Physiology amp Behavior 99 (2010) 91ndash99

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Adiponectin receptor 2 is regulated by nutritional status leptin and pregnancy in atissue-specific manner

Carmen Ruth Gonzaacutelez ae Jorge Eduardo Caminos ac Rosaliacutea Gallego be Sulay Tovar aeMariacutea Jesuacutes Vaacutezquez ae Mariacutea Fernanda Garceacutes c Miguel Lopez ae Tomaacutes Garciacutea-Caballero bManuel Tena-Sempere de Rubeacuten Nogueiras ae Carlos Dieacuteguez aea Department of Physiology School of Medicine University of Santiago de Compostela-Instituto de Investigacioacuten Sanitaria (IDIS) 15782 Santiago de Compostela Spainb Department of Morphological Science University of Santiago de Compostela School of Medicine 15705 Santiago de Compostela Spainc Department of Physiology ndash IGUN Faculty of Medicine and Direccioacuten de Investigaciones Sede Bogotaacute ndash Nacional University of Colombia Bogotaacute Colombiad Department of Cell Biology Physiology and Immunology University of Cordoba 14004 Cordoba Spaine CIBER Fisiopatologiacutea de la Obesidad y Nutricioacuten (CIBERobn) 15782 Santiago de Compostela Spain

Corresponding authors Department of Physiologyof Santiago de Compostela and CIBER Fisiopatologiacutea d(CIBERobn) S Francisco sn 15782 Santiago de CoTel +34981582658

E-mail addresses rubennogueirasusces (R Nogue(C Dieacuteguez)

0031-9384$ ndash see front matter copy 2009 Elsevier Inc Aldoi101016jphysbeh200910015

a b s t r a c t

a r t i c l e i n f o

Article historyReceived 20 April 2009Received in revised form 21 October 2009Accepted 22 October 2009

KeywordsAdiponectinAdiponectin receptor 2LeptinPregnancy

The aim of the present work was to study the regulation of circulating adiponectin levels and the expressionof adiponectin receptor 2 (Adipo-R2) in several rat tissues in relation to fasting leptin challenge pregnancyand chronic undernutritionUsing real-time PCR we found Adipo-R2 mRNA expression in the liver stomach white and brown adiposetissues (WAT and BAT) of adult rats Immunohistochemical studies confirmed protein expression in the sametissues Adipo-R2 mRNA levels were decreased in liver after fasting with no changes in the other tissuesLeptin decreased Adipo-R2 expression in liver and stomach but increased its expression in WAT and BATChronic caloric restriction in normal rats increased Adipo-R2 gene expression in stomach while it decreasedhepatic Adipo-R2 levels in pregnant rats Using radioimmunoassay we found that plasma adiponectin levelswere diminished by fasting and leptin Conversely circulating adiponectin was increased in food-restrictedrats whereas its levels decreased in food-restricted pregnant rats by the end of gestationIn conclusion our findings provide the first evidence that (a) Adipo-R2 mRNA is regulated in a tissue-specificmanner by fasting but leptin is not responsible for those changes (b) chronic caloric restriction in normaland pregnant rats also regulate Adipo-R2 mRNA in a tissue-specific manner and (c) Adipo-R2 mRNA doesnot show a clear correlation with plasma adiponectin levels

School of Medicine Universitye la Obesidad y la Nutricioacutenmpostela (A Coruntildea) Spain

iras) carlosdieguezusces

l rights reserved

copy 2009 Elsevier Inc All rights reserved

1 Introduction

Adiponectin is an adipokine discovered by four independent groups[1ndash3] which is mainly expressed in the white adipose tissue (WAT) ofboth humans and rodents but it is also detected in other tissues [45]Adiponectin is found at relatively high concentrations in the circulation[67] and its circulating levels are inversely correlated with obesity andits associate disorders such as cardiovascular diseases [89] hyperten-sion [10] and type 2 diabetes [11]whereas increased adiponectin serumlevels are associated with improved insulin sensitivity [12]

The receptors that mediate the actions of adiponectin have beencloned and named adiponectin receptor 1 (Adipo-R1) and 2 (Adipo-R2)

[13] The genes encoding these receptors are located on differentchromosomes and the gene products constitute a novel class of seven-transmembrane domain receptors [13] Adipo-R1 is abundantlyexpressed in skeletal muscle but was also ubiquitously found in severalmouse tissues whereas Adipo-R2 is predominantly located in liver [13]Adipo-R1 is a high-affinity receptor for globular adiponectin and also alow-affinity receptor for full-length adiponectin but Adipo-R2 is anintermediate-affinity receptor for full-length and globular adiponectin[13] Consistent with a beneficial role of adiponectin in insulinsensitivity simultaneous disruption of both Adipo-R1 and Adipo-R2abolished adiponectin binding and actions resulting in insulin resis-tance and marked glucose intolerance [14] Therefore in addition toadiponectin expression and plasma levels the expression of Adipo-R1and Adipo-R2 in target tissues may play a relevant role in the control ofinsulin sensitivity Although the immediate downstream signallingevents are largely unknown stimulation of the receptors activates AMP-activated protein kinase and induces peroxisome proliferator-activatedreceptor alpha signalling [131516] The physiological relevance ofthese two receptors is further supported by the fact that Adipo-R1

92 CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

deficiency resulted in increased adiposity associated with decreasedglucose tolerance physical activity and energy expenditure effects thatin part overlap the described phenotype of adiponectin-deficient miceOn the other hand Adipo-R2 deficiency resulted in resistance to HFD-induced obesity and glucose intolerance associated with increasedphysical activity and energy expenditure and decreased plasmacholesterol levels [17] Therefore it seems that Adipo-Rs have differentfunctions and it is plausible to hypothesize that there is a differentcorrelation between adiponectin levels and Adipo-R1 and Adipo-R2expressions

Only little is known about the regulation of these receptors indifferent tissues and the findings are controversial For instance it wasreported that skeletal muscle Adipo-R1 and Adipo-R2 levels werereduced in individuals with a family history of type 2 diabetes [18]However others failed to reproduce those data in the skeletal muscleof diabetic patients [1920] In mice it was found that Adipo-Rsexpression was decreased after insulin administration [2122] butinsulin had no effect on Adipo-R1 expression in human and murinemyotubes [23] In keeping with this several studies performed inhumans and rodents also showed a differential regulation ofadiponectin receptors between species For instance Adipo-R1 andAdipo-R2 were found in pancreatic β cells of humans and rats [24] butonly Adipo-R1 was located in pancreatic β cells of mice [25] Growthhormone (GH) has been shown to decrease Adipo-R2 mRNAexpression in human adipose tissue [26] whereas Adipo-R2 wasstimulated after GH treatment in 3T3-L1 adipocytes [27]

Taking into account the different phenotypes of Adipo-R1 andAdipo-R2 deficient-mice and the discrepancies regarding the regula-tion of Adipo-Rs in different species the aim of the present work wasto study the regulation of Adipo-R2 in rat tissues under differentphysiological conditions and to assess if circulating adiponectin levelswere correlated with Adipo-R2 expression More specifically we useddifferent physiological conditions such as caloric restriction preg-nancy and leptin treatment which are known to have an impact onenergy homeostasis and insulin sensitivity

It is important to note that we focused our analyses on Adipo-R2because we have previously shown that Adipo-R1 is expressed in ratblood cells [28] thus making it difficult to discriminate whether theexpression of Adipo-R1mRNAwas due to blood contamination or wasspecifically localized in the different tissues under analysis

2 Materials and methods

21 Animals

Male and female SpraguendashDawley rats (200ndash250 g) were housedin air-conditioned rooms (22ndash24 degC) under a 1212 h lightdark cycleand fed standard rat chow and water Depending of the experimentalparadigm the rats were fed ad libitum fasted or chronically restrictedAnimals were killed by decapitation and all tissues were rapidlyexplanted and snap frozen on dry ice or kept in formalin Tissues werecollected and frozen at minus80 degC until they were used All the animalprocedures were conducted according to the principles approved bythe Santiago de Compostela Medical School Animal Care ResearchCommittee

22 Experimental setting 1

To study its distribution Adipo-R2 was analyzed in variousperipheral tissues of adult male rats

23 Experimental setting 2

The effects of fasting upon liver stomachWAT and brown adiposetissue (BAT) Adipo-R2 mRNA expression were analyzed Adult maleswere subjected to 48 h food deprivation and tissues were collected at

the end of this fasting period In addition the effects of leptinadministration upon Adipo-R2 mRNA levels were evaluated Onegroup of animals that received the leptin treatment was fed ad libitumand the other group was fasted during the last 48 h of treatment Inthis setting vehicle or recombinant leptin (Leptin human recombi-nant expressed in E coli Sigmareg) was injected in rats at a dose of05 μggm BW ip every 6 h for 3 days [29]

24 Experimental setting 3

Virgin female SpraguendashDawley rats were used in this study Astable pattern of vaginal cyclicity was established during a 10-daybaseline period The rats were housed individually with free access towater Rats were randomly assigned to one of two dietary treatmentgroups A control group was fed ad libitum with a standard diet for21 days The restricted group was fed 30 of ad libitum intakedetermined by the amount of food consumed by the control group onthe previous days from day 1 until the end of the experiment(day 21) as previously reported [3031] Rat groups (each groupconsisted of at least six rats) were killed at days 12 16 19 and 21under anaesthesia

25 Experimental setting 4

Timed matings were performed in SpraguendashDawley rats bymonitoring the stage of oestrus of the rat before introducing themales Day 1 of pregnancy was determined by the presence ofspermatozoa after a vaginal smear After confirmation that mating hadoccurred rats were housed individually with free access to water Ratswere randomly assigned to one of two dietary treatment groups Acontrol group was fed ad libitum with a standard diet for 21 days Therestricted group was fed 30 of ad libitum intake determined by theamount of food consumed by the control group on the previous daysfrom day 1 until the end of the experiment (day 21) as previouslydescribed [3031] Rat groups (each group consisted of at least six rats)were killed at days 12 16 19 and 21 by decapitation

26 RNA isolation and Adipo-R2 mRNA expression analysis by RT-PCR

The expression of themRNAencodingAdipo-R2was assessed by RT-PCR Total RNA was extracted from the removed tissues using Trizolreg(Invitrogen Inc Carlsbad CA USA) according to the manufacturersinstructions Total RNA (2 microg) was reverse transcribed using randomprimer and M-MLV (Moloney Murine Leukemia Virus) ReverseTranscriptase (Invitrogen) As an internal standard hypoxanthinephosphoribosyl transferase (HPRT) cDNA was amplified For amplifica-tion of the different genes primers were selected to eliminate non-specific amplification of genomic DNA (Table 1) The PCR conditionswere 95 degC for 15 s 60 degC for 30 s and 72 degC for 1 min for 34 cycles forAdipo-R2 and 28 cycles forHPRT andfinally a step 72 degC for 10 min TheRT-PCR products were confirmed by sequencing andwere separated byelectrophoresis on 25 agarose gel and visualized by ethidiumbromidestaining with UV light using a Gel Doc 1000 Documentation System(Bio-Rad Laboratories Inc Richmond CA)

27 Immunohistochemistry

Samples of rat normal liver brown andwhite adipose tissue lungstomach (fundus) small bowel (duodenum and jejunum) colonadrenal gland and skeletal muscle were immersion-fixed in 10buffered formalin for 24 h dehydrated and embedded in paraffinwax using a standard procedure [3233] Sections 5 microm thick weremounted on Histobond adhesion Micro slides (Marienfeld Lauda-Koumlnigshofen Germany) dewaxed and rehydrated For immunohis-tochemical staining sections were consecutively incubated in (1)rabbit anti adiponectin receptor 2 (4ndash39 Phoenix Pharmaceuticals

Table 1Primers used for RT-PCR and Real-Time PCR analysis

Gene Primers and probes Product length bp GenBank accession no

Rat HPRT Forward 5prime-CAGTCCCAGCGTCGTGATTA-3prime 139 NM_013556Reverse 5prime-AGCAAGTCTTTCAGTCCTGTC-3prime

Rat 18S Forward 5prime-CGGCTACCACATCCAAGGAA-3prime 186 M11188Reverse 5prime-GCTGGAATTACCGCGGCT-3primeProbe Fam-5prime-GACGGCAAGTCTGGTGCCAGCA-3prime-TAMRA

Rat Adipo-R2 Forward 5prime-TTGCCACCCCTCAGTATCG-3prime 75 NM_001037979Reverse 5prime-AAGGTAGGGATGATTCCACTCAGA-3primeProbe Fam-5prime-AGAGCAGGAGTGTTCGTGGGCTTGG-3prime-TAMRA

Rat Cylophilin-A Forward 5prime-CAGACGCCGCTGTCTCTTTT-3prime 75 M19533Reverse 5prime-TCAGCCGTGATGTCGAAGAA-3primeProbe Fam-5prime-CCGCTTGCTGCAGACATGGTCAAC-3prime-TAMRA

93CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

Belmont CA) 1100 overnight at room temperature (2) 3hydrogen peroxide for 10 min (3) EnvisionregHRP rabbitmouse(Dako) for 30 min and (4) 3 3prime Diamino-benzidine (Dako) for10 min Between steps 1 and 4 the sections were washed twice for5 min in TBS (005 molL Tris buffer pH76 containing 03 molLNaCl) and after step 4 in distilled water Counterstaining was donewith haematoxylin Negative controls were performed replacing theprimary antibody with non-immune rabbit serum at the sameconcentration of the primary antibody in the stomach as previouslydescribed [34]

28 Real-time semiquantitative RT-PCR analysis

Real-time reverse-transcription polymerase chain reaction (RT-PCR)analyses were performed in a fluorescent temperature cycler (Taq-Manreg Applied Biosystems Foster City CA USA) following themanufacturers instructions [3536] 100 ng of total RNA was used foreachRT reaction of liver and stomach and 500 ngwasused forWATandBAT The PCR cycling conditions included an initial denaturation at 50 degCfor 10 min followed by 40 cycles at 95 degC for 15 s 60 degC for 1 min Theoligonucleotide specificprimers andprobes for ratAdipo-R2and18S aredescribed in Table 1 For the analysis of the data the input value ofAdipo-R2 was standardized to the 18S value for the sample group andwas expressed compared with the average value for the control groupExperiments were performed on six animals per group

29 Radioimmunoassay for rat adiponectin

Plasma levels of adiponectin were assayed using reagent kit andmethod provided by Linco Research (Linco Research St Charles MO)[37] Truncal vein plasma was obtained by decapitation from thedifferent groups of rats It was collected in tubes containing EDTA(1 mgml blood) and aprotinin (500 unitsml blood) (Sigma) centri-fuged immediately and the plasma adiponectin levels were assessed

Fig 1 Representative reverse-transcription polymerase chain reaction (RT-PCR) assay of thtissues A 50-bp mol wild-type marker was used The integrity and loading of RNA were co

by means of a double-antibody radioimmunoassay provided by LincoAll samples were assayed in duplicate within one assay and resultsexpressed in terms of the adiponectin standard The limit of eachassay sensitivity was 1 ngml the intra- and inter-assay levels wererespectively 411 and 656

210 Statistical analysis

Data are expressed as having a mean of plusmnSEM and analyzed usinga computerized package for statistical analysis Statistically significantdifference was determined by a nonparametric and unpaired test(Mann Whitney test) A p value lt005 was considered as beingsignificant

3 Results

31 Distribution of adiponectin receptor 2

In agreement with previous reports Adipo-R2 mRNA expressionwas detected in the liver stomach WAT and BAT (Fig 1) [1338] Tofurther explore Adipo-R2 protein expression in these tissues wecarried out an immunohistochemical study with a specific antibodyLiver (Fig 2A) and adipose tissue (white and brown Fig 2B) showedan intense immunostaining and thus constituted the positive stainingcontrols The digestive tract also showed positive staining for Adipo-R2 In the stomach (fundus-body region) Adipo-R2 was localized inthe epithelial mucous cells lining the surface of the stomach and thegastric pits and in the parietal cells of the gastric glands (Fig 2C)Epithelial lining of the villi of duodenum and jejunum (mainlyenterocytes) expressed the Adipo-R2 (Fig 2D) In the large intestine(colon) immunostaining for Adipo-R2 was intense in the enterocytesof the surface epithelia (Fig 2E) The specificity of the immunostain-ing was corroborated by replacing the primary antibody with non-immune rabbit serum at the same concentration of the primary

e expression levels of Adipo-R2 mRNA after 35 cycles of amplification in different ratnfirmed by amplification of rat hypoxanthine phosphoribosyl transferase (HPRT)

Fig 2 A Rat hepatocytes showed high Adipo-R2 immunostaining (original magnification times20) B White and brown adipose cells also were intensely immunostained for Adipo-R2(original magnification times40) C Adipo-R2 in the rat fundic mucosa Immunostaining was demonstrated in the surface mucous cells and parietal cells of gastric glands (originalmagnification times40) D This micrograph shows duodenal villi immunostained for Adipo-R2 Positivity wasmainly localized in enterocytes and goblet cells of the epithelial lining (originalmagnificationtimes20) E Rat colon localizationofAdipo-R2 expression showing apositive staining in the surface of epithelial cells (originalmagnificationtimes40) F Stomach sections incubatedwith non-immune rabbit serum at the same concentration of the primary antibody and no immunoreactivity was observed in this case (original magnification times20)

94 CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

antibody in the stomach No immunoreactivity was observed in thiscase (Fig 2F)

32 Influence of fasting and leptin

The response of the receptor to a fasting state was tissue-specificIn liver Adipo-R2 mRNA expression was decreased after 48 h offood deprivation (Fig 3C) Conversely in WAT Adipo-R2 mRNA levelswere increased after fasting (Fig 3C) while in stomach and BAT(p=00952) there were no significant differences (Fig 3C)

In order to study the influence of leptin onAdipo-R2 gene expressionwe used rats fed ad libitum and rats fasted during 48h treatedperipherally with leptin As shown in Fig 3 leptin treatment decreasedfood intake and body weight gain in rats fed ad libitum (Fig 3AndashB)However leptin did not modify body weight gain in rats fasted 48 h(Fig 3B) Leptin decreased Adipo-R2 mRNA expression in the stomachwhile liver Adipo-R2 expressionwas decreased by leptin only in fed but

not fasted rats (Fig 3C) Conversely leptin increased Adipo-R2 mRNAlevels in the WAT and BAT of rats fed ad libitum (Fig 3C) although inWAT it was not statistically significant (fed vehicle vs fed leptinplt0053) In fasted rats leptin inhibited Adio-R2 mRNA expression inthe stomach whereas it failed to exert significant effects in the othertissues (Fig 3C) Circulating adiponectin levels were decreased in ratsfasted 48 h (Fig 3D)Moreover leptindecreased circulating adiponectinlevels in rats fed ad libitum but not in rats fasted during 48 h (Fig 3D)Overall our data suggest that the effects of nutritional status on Adipo-R2 mRNA expression and circulating adiponectin levels were notmediated by leptin

33 Effect of chronic food restriction in normal rats

After food restriction normal-cycling female virgin rats exhibited amarked reduction in body weight compared with control rats [30] Asshown in Fig 4 the effect of chronic food restriction in normal rats

Fig 3 Effect of leptin (05 μggm BW ip every 6 h for 3 days) on food intake (A) and body weight (B) Analysis of Adipo-R2 mRNA expression by real-time RT-PCR in liver stomachWAT and BAT after leptin treatment in fed ad libitum and fasted rats (C) mRNA levels have been standardized by 18S mRNA levels and the results are expressed as arbitrary unitsMean values were obtained from six animals per group Plasma adiponectin levels in rats treated with leptin measured by RIA (D) Values are the meanplusmnSEM plt005 plt001

95CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

showed different profiles of Adipo-R2 mRNA expression in thedifferent tissues Liver Adipo-R2 mRNA levels were increased after12 days of food restriction and decreased thereafter reaching thesame levels as the fed ad libitum group when the rats were foodrestricted during 21 days (Fig 4A) In the stomach and BAT Adipo-R2mRNA expression progressively increased during chronic caloricrestriction even though in BAT its expression failed to reach statisticalsignificance (Fig 4BndashC) Finally plasma adiponectin levels were alsoincreased during chronic food restriction (Fig 4D) WAT was notanalyzed in this experiment because we were unable to get enoughtissue from food-restricted rats

34 Influence of pregnancy and chronic caloric restriction

We investigated the influence of pregnancy a physiologicalmodel ofhyperleptinemia and decreased insulin sensitivity on the Adipo-R2mRNA expression Our results showed that the regulation of Adipo-R2mRNA expression throughout gestation was tissue-specific In liver wedetected higher levels of Adipo-R2 at day 12 of gestation comparedwithcontrol non-pregnant (CNP) rats and those levels were progressivelydecreased thereafter (Fig 5A) However when Adipo-R2 mRNA levelswere compared to CNP rats there were no significant differencesthroughout gestation In contrast to the liver theexpressionof Adipo-R2

in the stomach was increased at the end of the gestation whencompared to the beginning of this period (Fig 5B) Similarly in the liverAdipo-R2 gene expression remained unaltered in the stomach through-out gestationwhen compared to non-pregnant rats (Fig 5B) In BATwefailed to detect any significant change during pregnancy (CNP vs 12dp=04286 CNP vs 16d p=02222 CNP vs 19d p=06905 CNP vs21d p=06905) (Fig 5C)

As we have previously shown [30] chronic caloric restrictionduring pregnancy resulted in a marked reduction in maternal bodyweight from day 1 to day 16 From day 16 until the end of theexperiment the food-restricted pregnant rats slightly recoveredweight without recovering premating weights Our results indicatedthat the effect of chronic caloric restriction on Adipo-R2 mRNA levelsthroughout gestation was tissue-specific As a matter of fact foodrestriction decreased Adipo-R2 mRNA expression in liver of pregnantrats (Fig 5A) but not in the stomach (Fig 5B) BAT mRNA expressionwas slightly increased in caloric-restricted pregnant rats but failed tobe statistically significant (CNP vs 12d p=04127 CNP vs 16dp=01255 CNP vs 19d p=00159 CNP vs 21d p=00823) (Fig 5C)WAT was not analyzed in this experiment because we were unable toget enough tissue from food-restricted pregnant rats Plasma adipo-nectin levels during gestation were measured previously [39] and areshown in Fig 5D

Fig 4 Effect of food restriction (30 ad libitum) on Adipo-R2 mRNA expression in liver (A) stomach (B) and BAT (C) of normal female rats Adipo-R2 mRNA levels have beenstandardized by 18S mRNA levels and the results are expressed as arbitrary units Plasma adiponectin levels in those animals were measured by RIA (D) Mean values were obtainedfrom six animals per group Values are the meanplusmnSEM ab and c denote groups that are statistically different

96 CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

4 Discussion

In order to gain further insight into the physiological role of Adipo-Rs in rat we carried out an analysis of their expression and regulationin different rat tissues using real-time PCR and immunohistochem-istry Since our group has previously shown that Adipo-R1 isexpressed in blood cells [40] we focused our attention on thedistribution and regulation of Adipo-R2 Consistent with previousdata obtained in mice our work shows that the highest amount ofAdipo-R2 mRNA was found in the rat liver In addition we confirmedits location in the gastrointestinal tract WAT and BAT anddemonstrated for the first time that Adipo-R2 is regulated in the ratin a tissue-specific fashion under several physiological conditions

It is well established that adiponectin increases insulin sensitivityand improves glucose tolerance but in addition to those actions thereare several reports suggesting that adiponectin and Adipo-Rs areimportantmodulators of energymetabolism even though this is still acontroversial topic One study has shown that adiponectin acts in thebrain increasing the expression of corticotrophin-releasing hormone(CRH) and UCP-1 thereby stimulating energy expenditure andleading to a decrease in body weight [41] In contrast another studyhas indicated that adiponectin stimulates food intake and decreasesenergy expenditure through its effects in the central nervous system[42] Opposing effects on energy metabolism have been also observedfor Adipo-Rs [17] Adipo-R1 ko mice showed increased adiposity andlower locomotor activity and energy expenditure whereas Adipo-R2ko mice were lean and resistant to high fat diet and had higherlocomotor activity [1743] In the present study we looked at theeffects of nutritional status and chronic leptin administration onplasma adiponectin levels and Adipo-R2 mRNA expression Ourresults showed that circulating adiponectin levels were decreasedafter 48 h of fasting Moreover adiponectin levels are also decreasedafter leptin treatment in rats fed ad libitum but not in fasted ratstreated with leptin probably due to the very low levels of basal leptin

in fasted rats When leptin is administered to fed rats circulatingleptin levels at the end of the treatment will be higher than in fastedrats treated with leptin Taken together these data imply that leptinlevels need to reach a certain set-point to decrease adiponectinsomething that is obtained in fed but not in fasted rats treated withleptin In any event our results support previous findings showingthat adiponectin mRNA expression inWAT decreases after fasting andafter leptin treatment [44] Overall the data suggest that adiponectinis regulated by nutritional signals

Regarding the effect of nutrient signals on Adipo-Rs there areseveral reports indicating that in mice Adipo-R1 and Adipo-R2 areregulated in a tissue-specific manner and could exert different actionsin each tissue For instance the mRNA levels of both receptors areincreased in the liver and skeletal muscle of mice after 48 h fasting[21] Conversely in the BAT of mice only Adipo-R1 was increasedafter fasting with no changes in Adipo-R2 [45] Interestingly theresponse of each receptor to food deprivation was also differentdepending of the fat depot [45] In the study presented herein weobserved that Adipo-R2 mRNA expression was down-regulated inliver after fasting whereas it was unchanged in the remaining tissues

Interestingly leptin infusion decreased Adipo-R2 mRNA levels inliver and stomach in rats fed ad libitum Conversely leptin inducedAdipo-R2 levels in the BAT of rats fed ad libitum Therefore ourfindings indicate that the modulation of Adipo-R2 mRNA expressionby nutritional status is not mediated by leptin Our data are inagreement with previous reports demonstrating that Adipo-Rs areregulated in a tissue-specific fashion in rodents and humans underdifferent nutritional conditions [46ndash48] The differences between ourresults and previous reports might be explained by differencesbetween species andor genetic background As we stated aboveother reports performed in humans and rats have also found adifferential regulation of adiponectin receptors between species [24ndash27] Overall the relevance of these results is still unknown but ourdata indicate that nutritional status and leptin regulate Adipo-R2

Fig 5 Effect of food restriction (30 ad libitum) on Adipo-R2 mRNA expression in liver (A) stomach (B) and BAT (C) of pregnant rats Adipo-R2 mRNA levels have been standardizedby 18S mRNA levels and the results are expressed as arbitrary units Plasma adiponectin levels in those animals were measured by RIA (D) Mean values were obtained from sixanimals per group Values are the meanplusmnSEM ab and c denote groups that are statistically different

97CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

mRNA expression in a tissue-specific manner suggesting differentroles for this receptor in the different tissues where it is located

It is well known that metabolic adaptations to undernutrition arelinked to changes in the endocrine environment [49] includingincreased insulin sensitivity [5051] We have used a chronic food-restriction model in normal-cycling female rats and a maternalnutritional restriction throughout pregnancy as an experimentalapproach to study the effect of long-term undernutrition onadiponectin levels and Adipo-R2 mRNA expression As expected in astate of improved insulin sensitivity we detected that plasmaadiponectin levels were significantly increased in normal femalefood-restricted rats Consistent with circulating adiponectin levelsAdipo-R2 mRNA expression in stomach and BAT was also increasedafter chronic food restriction whereas an oscillatory pattern was

detected in liver Hepatic Adipo-R2 was significantly increased after12 days of food restriction and decreased afterwards This oscillatorypattern might be explained by an adaptive metabolic response of theliver after two weeks on chronic caloric restriction Therefore ourresults indicated that chronic caloric restriction regulated Adipo-R2gene expression in a tissue-specific manner Moreover these findingssuggested that only stomach and BAT Adipo-R2 gene expression butnot liver Adipo-R2 were correlated with circulating adiponectin levels

Pregnancy is characterized by a series of metabolic changesincluding a marked increase in food intake and changes in severalmechanisms related to glucose homeostasis [5253] As a matter offact it is established that gestational diabetes is the most commonmetabolic abnormality occurring during gestation [54ndash56] In ourstudy we detected once again that Adipo-R2was regulated in a tissue-

98 CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

specific manner during pregnancy showing a significant decrease atthe end of gestation only in the liver without significant changes inother tissues Although it is well known that food restriction improvesinsulin sensitivity [5051] our data failed to detect a significantincrease of Adipo-R2 mRNA levels in any tissue Unexpectedly wefound that Adipo-R2 gene expression was decreased in the liver ofpregnant food-restricted rats whereas no changes were detected instomach or BAT Overall our findings indicate that Adipo-R2expression is not related with alterations in insulin sensitivity orglucose homeostasis during pregnancy

In summary this study provides the first evidence that (a) Adipo-R2mRNA is regulated in a tissue-specific manner by fasting but leptinis not responsible of those changes (b) chronic caloric restriction andgestation also regulate Adipo-R2 mRNA in a tissue-specific mannerand (c) Adipo-R2 mRNA does not show a clear correlation withplasma adiponectin levels

Acknowledgements

This work has been supported by grants from Xunta de Galicia(ML GRC200666) Fondo Investigationes Sanitarias (ML PI061700)Ministerio de Educacion y Ciencia (CD BFU2008 ML RYC-2007-00211 RN RYC-2008-02219) Muacutetua Madrilentildea (CD and ML)European Union (Health-F2-2008-223713) CIBER de Fisiopatologiacuteade la Obesidad y Nutricioacuten is an initiative of ISCIII

Appendix A Supplementary data

Supplementary data associated with this article can be found inthe online version at doi101016jphysbeh200910015

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[2] Scherer PE Williams S Fogliano M Baldini G Lodish HF A novel serum proteinsimilar to C1q produced exclusively in adipocytes J Biol Chem 199527026746ndash9

[3] Nakano Y Tobe T Choi-Miura NH Mazda T Tomita M Isolation andcharacterization of GBP28 a novel gelatin-binding protein purified from humanplasma J Biochem (Tokyo) 1996120803ndash12

[4] Chabrolle C Tosca L Dupont J Regulation of adiponectin and its receptors in ratovary by human chorionic gonadotrophin treatment and potential involvement ofadiponectin in granulosa cell steroidogenesis Reproduction 2007133719ndash31

[5] Caminos JE Nogueiras R Gaytan F Pineda R Gonzalez CR Barreiro ML et al Novelexpression and direct effects of adiponectin in the rat testis Endocrinology 20081493390ndash402

[6] Kadowaki T Yamauchi T Kubota N Hara K Ueki K Tobe K Adiponectin andadiponectin receptors in insulin resistance diabetes and the metabolic syndromeJ Clin Invest 20061161784ndash92

[7] Scherer PE Adipose tissue from lipid storage compartment to endocrine organDiabetes 2006551537ndash45

[8] Hu E Liang P Spiegelman BM AdipoQ is a novel adipose-specific genedysregulated in obesity J Biol Chem 199627110697ndash703

[9] Hotta K Funahashi T Arita Y Takahashi M Matsuda M Okamoto Y et al Plasmaconcentrations of a novel adipose-specific protein adiponectin in type 2 diabeticpatients Arterioscler Thromb Vasc Biol 2000201595ndash9

[10] Ouchi N Ohishi M Kihara S Funahashi T Nakamura T Nagaretani H et alAssociation of hypoadiponectinemia with impaired vasoreactivity Hypertension200342231ndash4

[11] Weyer C Funahashi T Tanaka S Hotta K Matsuzawa Y Pratley RE et alHypoadiponectinemia in obesity and type 2 diabetes close association withinsulin resistance and hyperinsulinemia J Clin Endocrinol Metab 2001861930ndash5

[12] Yamauchi T Kamon J Waki H Terauchi Y Kubota N Hara K et al The fat-derivedhormone adiponectin reverses insulin resistance associated with both lipoatrophyand obesity Nat Med 20017941ndash6

[13] Yamauchi T Kamon J Ito Y Tsuchida A Yokomizo T Kita S et al Cloning ofadiponectin receptors that mediate antidiabetic metabolic effects Nature 2003423762ndash9

[14] Yamauchi T Nio Y Maki T Kobayashi M Takazawa T Iwabu M et al Targeteddisruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding andmetabolic actions Nat Med 200713332ndash9

[15] Yamauchi T Kamon J Minokoshi Y Ito Y Waki H Uchida S et al Adiponectinstimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase Nat Med 200281288ndash95

[16] Tomas E Tsao TS Saha AK Murrey HE Zhang CC Itani SI et al Enhanced musclefat oxidation and glucose transport by ACRP30 globular domain acetyl-CoAcarboxylase inhibition and AMP-activated protein kinase activation Proc NatlAcad Sci U S A 20029916309ndash13

[17] Bjursell M Ahnmark A Bohlooly Y William-Olsson L Rhedin M Peng XR et alOpposing effects of adiponectin receptors 1 and 2 on energy metabolism Diabetes200756583ndash93

[18] Civitarese AE Jenkinson CP Richardson D Bajaj M Cusi K Kashyap S et alAdiponectin receptors gene expression and insulin sensitivity in non-diabeticMexican Americans with orwithout a family history of Type 2 diabetes Diabetologia200447816ndash20

[19] Tan GD Debard C Funahashi T Humphreys SM Matsuzawa Y Frayn KN et alChanges in adiponectin receptor expression in muscle and adipose tissue of type 2diabetic patients during rosiglitazone therapy Diabetologia 2005481585ndash9

[20] McAinch AJ Steinberg GR Mollica J OBrien PE Dixon JB Macaulay SL et alDifferential regulation of adiponectin receptor gene expression by adiponectinand leptin in myotubes derived from obese and diabetic individuals Obesity(Silver Spring) 2006141898ndash904

[21] Tsuchida A Yamauchi T Ito Y Hada Y Maki T Takekawa S et al InsulinFoxo1pathway regulates expression levels of adiponectin receptors and adiponectinsensitivity J Biol Chem 200427930817ndash22

[22] Inukai K Nakashima YWatanabeM Takata N Sawa T Kurihara S et al Regulationof adiponectin receptor gene expression in diabetic mice Am J Physiol EndocrinolMetab 2005288E876ndash82

[23] Staiger H Kaltenbach S Staiger K Stefan N Fritsche A Guirguis A et al Expressionof adiponectin receptor mRNA in human skeletal muscle cells is related to in vivoparameters of glucose and lipid metabolism Diabetes 2004532195ndash201

[24] Kharroubi I Rasschaert J Eizirik DL Cnop M Expression of adiponectin receptorsin pancreatic beta cells Biochem Biophys Res Commun 20033121118ndash22

[25] Winzell MS Nogueiras R Dieguez C Ahren B Dual action of adiponectin on insulinsecretion in insulin-resistant mice Biochem Biophys Res Commun 2004321154ndash60

[26] Nilsson L Binart N Bohlooly Y Bramnert M Egecioglu E Kindblom J et alProlactin and growth hormone regulate adiponectin secretion and receptorexpression in adipose tissue Biochem Biophys Res Commun 20053311120ndash6

[27] Fasshauer M Klein J Kralisch S Klier M Lossner U Bluher M et al Growthhormone is a positive regulator of adiponectin receptor 2 in 3T3-L1 adipocytesFEBS Lett 200455827ndash32

[28] Caminos JE Nogueiras R Gallego R Bravo S Tovar S Garcia-Caballero T et alExpression and regulation of adiponectin and receptor in human and rat placentaJ Clin Endocrinol Metab 2005904276ndash86

[29] LegradiGEmersonCHAhimaRS Flier JS LechanRM Leptinprevents fasting-inducedsuppression of prothyrotropin-releasing hormone messenger ribonucleic acid inneurons of the hypothalamic paraventricular nucleus Endocrinology 19971382569ndash76

[30] Gualillo O Caminos JE Nogueiras R Seoane LM Arvat E Ghigo E et al Effect offood restriction on ghrelin in normal-cycling female rats and in pregnancy ObesRes 200210682ndash7

[31] Nogueiras R Gualillo O Caminos JE Casanueva FF Dieguez C Regulation of resistinby gonadal thyroid hormone and nutritional status Obes Res 200311408ndash14

[32] Caminos JE Nogueiras R Gallego R Bravo S Tovar S Garcia-Caballero T et alExpression and regulation of adiponectin and receptor in human and rat placentaJ Clin Endocrinol Metab 2005904276ndash86

[33] Pineiro R Iglesias MJ Gallego R Raghay K Eiras S Rubio J et al Adiponectin issynthesized and secretedbyhuman andmurine cardiomyocytes FEBS Lett 20055795163ndash9

[34] Caminos JE Bravo SB Garces MF Gonzalez CR Cepeda LA Gonzalez AC et alVaspin and amylin are expressed in human and rat placenta and regulated bynutritional status Histol Histopathol 200924979ndash90

[35] Vazquez MJ Gonzalez CR Varela L Lage R Tovar S Sangiao-Alvarellos S et alCentral resistin regulates hypothalamic and peripheral lipid metabolism in anutritional dependent fashion Endocrinology 2008 1494534ndash43

[36] Gonzalez CR Vazquez MJ Lopez M Dieguez C Influence of chronic undernutritionand leptin on GOAT mRNA levels in rat stomach mucosa J Mol Endocrinol 200841415ndash21

[37] Caminos JE Nogueiras R Gallego R Bravo S Tovar S Garcia-Caballero T et alExpression and regulation of adiponectin and receptor in human and rat placentaJ Clin Endocrinol Metab 2005904276ndash86

[38] Ishikawa M Kitayama J Yamauchi T Kadowaki T Maki T Miyato H et alAdiponectin inhibits the growth and peritoneal metastasis of gastric cancerthrough its specific membrane receptors AdipoR1 and AdipoR2 Cancer Sci200798 1120ndash7

[39] Caminos JE Nogueiras R Gallego R Bravo S Tovar S Garcia-Caballero T et alExpression and regulation of adiponectin and receptor in human and rat placentaJ Clin Endocrinol Metab 2005904276ndash86

[40] Caminos JE Nogueiras R Gallego R Bravo S Tovar S Garcia-Caballero T et alExpression and regulation of adiponectin and receptor in human and rat placentaJ Clin Endocrinol Metab 2005904276ndash86

[41] Qi Y Takahashi N Hileman SM Patel HR Berg AH Pajvani UB et al Adiponectinacts in the brain to decrease body weight Nat Med 200410524ndash9

[42] Kubota N Yano W Kubota T Yamauchi T Itoh S Kumagai H et al Adiponectinstimulates AMP-activated protein kinase in the hypothalamus and increases foodintake Cell Metab 2007655ndash68

[43] Liu Y Michael MD Kash S Bensch WR Monia BP Murray SF et al Deficiency ofadiponectin receptor 2 reduces diet-induced insulin resistance but promotes type2 diabetes Endocrinology 2007148683ndash92

99CR Gonzaacutelez et al Physiology amp Behavior 99 (2010) 91ndash99

[44] Zhang Y Matheny M Zolotukhin S Tumer N Scarpace PJ Regulation ofadiponectin and leptin gene expression in white and brown adipose tissuesinfluence of beta3-adrenergic agonists retinoic acid leptin and fasting BiochimBiophys Acta 20021584115ndash22

[45] Bluher M Fasshauer M Kralisch S Schon MR Krohn K Paschke R Regulation ofadiponectin receptor R1 and R2 gene expression in adipocytes of C57BL6 miceBiochem Biophys Res Commun 20053291127ndash32

[46] Barnea M Shamay A Stark AH Madar Z A high-fat diet has a tissue-specific effect onadiponectin andrelatedenzymeexpressionObesity (Silver Spring)200614 2145ndash53

[47] Beylot M Pinteur C Peroni O Expression of the adiponectin receptors AdipoR1and AdipoR2 in lean rats and in obese Zucker rats Metabolism 200655396ndash401

[48] Rasmussen MS Lihn AS Pedersen SB Bruun JM Rasmussen M Richelsen BAdiponectin receptors in human adipose tissue effects of obesity weight loss andfat depots Obesity (Silver Spring) 20061428ndash35

[49] Woodall SM Breier BH Johnston BM Gluckman PD A model of intrauterinegrowth retardation caused by chronic maternal undernutrition in the rat effectson the somatotrophic axis and postnatal growth J Endocrinol 1996150231ndash42

[50] Friedman JE Dohm GL Leggett-Frazier N Elton CW Tapscott EB Pories WP et alRestoration of insulin responsiveness in skeletal muscle of morbidly obese patients

afterweight loss Effect onmuscle glucose transport and glucose transporterGLUT4 JClin Invest 199289701ndash5

[51] Weiss EP Holloszy JO Improvements in body composition glucose tolerance andinsulin action induced by increasing energy expenditure or decreasing energyintake J Nutr 20071371087ndash90

[52] Sutter-DubMT Sfaxi A Strozza P Glucosemetabolism in the female rat adipocytelipid synthesis from glucose during pregnancy and progesterone treatmentJ Endocrinol 198397207ndash12

[53] Tamas G Kerenyi Z Gestational diabetes current aspects on pathogenesis andtreatment Exp Clin Endocrinol Diabetes 2001109(Suppl 2)S400ndash11

[54] Chien EK Hara M Rouard M Yano H Phillippe M Polonsky KS et al Increase inserum leptin and uterine leptin receptor messenger RNA levels during pregnancyin rats Biochem Biophys Res Commun 1997237476ndash80

[55] Pucci E Chiovato L Pinchera A Thyroid and lipid metabolism Int J Obes RelatMetab Disord 200024(Suppl 2)S109ndash12

[56] Pujol E Proenza AM Roca P Llado I Changes inmammary fat pad composition andlipolytic capacity throughout pregnancy Cell Tissue Res 2006323505ndash11