from cachexia to obesity by TNF. FASEB J 11:743–751[Abstract]
  44. Hirsch D, Stahl A, Lodish HF 1998 A family of fatty acid transporters conserved from mycobacterium to man. Proc Natl Acad Sci USA 95:8625–8629[Abstract/Free Full Text]
  45. Coe NR, Smith AJ, Frohnert BI, Watkins PA, Bernlohr DA 1999 The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase. J Biol Chem 274:36300–36304[Abstract/Free Full Text]
  46. Hamilton JA, Kamp F 1999 How are free fatty acids transported in membranes? Diabetes 48:2255–2269[Abstract]
  47. Kishada K, Kuriyama H, Funahashi T, et al. 2000 Aquaporin adipose, a putative glycerol channel in adipocytes. J Biol Chem 275:20896–20902[Abstract/Free Full Text]
  48. Petit L, Lacroix I, de Coppet P, Strosberg AD, Jockers R 1999 Differential signaling of human Mel 1a and Mel 1b melatonin receptors through the cyclic guanosine 3'-5'-monophosphate pathway. Biochem Pharmacol 58:633–639[CrossRef][Medline]
  49. Ostman J, Arner P, Engfeldt P, Karger L 1979 Regional differences in the control of lipolysis in human adipose tissue. Metabolism 28:1198–1205[CrossRef][Medline]
  50. Morimoto C, Tsujita T, Okuda H 1997 Norepinephrine-induced lipolysis in rat fat cells from visceral and subcutaneous sites: role of hormone-sensitive lipase and lipid droplets. J Lipid Res 38:132–138[Abstract]
  51. Robidoux J, Pirouzi P, Lafond J, Savard R 1995 Site-specific effects of sympathectomy on the adrenergic control of lipolysis in hamster fat cells. Can J Physiol Pharmacol 73:450–458[Medline]
  52. Lacasa D, Agli B, Pecquery R, Giudicelli Y 1991 Influence of ovariectomy and regional fat distribution on the membraneous transducing system controlling lipolysis in rat fat cells. Endocrinology 128:747–753[Abstract]
  53. Goldman BD, Nelson RJ 1993 Melatonin and seasonality in mammals. In: Yu H-S, Reiter RJ, eds. Melatonin: biosynthesis, physiological effects, and clinical applications. Boca Raton, FL: CRC Press; 225–252
  54. Bartness TJ, Goldman BD 1988 Peak duration of serum melatonin and short-day responses in adult Siberian hamsters. Am J Physiol 255:R812–R822
  55. Bartness TJ, Goldman BD 1988 Effects of melatonin on long-day responses in short-day housed adult Siberian hamsters. Am J Physiol 255:R823–R830
  56. Bartness TJ, Hamilton JM, Wade GN, Goldman BD 1989 Regional differences in fat responses to short days in Siberian hamsters. Am J Physiol 257:R1533–R1540



This article has been cited by other articles:


Home page
 J EndocrinolHome page
D A Zieba, M Szczesna, B Klocek-Gorka, E Molik, T Misztal, G L Williams, K Romanowicz, E Stepien, D H Keisler, and M Murawski
Seasonal effects of central leptin infusion on secretion of melatonin and prolactin and on SOCS-3 gene expression in ewes
J. Endocrinol., July 1, 2008; 198(1): 147 - 155.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
D. E. Blask, G. C. Brainard, R. T. Dauchy, J. P. Hanifin, L. K. Davidson, J. A. Krause, L. A. Sauer, M. A. Rivera-Bermudez, M. L. Dubocovich, S. A. Jasser, et al.
Melatonin-Depleted Blood from Premenopausal Women Exposed to Light at Night Stimulates Growth of Human Breast Cancer Xenografts in Nude Rats
Cancer Res., December 1, 2005; 65(23): 11174 - 11184.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. I. C. Alonso-Vale, S. Andreotti, S. B. Peres, G. F. Anhe, C. das Neves Borges-Silva, J. C. Neto, and F. B. Lima
Melatonin enhances leptin expression by rat adipocytes in the presence of insulin
Am J Physiol Endocrinol Metab, April 1, 2005; 288(4): E805 - E812.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zalatan, F.
Right arrow Articles by Blask, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zalatan, F.
Right arrow Articles by Blask, D. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals