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2008 Alonso-Vale JPR - Mel and the Circadian Entrainment of Metabolic and Horm Activities in Adip

2008 Alonso-Vale JPR - Mel and the Circadian Entrainment of Metabolic and Horm Activities in Adip

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Published by Franco Pecoraro
J. Pineal Res. 2008; 45:422–429
Doi:10.1111/j.1600-079X.2008.00610.x

Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Munksgaard

Journal of Pineal Research

Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes
Abstract: The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simul
J. Pineal Res. 2008; 45:422–429
Doi:10.1111/j.1600-079X.2008.00610.x

Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Munksgaard

Journal of Pineal Research

Melatonin and the circadian entrainment of metabolic and hormonal activities in primary isolated adipocytes
Abstract: The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To simul

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

Circadian molecular clocks driven by autoregulatory tran- scription–translation feedback loops consist of a pair of activator proteins [circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL-1) in mammals] that induce the transcription of a pair of repressor genes [Period (Per) and Cryptochrome (Cry)], additionally regulated by modifiers [1]. These proteins have been found in the neural circadian master clock, the suprachiasmatic nuclei (SCN), and in several peripheral tissues including the adipose [2]. These peri- pheral clock genes are similar to those present in the SCN neurons, although only the latter seem to be self-sustained. It is still unclear how these peripheral clocks are synchro- nized by the central SCN clock.

Among the many circadian rhythms in the body that are driven by SCN output, melatonin, produced by the pineal gland, functions as messager encoding the duration of darkness. Dissemination of at least some of this circadian information relies on the activation of melatonin receptors, which are expressed in almost all cells of the organism. A deficient melatonin synthesis or receptor expression should therefore have consequences for circadian biology.

The role of clock genes is to control the transcription of other genes, named clock-controlled genes. Recent molecu- lar studies revealed the direct coupling of clock genes and the regulation of metabolism [3]. Genetic mutations or deletions have implicated the peripheral clock genes in the regulation of some aspects of cellular function, including the control of glucose homeostasis [4], lipid synthesis [5] and adipogenesis [6], which are associated with obesity and type 2 diabetes mellitus. On the other hand, melatonin also plays an important role in regulating body mass and energy balance [7–10]. Previous studies have reported that young people with nocturnal lifestyle, whose melatonin secretion was shown to be altered, increased their risk of health problems, including eating disorders, obesity and diabetes [11].

We previously showed that the circadian pattern of metabolic responses observed in adipose tissue of intact animals is altered and/or impaired in pinealectomized rats, in addition to a persistent picture of insulin (Ins) resistance [12–15]. Moreover, we found that melatonin also influences the adipose tissue endocrine activity by modulating the leptin synthesis and secretion [16], which exhibits a circadian expression. This effect was the result of melatonin binding to its Gi-protein-coupled MT1membrane receptor. Addition- ally and as important as the direct effect on leptin secretion,

Abstract: The aim of this work was to investigate the effect of the in vitro circadian-like exposure to melatonin [in the presence or absence of insulin (Ins)] on the metabolism and clock gene expression in adipocytes. To

simulate the cyclic characteristics of the daily melatonin profile, isolated rat adipocytes were exposed in a circadian-like pattern to melatonin added to the incubating medium for 12 hr (mimicking the night), followed by an equal period without melatonin (mimicking the day) combined or not with Ins. This intermittent incubation was interrupted when four and a half 24-hr cycles were fulfilled. At the end, either during the induced night (melatonin present) or the induced day (melatonin absent), the rates of lipolysis and D-[U-14C]-glucose incorporation into lipids were estimated, in addition to the determination of lipogenic [glucose-6-phosphate dehydrogenase and fatty acid synthase (FAS)] and lipolytic (hormone sensitive lipase) enzymes and clock gene (Bmal-1b,Clock,Per-1 andCry-1) mRNA expression. The leptin release was also measured. During the induced night, the following effects were observed: an increase in the mRNA expression ofClock,Per-1 and FAS; a rise in lipogenic response and leptin secretion; and a decrease in the lipolytic activity. The intermittent exposure of adipocytes to melatonin

temporally and rhythmically synchronized their metabolic and hormonal
function in a circadian fashion, mimicking what is observed in vivo in
animals during the daily light–dark cycle. Therefore, this work helps to
clarify the physiological relevance of the circadian pattern of melatonin

secretion and its interactions with Ins, contributing to a better understanding
of the adipocyte biology.

Maria Isabel Cardoso Alonso-
Vale, Sandra Andreotti, Paula
Yuri Mukai, Cristina das Neves
Borges-Silva, Sidney Barnabe´
Peres, Jose´ Cipolla-Neto and
Fabio Bessa Lima

Department of Physiology and Biophysics,
Institute of Biomedical Sciences, University of
Sao Paulo, Sao Paulo, SP, Brazil

Key words: clock genes, insulin, leptin, lipogenesis, lipolysis, primary culture of adipocytes

Address reprint requests to Fabio Bessa Lima

MD, PhD, Department of Physiology and
Biophysics, Institute of Biomedical Sciences,
University of Sao Paulo, 1524 Prof. Lineu
Prestes Ave., 05508-900, Sao Paulo, SP,

Brazil.
E-mail: fabio@icb.usp.br
Received April 4, 2008;
accepted June 3, 2008.
J. Pineal Res. 2008; 45:422–429
Doi:10.1111/j.1600-079X.2008.00610.x
Ó2008 The Authors
Journal compilationÓ 2008 Blackwell Munksgaard
Journal of Pineal Research
422

it was observed that the chronic modulatory action of melatonin occurred only when adipocytes were intermit- tently but not continuously exposed to melatonin [17]. The nature of the temporal information transmitted by the intermittent melatonin treatment to the adipose cells is not known but our main hypothesis is that the adipose tissue is a target for melatonin and that the circadian oscillation of its endocrine function is driven by the indoleamine.

Here, we report the effects of the in vitro circadian-like exposure of adipocytes to melatonin (and its interaction with Ins) on clock genes expression, emphasizing the different responses that occur in the induced night and induced day periods, indicated by the in vitro melatonin presence or absence, respectively. Additionally, the metabolic function of these cells was assessed by the quantification of lipolytic and lipogenic responses, as well as the expression of important enzymes involved in these process. Leptin secre- tion was also characterized. The results of this work help to clarify the physiological relevance and the repercussions of the in vivo circadian pattern of melatonin secretion.

Materials and methods
Adipocyte preparation

Male adult Wistar rats (8-wk old) from the Animal Resource of the Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil, were kept on a 12 h/12 h light–dark cycle (lights on at 0700 hr) in a temperature controlled room (23 ± 1°C). Animals were housed in groups of three to four in plastic cages with food (balanced chow pellet diet; Nuvilab CR1, Nuvital SA, Colombo, PR, Brazil) and water ad libitum. Rats were killed at 18:00 hr by decapitation. The abdominal wall was opened and the epididymal fat pads were excised and processed for adipocyte isolation. All the procedures followed the insti- tutionally approved protocol in agreement with the Ethical Principles in Animal Research adopted by the Ethical Committee for Animal Research (CEEA) of the Institute of Biomedical Sciences (no. 180/01).

The adipocyte isolation was performed according to Rodbell [18] with some slight modifications. Briefly, epididymal fat pads were minced with fine scissors and added to a flask (approximately 4 g per flask) containing 10 mL of sterile DulbeccoÕs modified EagleÕs medium (DMEM) (Invitrogen, Carlsbad, CA, USA) with 20 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, 5 mM glucose, 1% bovine serum albumin (BSA) (fraction V; Sigma Chemical Co., St. Louis, MO, USA), antibiotics (penicillin 100lU/mL; streptomycin 100lg/mL – Invitro- gen) and 1 mg/mL collagenase type II (Sigma), pH 7.4. The mixture was incubated for 30 min at 37°C in an orbital shaker (New Brunswick Scientific, Edison, NJ, USA) at 150 rpm. The isolated adipocytes were filtered through a fine plastic mesh (150lm), transferred to a conical (50 mL) plastic tube, and washed three times in the same buffer without collagenase (25 mL per wash). After the final wash, the medium was thoroughly aspirated, and the cells were harvested as described later. Adipocyte viability was tested with trypan blue and cell number was determined as previously described [19].

Adipocyte incubation

Incubations were performed in culture flasks (25 cm2of free surface area). From the resultant adipocyte pack, an aliquot of cells was added to the flasks containing 10 mL of incubation buffer (similar to the digestion buffer except that collagenase was absent and 10% fetal bovine serum was added) in order to obtain a final cell concentration close to 4· 105cells per mL, followed by the addition of 1 nM melatonin (similar to the physiological plasma content found at its nocturnal peak in living rats [20]). Low or basal concentrations (0.5 nM) of Ins was also added in the beginning of the incubations in the groups denominated

ÔInsÕin the figures, and was continuously present in the

incubations until being replaced by another medium con- taining higher concentration of Ins (2.5 nM) for the last 6-hr period that precedes the experiments, as described in the following. All drugs were purchased from Sigma.

Incubations were carried out at 37°C in a 5% CO2 atmosphere up to 114 hr. All the incubations started at 20:00 hr. The medium and the hormone content were changed every 24 hr. Melatonin was added intermittently for 12 hr in a 24-hr period. Two incubation protocols were followed: (i) 12h+/12h-, cells started the incubation in the presence of melatonin; and (ii) 12h-/12h+, in its absence for the next 12 hr. After this period, the medium containing melatonin was washed out and replaced by the same medium without the hormone, while in the other group, melatonin was added to the flask. Every 12 hr, the procedure of washing out and adding melatonin was repeated in order to fulfill a complete 24 hr melatonin+/melatonin- cycle. This procedure was interrupted when four and a half cycles in the intermittent presence of melatonin (alone or in combination of low concentrations of Ins) were achieved (108 hr of incubation). At this moment, the adipocytes were washed, the medium was replaced and an additional 6-hr period of incubation pro- ceeded (in the presence of melatonin for cells, which in the preceding 12 hr were not incubated with the hormone, while for the other group bathed with melatonin, the medium was replaced without it). Therefore, mimicking the night with the addition of melatonin or the day with its absence, we intended to induce changes in the cell metabolism. The expressions

Ôinduced nightÕand Ôinduced dayÕestablished here will describe

the events occurring in the presence or absence of melatonin, respectively. Higher concentration of Ins (2.5 nM) also replaced the low or basal concentrations in the corresponding groups of treatment for the last 6 hr period. In the end, cells were prepared for metabolic studies (lipogenesis and lipolysis), and samples were also removed for evaluation of the glucose- 6-phosphate dehydrogenase (G6PDH), fatty acid synthase (FAS), hormone sensitive lipase (HSL),Bmal-1b,Clock,Per-1 andCry-1 mRNA expression. Aliquots of the medium were collected for the determination of leptin released by radioimmunoassay.

Reverse transcriptase-polymerase chain reaction
(RT-PCR) assay for evaluation of clock genes and
enzyme expression

All reagents were purchased from Invitrogen. Total RNA
was extracted from isolated adipocytes using guanidine
Melatonin and adipocyte circadian metabolism
423

isothiocyanate based TRIzolÒ reagent according to the manufacturerÕs specifications, and quantified spectrophoto- metrically at 260 nm with acceptable 260/280 ratios between 1.7 to 2.0. The RNA quality was also checked by 1% agarose gel electrophoresis, stained with 1lg/mL ethidium bromide. Superscript II RT was used to reversely transcribe 5lg of total RNA isolated using an oligo- (dT)10nprimer in a total reaction volume of 50lL. After the RT reaction, 4lL aliquots were used as cDNA template for PCR amplifications with Taq DNA polymer- ase and 2.5 mM MgCl2. The thermal cycling parameters used were: 25 cycles, 45 s at 95°C, 45 s at 60°C and 30 s at 72°C using a Gradient Mastercycler (Eppendorf AG, Hamburg, Germany). The primer sequences used and the respective fragments obtained were as follows: rat G6PDH: sense, 5Õ-CCA TAG ACA TAC GGG ATG GG-3Õ; and antisense, 5Õ-CAA CCC TGA GGA GTC TGA GC-3Õ, 216 base pair (bp); rat FAS: sense, 5Õ-AAG CCA GGA AGA GTG GGA GAG C-3Õ; and antisense, 5Õ-GGT TGG CAG CAG GAT ACA CCG-3Õ, 311 bp; rat HSL: sense, 5Õ-AGC CTA CCC AGT TAC CAC CCT G-3Õ; and antisense, 5Õ-AAG GAG TTG AGC CAT GAG GAG G-3Õ, 240 bp; ratBMAL-1b: sense, 5Õ-TGC CGA GGA AAT CAT GGG AAT C-3Õ; and antisense, 5Õ-CGC ATC TGC TTC CAA GAG GCT CG-3Õ, 337 bp; ratClock: sense, 5Õ-TCC CGA TTC CAT CCA GTA TGC C-3Õ; and antisense, 5Õ-CTG TGC CTG CTG CTG TTG GTG-3Õ, 221 bp; ratPer-1: sense, 5Õ-ACC CAA GGA CCG AGA CCA TCA C-3Õ; and antisense, 5Õ-CCA GTT TCG ACA GTC TGT GGC-3Õ, 461 bp; ratCry-1: sense, 5Õ-AAC GGA GGG CTC ATG GGC TAT G-3Õ; and antisense, 5Õ-TGC TCT GTC GCT GGA CTT TGG G-3Õ, 232 bp. As an internal control for the integrity of the mRNA in each sample, additional oligonucleotide primers for rat RPL-37a: sense, 5Õ-CAA GAA GGT CGG GAT CGT-3Õ; and antisense, 5Õ-ACC AGG CAA GTC TCA GGA GGT G-3Õ, resulting in a product of 290 bp, were included in the PCR reactions. Aliquots (5lL) from the RT reactions were used in the quantitative measurements. The reaction products were separated by agarose gel (2.0%) electrophoresis, stained with ethidium bromide, and analyzed by scanning densi- tometry [Eagle Eye – StratageneÒ, model 401304, software Eagle Sight 3.2 (La Jolla, CA, USA)]. Samples were normalized to the quantity of RPL-37a signal produced by RT-PCR and presented as arbitrary units of the various genes mRNA relative to control. The DNA amplification product levels were carefully measured on the linear portion of the amplification curve.

D-[U-14C]-glucose incorporation into lipids

From a 10% adipocyte suspension in Krebs/Ringer/phos- phate buffer pH 7.4, with 1% BSA and 2 mmol/L glucose, at 37°C and saturated with a gas mixture of CO2(5%)/O2 (95%), 450lL aliquots were transferred to polypropylene test tubes containing 5lL (0.05lCi per tube) ofd-[U-14C]- glucose, in the presence or absence of Ins (10 nmol/L). These samples were then incubated for 1 hr at 37°C in a water bath (final volume = 500lL). After incubation, the mixture was acidified with 0.2 mL H2SO4(8 N) and incubated for additional 30 min. At the end of incubation, the reaction

mixture was treated with 2.5 mL of DoleÕs reagent (isopro- panol:n-heptane: H2SO4, 4:1:0.25 v/v/v) for lipid extraction [18]. The results were expressed as nanomoles of glucose incorporated into lipids per 106cells per hr.

Measurement of lipolysis

From a 20% adipocyte suspension in Krebs/Ringer/Phos- phate buffer pH 7.4, with 1% BSA, 170lL aliquots were transferred to microfuge tubes (1.5 mL) containing 20lL of adenosine deaminase (0.2 U/mL) and were incubated at 37°C for 5 min to allow the degradation of endogenous released adenosine, which is a potent inhibitor of lipolysis [21]. After this period, these samples were incubated for 1 hr at 37°C in the presence or absence of 10lL of isoproterenol (10

)5mol/L), in a final volume of 200lL. At

the end of incubation, the reaction was blocked by moving the tubes to a cold water bath followed by centrifugation at 3500 g for 5 min at 4°C. The infranatant was carefully transferred to microtubes containing 150lL of silicone oil and re-centrifuged at 3500 g for 2 min. The glycerol content of the incubation medium was measured using an enzy- matic-colorimetric assay (Free glycerol determination kit; Sigma). It was used as an index of lipolysis and was corrected to be expressed as nanomoles per 106cells per hr.

Leptin secretion

The leptin levels were measured in the culture medium by radioimmunoassay (RIA) using a commercially available Rat leptin kit (Linco Research Inc., St. Charles, MO, USA). The test sensitivity is 0.5 ng/mL and the intra-assay coefficient of variation was less than 5%. The secretion rate was expressed as picograms of leptin released per 106cells per 6 hr.

Statistical analysis

Statistical analysis was performed by two-way ANOVA followed by Bonferroni post hoc tests using GraphPad Prism version 4.03 for Windows (GraphPad Software, Inc., San Diego, CA, USA). The level of significance was set at

P< 0.05. Data were presented as mean ± SEM.
Results

Figure 1 illustrates the clock gene mRNA expression during the induced night and the induced day in adipocytes cultured for 114 hr in the intermittent presence or absence of melatonin (alone or in combination with Ins). The results show an increase (approximately 55%, n = 14,P < 0.05) on theClock mRNA expression during the induced night (Fig. 1A). In the presence of Ins, this effect was not observed. A similar pattern of response (however not statistical significantly,P = 0.06) was observed toBmal-1b clock gene expression (Fig. 1B). On the other hand, the clock genePer1 mRNA expression was higher (60%, n = 14,P < 0.05) in the presence of Ins during the induced night (Fig. 1C). Finally, the synchronization of the adipocytes culture by melatonin did not modulate the expression of the clock geneCry1 (Fig. 1D).

Alonso-Vale et al.
424

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