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Centre National de la Recherche Scientifique-UPR 0415 and Université Paris VII, Institut Cochin de Génétique Moléculaire (L.B., L.P., A.D.S., R.J.), F-75014 Paris, France; Rowett Research Institute (L.B.), Bucksburn, Aberdeen, United Kingdom AB2 9SB; and Institut de Recherches Internationales Servier (P.D.), F-92415 Courbevoie, France
Address all correspondence and requests for reprints to: Dr. Ralf Jockers, Centre National de la Recherche Scientifique, UPR415, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, F-75014 Paris, France. E-mail: jockers{at}cochin.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For several years, melatonin has been known to also play a role in energy expenditure and body mass regulation in mammals (2). Hibernating species, for example, undergo dramatic changes in body weight, particularly in fat mass, in response to photoperiodic changes. These responses are mainly mediated by the length of nocturnal melatonin release (3). Direct evidence for melatonin’s effect on body weight comes from infusion experiments with pinealectomized hamsters (4) and injection of melatonin receptor agonists or antagonists into Garden dormice (5).
Variations in melatonin secretion patterns and body mass have also been observed in species that exhibit less dramatic seasonality. In rats and humans, for example, visceral fat levels increase with age, whereas nocturnal plasma melatonin peak concentrations decline (6, 7, 8). Daily melatonin supplementation to middle-aged rats restored melatonin levels to those observed in young rats and suppressed the age-related gain in visceral fat (9).
Clinical data also support a link between photoperiodic changes and control of energy balance in man. For example, certain human pathologies associated with desynchronized circadian rhythms and melatonin secretion pattern such as seasonal affective disorder share features common to the prehibernating mammal, such as oversleeping, carbohydrate craving, and overall weight gain (10). Notably, these changes may be triggered by seasonal changes in light, and light treatment has proven useful for correcting disturbances in melatonin rhythm (11, 12).
Mechanisms underlying melatonin’s effect on body weight regulation have yet to be characterized. In Djungarian and Syrian hamsters, the melatonin-induced decrease in fat mass has been associated with thermogenic activation of brown adipose tissue (BAT) (13, 14). In contrast, in other species, such as the dormouse, ground squirrel, or Syrian hamster, melatonin has been shown to induce an increase in fat mass (5) and to induce BAT hypertrophy (2).
Effects of melatonin on adipose tissue may also vary during development. Nocturnal melatonin levels are high in young children and decline with age. Interestingly, human BAT is best developed during early childhood, when melatonin levels are high, and is largely reduced in adults (7, 15). The role of melatonin in BAT may therefore be most significant during the early stages of development.
Melatonin mediates its effects through high affinity G protein-coupled receptors. In mammals, two distinct receptor subtypes have been cloned and named MT1 (Mel1a) and MT2 (Mel1b) (16, 17, 18). Both melatonin receptor subtypes inhibit adenylyl cyclase via pertussis toxin-sensitive Gi proteins. In addition, MT1 receptors have been shown to stimulate calcium mobilization through pertussis toxin-insensitive Gq/11 proteins, and MT2 receptors to couple to cGMP inhibition (19, 20). The MT1 receptor is localized in the hypothalamic SCN and hypophyseal pars tuberalis and is thought to mediate circadian and reproductive responses to melatonin. MT2 is localized in the SCN and retina and is thought to mediate melatonin’s effects on circadian rhythms and retinal physiology (21). It is therefore possible that melatonin’s effect on body mass regulation is mediated through activation of central receptors, resulting in changes in metabolic rate via sympathetic nervous activity or altered feeding behavior. However, a direct effect of melatonin on peripheral tissues, such as adipose tissue, is also possible. Indeed, there is now a substantial amount of evidence supporting the expression of 2-[125I]iodomelatonin-binding sites in several peripheral tissues, including lymphocytes, arteries, kidneys, and gastrointestinal tract (22 ; see Ref. 23 for review). However, in most cases, the subtype of melatonin receptor and the functional effects of melatonin in these tissues remain unclear.
In the present study we investigated whether there are functional melatonin receptors present in human adipocytes. The presence of these receptors would suggest that melatonin could act directly at adipose tissue and regulate its physiology. We used as a model, PAZ6 adipocytes, an immortalized human brown preadipocyte cell line, which retains the ability to differentiate into mature adipocytes and to express adipocyte-specific marker genes (24).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RT-PCR
Total RNAs were extracted from PAZ6 preadipocytes, PAZ6
adipocytes, and BAT from three patients with pheochromocytoma and from
white adipose tissue (WAT) from two patients undergoing surgery to
remove mammary fat. RT-PCR reactions were performed as described
previously (24, 25). Polyadenylated
[poly(A)+] mRNA from 63 pooled human
hypothalami was obtained from CLONTECH Laboratories, Inc.
(Palo Alto, CA). Analysis of mRNA levels was performed during the
exponential phase of the amplification, which was assessed in
preliminary experiments for each pair of primers. Briefly, 100 ng
reverse transcribed RNA were used in a PCR reaction where the number of
cycles was gradually increased (usually from 20 to 37 cycles). The
products of the reaction were visualized on a gel by ethidium bromide
staining and analyzed by densitometric scanning. The exponential phase
of the reaction was determined by plotting, on a logarithmic scale, the
intensity of the signal against the number of amplification cycles.
During this phase, when the reaction was performed with variable
amounts of reverse transcribed mRNA (from 50–200 ng), the signal
obtained was proportional to the amount of cDNA used in the reaction.
The same procedure was used for the poly(A)+ mRNA
from human hypothalamus. Samples containing total mRNA and
poly(A)+ mRNA were normalized to the same amount
of the cyclophilin amplification product. Primers for the following
cDNAs were designed with the program Oligo4, using the following
GenBank entries: MT1 receptor (accession no. U14108): sense,
5'-TCAACCGCTACTGCTACATC-3', 5'-annealing position 400; antisense,
5'-TCATCAGTGGAGACGGTTTC-3', 5'-annealing position 1031; MT2
receptor (accession no. U25341): sense, 5'-TCATCGGCTCTGTCTTCAATA-3',
5'-annealing position 383; antisense, 5'-ACTGGGTGCTGGCGGTCTGGA-3',
5'-annealing position 611; Mel1c receptor (accession no. U67882):
sense, 5'-CTTCAACATAACAGCCATAGC-3', 5'-annealing position 360;
antisense, 5'-TGCTTGATTGTTGTTGGTTAC-3', 5'-annealing position 1051;
melatonin receptor-related gene (accession no. U52219): sense,
5'-GAAGGAGATGGCAGGCAAGA-3', 5'-annealing position 861; antisense,
5'-TGGTGGGTAGAGGCAGATTT-3', 5'-annealing position 1281; PPAR
(accession no. L40904): sense, 5'-AGACAACAGACAAATCACCAT-3', 5'
annealing position 894; antisense 5'-CTTCACAGCAAACTCAAACTT-3',
5'-annealing position 1294; cyclophilin (accession no. Y00052): sense,
5'-AGCACTGGAGAGAAAGGATT-3', 5' annealing position 132; antisense
5'-GGAGGGAACAAGGAAAACAT-3', 5'-annealing position 659; Glut 4
(accession no. M20747): sense, 5'-TCCTGCTGCCCTTCTGTC, 5'-annealing
position 653; antisense, 5'-GGCCTACCCCTGCTGTCT, 5'-annealing position
961; leptin (accession no. U43653): sense, 5'-GCTGTGCCCATCCAAAAAGT,
5'-annealing position 61; antisense, 5'-ACTGCCAGTGTCTGGTCCAT,
5'-annealing position 242; uncoupling protein 1 (UCP1; accession no.
XM011103): sense, 5'-TTAGGAAGCAAGATTTTAGC-3', 5'-annealing position
337; antisense, 5'-AAGTCGCAAGAAGGAAGGTA-3', 5'-annealing position 835;
UCP2 (accession no. U76367): sense, 5'-TGTGCTGAGCTGGTGACCTATGAC-3',
5'-annealing position 571; antisense,
5'-AAGGGAGCCTCTCGGGAAGTGCAG-3', 5'-annealing position 926; lipoprotein
lipase (LPL; accession no. M76722): sense, 5'-GAGATTTCTCTGTATGGCACC-3',
5'-annealing position 267; antisense, 5'-CTGCAAATGAGACACTTTCTC-3',
5'-annealing position 391; and TNF
(accession no. X01394):
sense, 5'-CAGAGGGAAGAGTTCCCCAG, 5'-annealing position 327; antisense,
5'-CCTTGGTCTGGTAGGAGACG, 5'-annealing position 651.
Radioligand binding assays and competition experiments
Binding assays were performed on PAZ6 adipocytes after 14 d
of differentiation. The medium was aspirated and replaced by 0.25 ml
binding buffer [DMEM-Ham’s F-12 (1:1 vol/vol) supplemented with 15
mM HEPES and 0.5% fatty acid-free BSA], containing
16–1200 pM125I-Mel
(DuPont-NEN Life Science Products, Boston, MA) as
radioligand. Specific binding was defined as binding displaced by 1
µM melatonin (Sigma, St. Louis, MO). Culture
plates were incubated at 25 C for 90 min, and the reaction was
terminated by transferring plates to ice for 10 min. The medium was
aspirated, and each well was rinsed twice with 1 ml ice-cold PBS. Cells
were solubilized with 0.5 ml NaOH (1 M) and transferred to
Eppendorf tubes, and radioactivity was counted with a
-counter. Competition experiments were performed as described above
with approximately 300 pM 125I-Mel
and varying concentrations of cold ligands: melatonin,
5-methoxycarbonylamino-N-acetyltryptamine (GR 135,531), and
4-phenyl-2-propionamidotetraline (4-P-PDOT) (Tocris Cookson Ltd.,
Bristol, UK).
Determination of intracellular cAMP levels
PAZ6 cells differentiated in six-well dishes were incubated for
15 min at 37 C in medium 1 with or without forskolin (1
µM Sigma) and increasing concentrations of
melatonin. The incubation buffer was discarded, and cells were
extracted in 0.5 ml ice-cold 65% ethanol. Cell extracts were
centrifuged in a microcentrifuge at 17,000 x g for 10
min. The supernatant was concentrated in a Speed-Vac (Savant,
Farmingdale, NY), and the pellet was diluted in 10
mM Tris (pH 7.4) and 1 mM
EDTA. cAMP concentrations were determined using a
[3H]cAMP assay system (Amersham Pharmacia Biotech, Arlington Heights, IL).
Determination of intracellular cGMP levels
PAZ6 cells differentiated in six-well dishes were incubated for
15 min at 37 C in medium 1 with IBMX (1 mM) and increasing
concentrations of melatonin. The medium was then discarded, and cells
were extracted in 0.5 ml ice-cold 65% ethanol. Cell extracts were
centrifuged at 20,000 x g for 15 min at 4 C.
Supernatants were dried using a Speed-Vac, and pellets were resuspended
in 0.25 ml assay buffer, acetylated, and assayed for cGMP, according to
the instructions of the manufacturer of the enzyme immunoassay kit
(Amersham Pharmacia Biotech).
Membrane preparation, SDS-PAGE, and immunoblotting
Crude membranes were prepared as described recently
(26) and denatured in 25 mM Tris (pH 6.8), 4
M urea, 180 mM dithiothreitol, 2.5%
SDS, 5% glycerol, and 0.05% bromophenol blue overnight at room
temperature. Proteins were separated by 10.5% SDS-PAGE and transferred
to nitrocellulose. Immunoblot analysis was carried out with a rabbit
anti-Glut4-specific antibody (1:10,000). Immunoreactivity was revealed
using appropriate secondary antibodies coupled to horseradish
peroxidase and the ECL chemiluminescent reagent (Amersham Pharmacia Biotech).
Glucose uptake
PAZ6 cells were differentiated in the differentiation medium in
the presence or absence of 10 nM melatonin. Twenty-four
hours before the experiment cells were incubated in differentiation
medium without insulin and with 2% FCS. Cells were then washed twice
with KREBS puffer (supplemented with 2% fatty acid-free BSA, 1
mM pyruvate, and 0.1 mM glucose) and incubated
in this buffer for 2 h at 37 C. Stimulation was performed in the
same buffer supplemented with 0.5 µCi/ml
2-[1,2-3H]deoxy-D-glucose
(Amersham Pharmacia Biotech) at 37 C for 5 min in the
presence or absence of 200 nM insulin. Cells were washed
twice with ice-cold PBS and counted in a ß-counter.
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Results |
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Semiquantitative RT-PCR experiments were used to study the expression
of melatonin receptor mRNA in PAZ6 preadipocytes, PAZ6 adipocytes, and
human BAT, WAT, and hypothalamic tissue (Fig. 1
). BAT was obtained from patients with
pheochromocytoma. In this disease, BAT hyperplasia develops in fat
tissue surrounding the tumor (27). WAT was obtained from
patients undergoing surgery to remove mammary fat.
Poly(A)+ mRNA pooled from different human
hypothalami was used as a reference for melatonin receptor expression.
Amplification products corresponding to both receptor subtypes were
detected in PAZ6 adipocytes as well as in WAT, BAT, and hypothalamus
(Fig. 1). Expression of the MT1 receptor transcript was most abundant
in the hypothalamus and much weaker in BAT and PAZ6 adipocytes, which
expressed equivalent amounts. WAT expressed less MT1 transcript than
BAT. The MT2 transcript was most abundant in BAT, slightly less
abundant in hypothalamus and PAZ6 adipocytes, and barely detectable in
WAT. MT1 receptor transcripts were weakly expressed in PAZ6
preadipocytes, whereas MT2 transcripts were undetectable.
Expression of both subtypes clearly increased with adipocyte
differentiation. Melatonin receptor transcripts were not detected in
other human peripheral tissues tested (myometrium, gallbladder, and
liver; data not shown), supporting the specificity of melatonin
receptor expression in human adipose tissue. Primers specific for two
additional members of the melatonin receptor family, the
Xenopus Mel1c receptor and the human melatonin-related
receptor, failed to amplify the corresponding or closely related
amplification products (data not shown). These data show that both
melatonin receptor subtypes are expressed in human adipocytes at higher
levels in BAT compared with WAT. Expression levels of the MT2
transcript in BAT are comparable to those in the hypothalamus, the
principal expression site of central melatonin receptors. Comparable
results were obtained in PAZ6 adipocytes and BAT, suggesting that PAZ6
adipocytes are an appropriate model for studying the role of melatonin
receptors in human fat tissue.
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). 125I-Mel
binding to PAZ6 adipocytes was further characterized in competition
experiments. Melatonin displaced 125I-Mel binding
with a Ki of approximately 3 x
10-11 M (Fig. 2B), confirming the
expression of high affinity melatonin receptors. Recently, an atypical
125I-Mel-binding site was identified in Siberian
hamster brown adipocytes (28). A key feature of this
binding site was its sensitivity to GR135531, a compound that has no
affinity for MT1 and MT2 receptors (28). In PAZ6
adipocytes 125I-Mel binding was not competed by
GR135531 at concentrations of up to 1 µM, suggesting that
this binding site is different from that described in Siberian hamster
brown adipocytes. To discriminate between MT1 and MT2 receptors we used
the melatonin receptor antagonist 4P-PDOT, which has a more than 1000
times higher affinity for MT2 receptors than for MT1 receptors
(29). The competition curve for 4P-PDOT was monophasic
with a Ki of approximately 1.5 x
10-11 M, which is in good agreement
with values reported for human MT2 receptors expressed in CHO cells
(29). Combined results from both molecular and
pharmacological analyses, support the presence of high affinity
melatonin receptors in PAZ6 adipocytes, predominantly corresponding to
the MT2 subtype.
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). Melatonin alone had no effect on
basal cAMP levels.
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). Both parameters
are in good agreement with those previously observed for the cloned
human MT2 receptor (20) and support functional expression
of this receptor subtype in PAZ6 adipocytes.
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). In
contrast, melatonin had no significant effect on the expression of
genes encoding for other adipocyte markers: uncoupling proteins UCP1
and UCP2, PPAR, TNF, leptin (ob), LPL (Fig. 5), and
hormone-sensitive lipase (not shown). Melatonin also had no effect on
the amount of leptin protein secreted into the culture medium,
confirming the RT-PCR data (not shown). These results demonstrate that
melatonin can specifically modify the expression of Glut4, a gene
important for adipocyte homeostasis.
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). One-day melatonin treatment did not
change either Glut4 protein levels or glucose uptake significantly,
whereas long-term melatonin treatment markedly decreased both
parameters.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several lines of evidence support functional expression of the MT2 receptor subtype in human adipocytes. MT2 receptor mRNA has been detected in PAZ6 adipocytes, BAT, and WAT, but not in several other peripheral tissues. The quantity of MT2 receptor mRNA in BAT is comparable to that in human hypothalamus, a well established expression site of melatonin receptors. Pharmacological analysis indicated the expression of a single high affinity 125I-Mel-binding site in PAZ6 adipocytes. The Kd of 450 pM for 125I-Mel binding fits best with expression of the MT2 subtype, as Kd values are typically lower for MT1 (20–150 nM) than for MT2 receptors (100–500 nM) (18, 21, 30). The binding capacity of 7.5 fmol/mg protein corresponds to receptor densities reported for high affinity melatonin receptors identified at other peripheral sites, such as lymphocytes and prostate epithelial cells (23, 31). Importantly, 125I-Mel binding in PAZ6 adipocytes was competed by the MT2-selective melatonin receptor ligand 4P-PDOT with high affinity. Furthermore, melatonin inhibited forskolin-stimulated cAMP levels with an IC50 within the range of physiological melatonin concentrations (32). Maximal inhibition levels were weak (20%). However, considerable variation has been observed for this parameter (10–80%) in transfected cells as well as in cells expressing endogenous receptors (1, 20, 23, 33), reflecting the variable importance of this pathway depending on the cellular background. Importantly, melatonin also modulated a further second messenger, cGMP, a phenomenon that has been shown to be selective for the MT2 receptor subtype in humans (20). The IC50 value for cGMP modulation was within the range of circulating melatonin levels. Maximal inhibition levels (60%) fitted well with those observed in HEK293 cells transfected with the human MT2 receptor (20).
Expression of 125I-Mel-binding sites has previously been reported in BAT of Siberian hamsters, a species known to exhibit dramatic body weight changes in response to changing photoperiod (28). Pharmacological analysis suggested expression of an atypical binding site, different from cloned melatonin receptors and different from pharmacologically characterized 125I-Mel-binding sites in hamster brain (30). In agreement with this observation, melatonin did not inhibit cAMP accumulation, and no MT1 receptor transcript was detected in these adipocytes. Expression of the MT2 subtype could be ruled out, because Siberian hamsters have been reported to be natural knockouts for this receptor (34). Taken together, these results suggest that melatonin receptors expressed in BAT are different in humans and Siberian hamsters.
Identification of functional MT2 receptors in human adipocytes raises
the question of the physiological role of melatonin in this tissue. To
address that question, we investigated whether treatment of PAZ6
adipocytes with melatonin could alter the expression of key genes
involved in adipocyte homeostasis. Long-term melatonin treatment of
PAZ6 adipocytes markedly decreased Glut4 mRNA levels. This response
appears to be specific, as the expression of several other adipocyte
marker genes (UCP1, UCP2, PPAR, TNF, leptin, hormone-sensitive
lipase, and LPL) was not altered by melatonin. The functional relevance
of melatonin’s effect on Glut4 mRNA levels was confirmed by
measurements showing that long-term melatonin treatment reduces Glut4
protein levels and glucose uptake in PAZ6 adipocytes.
Recently, melatonin has been shown to regulate the expression of another gene in BAT. Treatment of isolated hamster adipocytes with melatonin specifically induced the mitochondrially encoded cytochrome b mRNA (35). The functional importance of this effect is to date unknown.
Glut4 plays a predominant role in insulin-mediated glucose transport. Transgenic mice bearing only one allele of the Glut4 gene were shown to express reduced Glut4 protein levels, resulting in a progressive diabetic phenotype, characterized by impaired glucose homeostasis (36). Adipocyte-selective targeting of the Glut4 gene impaired insulin action in muscle and liver, demonstrating that the expression level of Glut4 in adipose tissue has major effects not only on the adipose tissue but also on the glucose metabolism of the whole body (37). Overexpression of Glut4 in adipose tissue leads to a selective increase in fat cell number and a 2- to 3-fold increase in total body lipids. Changes in Glut4 gene expression have been observed under different physiological and pathological situations. In general, Glut4 mRNA expression is down-regulated in states of relative insulin deficiency, such as streptozotocin-induced diabetes and chronic fasting. In diabetic rodents and humans, Glut4 expression is suppressed in adipocytes in association with insulin resistance. In contrast, exercise has been shown to cause a 1.5-fold increase in Glut4 mRNA expression (38).
Melatonin decreased Glut4 mRNA levels in PAZ6 adipocytes when applied
during the whole differentiation process. Two important transcription
factors for adipocyte differentiation, PPAR and C/EBP, are
required for expression of the Glut4 gene during adipocyte
differentiation (39). Our results suggest that melatonin
has a dominant effect on the increase of Glut4 mRNA levels during
differentiation, even in the presence of the differentiation cocktail.
A direct effect of melatonin on PPAR expression can be excluded,
because PPAR mRNA levels were not modified by melatonin
treatment.
Incubation of fully differentiated PAZ6 adipocytes with melatonin also
decreases Glut4 mRNA levels. Several molecules, such as insulin, cAMP,
TNF, and arachidonic acid, were shown to decrease Glut4 mRNA levels
in differentiated adipocytes (40, 41, 42, 43). As melatonin
receptors are negatively coupled to the adenylyl cyclase system, cAMP
is unlikely to be involved in the effect of melatonin on Glut4 mRNA
levels. TNF was shown to decrease Glut4 mRNA levels in PAZ6
adipocytes (44). However, melatonin treatment did not
change TNF mRNA levels, arguing against the involvement of this
mechanism. A 48-h treatment with arachidonic acid was shown to suppress
Glut4 mRNA accumulation in differentiated 3T3-L1 adipocytes by several
different mechanisms (42). Melatonin receptors stimulate
the inositol phosphate pathway and potentialize arachidonic acid
release in transfected NIH-3T3 cells (45). Thus, a
plausible scenario may involve the stimulation of arachidonic acid
production by melatonin in PAZ6 adipocytes, followed by a decrease in
Glut4 mRNA levels. Further possibilities include new, but still poorly
characterized, transcription factors that have been reported to
specifically regulate the human Glut4 promoter in transgenic mice
(46).
In conclusion, we have shown that melatonin receptors of the MT2 subtype are functionally expressed in human adipocytes. PAZ6 adipocytes constitute the first human cell line expressing endogenous MT2 receptors. Additional studies will be necessary to further define the physiological role of melatonin receptors and their spacio-temporal regulation in human adipocytes.
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Acknowledgments |
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Footnotes |
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Abbreviations: BAT, Brown adipose tissue; IBMX, 3-isobutyl-1-methylxanthine; LPL, lipoprotein lipase; 125I-Mel, 2-[125I]iodomelatonin; 4P-PDOT, 4-phenyl-2-propionamidotetraline; poly(A)+, polyadenylated; SCN, suprachiasmatic nuclei; UCP1, uncoupling protein 1; WAT, white adipose tissue.
Received March 12, 2001.
Accepted for publication June 13, 2001.
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and C/EBP. J Biol Chem 274:7946–7951This article has been cited by other articles:
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  J.-L. Guillaume, A. M. Daulat, P. Maurice, A. Levoye, M. Migaud, L. Brydon, B. Malpaux, C. Borg-Capra, and R. Jockers The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor J. Biol. Chem., June 13, 2008; 283(24): 16762 - 16771. [Abstract] [Full Text] [PDF]
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), melatonin (
), and 4-P-PDOT (
). Data represent the
mean values of a single experiment, performed in triplicate, that is
representative of three independent experiments. Data were fitted using
the GraphPad Plot program version 4.04 (San Diego, CA).
