Reprod Dom Anim 49, 378–386 (2014); doi: 10.1111/rda.12282 ISSN 0936–6768
Expression of Adiponectin and its Receptors in the Porcine Hypothalamus During the Oestrous Cycle T Kaminski, N Smolinska, A Maleszka, M Kiezun, K Dobrzyn, J Czerwinska, K Szeszko and A Nitkiewicz Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Contents Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14–16, whereas in SME, the most pronounced gene expression was found on days 2–3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17–19 and in POA on days 2–3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14–16. AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2–3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2–14 of the cycle), and they decreased on days 17–19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2–3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2–3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2–3 (p < 0.05), while in POA on days 17–19 and in SME on days 10–12 and 14–16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig’s endocrine status related to the oestrous cycle.
Introduction Adiponectin is one of the adipocytokines highly expressed in the adipose tissue and is one of the most abundant plasma proteins (2–25 lg/ml) in mammals (Nishizawa et al. 2002; B€ ottner et al. 2004). Adiponectin circulates in the serum in different multimeric forms, including trimers, hexamers and high-molecular-weight multimers, and as the globular fraction (globular adiponectin) generated by the proteolytic cleavage of multimers. Adiponectin controls energy homeostasis and insulin sensitivity, and it affects lipid synthesis, vasodilatation and atherogenic activity (Yamauchi et al. 2002; Trujillo and Scherer 2005). Plasma adiponectin concentrations are inversely correlated with the adipose tissue reservoir (Arita et al. 1999). Adiponectin is also identified in cerebrospinal fluid (Kos et al. 2007), and the hormone levels increase after its intravenous injection (Qi et al. 2004).
Adiponectin actions are mediated by two types of receptors: AdipoR1 and AdipoR2. Unlike G-coupled protein receptors, AdipoR1 and AdipoR2 are seventransmembrane domain receptors with an extracellular carboxyl terminus and an intracellular amino terminus. AdipoR1 shows high affinity for the globular form of adiponectin, and AdipoR2 has an intermediate binding affinity for both full-length and globular species. The receptors are highly related and share 66.7% sequence identity in mice (Yamauchi et al. 2003). AdipoR1 and AdipoR2 are widely expressed in humans and rodents, which suggests that adiponectin has pleiotropic effects. The highest levels of AdipoR1 expression are observed in skeletal muscles, whereas AdipoR2 is most abundantly expressed in the liver (Yamauchi et al. 2003; Lord et al. 2005). The presence of the adiponectin receptors was observed in human and rodent hypothalami, including in the paraventricular nucleus (Kos et al. 2007; Guillod-Maximin et al. 2009), in periventricular areas and the pituitary (Yamauchi et al. 2003; RodriguezPacheco et al. 2007; Psilopanagioti et al. 2009). Both types of receptors were also expressed in GT1-7 hypothalamic GnRH neuron cells (Wen et al. 2008). The expression of adiponectin and its receptors in species other than humans and rodents remains largely unexplored. Adiponectin seems to be a hormonal link between obesity and reproduction. It was described as a factor affecting ovarian steroidogenesis, oocyte maturation, embryo implantation and development (Palin et al. 2012). Several lines of evidence also suggest that adiponectin influences the reproductive system by exerting central effects on the highest branch of the hypothalamic–pituitary–ovarian axis, inhibiting GnRH (Wen et al. 2008) and GnRH-induced LH secretion (Rodriguez-Pacheco et al. 2007; Lu et al. 2008). To date, there has been no research investigating the gene and protein expression patterns of adiponectin and its receptors in porcine hypothalamic structures responsible for GnRH production and secretion, including the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME), or the effect of the oestrous cycle on transcript and protein levels in animals. The objective of this study was to compare the expression levels of adiponectin, AdipoR1 and AdipoR2 mRNAs and proteins in selected areas of the porcine hypothalamus during the oestrous cycle.
Materials and Methods Animals and tissue collection This study was carried out in accordance with ethical standards of the Animal Ethics Committee at the © 2014 Blackwell Verlag GmbH
Adiponectin and its Receptors in Pig Hypothalamus
University of Warmia and Mazury in Olsztyn. Mature gilts (Large White 9 Polish Landrace), 7–8 months old, weighing 120–130 kg, descended from private breeding were used. Diets were balanced (crude protein, metabolisable energy, exogenous amino acids and minerals) in accordance with the nutrient requirements of domestic pigs. Twenty gilts were divided into four experimental groups (n = 5 per group) as follows: days 2–3 (the early luteal phase), 10–12 (the mid-luteal phase), 14–16 (the late luteal phase) and 17–19 (the follicular phase) of the oestrous cycle. The analysed days are representative of the stages of the oestrous cycle characterized by major hormonal and physiological changes observed throughout the cycle (Akins and Morrissette 1968; Henricks et al. 1972). Females were monitored daily for oestrous behaviour in the presence of an intact boar. The day of the second oestrous onset was designated as day 0 of the oestrous cycle. The phase of the oestrous cycle was also confirmed on the basis of ovarian morphology, and highly characteristic ovarian morphology was noted in each of the analysed periods with the presence of corpora haemorrhagica on days 2–3, fully active corpora lutea on days 10–12, corpora lutea with clear signs of luteolysis on days 14–16 and only corpora albicantia with pre-ovulatory follicles on days 17–19 (Akins and Morrissette 1968). Within a few minutes after the slaughter, the hypothalamus, muscle, liver and subcutaneous adipose tissue were removed. The hypothalamus has been divided into the pre-optic area, medial basal hypothalamus (the structures responsible for GnRH production) and stalk median eminence (the structure responsible for GnRH release) according to method by Sesti and Britt (1993). All the samples were frozen in liquid nitrogen and stored at 80°C until processing for RNA and protein analysis. Total RNA isolation and cDNA synthesis Total RNA was extracted from hypothalamic tissues using the Absolutety RNA Miniprep Kit (Stratagene, La Jolla, CA, USA). RNA concentration and quality were determined spectrophotometrically (NanoDrop ND-1000, NanoDrop Technologies Inc., Wilmington, DE, USA). Approximately 1 lg of RNA was reverse transcribed into cDNA in a total volume of 20 ll with 0.5 lg oligo(dt)15 primer (Roche, Berlin, Germany) using the Omniscript RT Kit (Qiagen, Valencia, CA, USA) at 37°C for 1 h and was terminated by incubation at 93°C for 5 min.
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Quantitative real-time PCR Quantitative real-time PCR analysis was performed using a PCR System 7300 (Applied Biosystems, Grand Island, NY, USA). The PCR included 20 ng cDNA, one of five primer pairs: adiponectin forward – 900 nM and adiponectin reverse – 300 nM, AdipoR1 forward – 300 nM and AdipoR1 reverse – 50 nM, AdipoR2 forward and reverse – 50 nM, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward and reverse – 500 nM, cyclophilin A forward and reverse – 300 nM as well as 12,5 ll SYBR Green PCR Master Mix (Applied Biosystems) and RNase free water in a final volume of 25 ll. The details concerning primers used in the study are described in Table 1. The constitutively expressed genes, cyclophilin A and GAPDH, were used as the internal control to verify the quantitative real-time PCR. During the preliminary experiments, it was found that expression of cyclophilin A and GAPDH was very similar in MBH, POA and SME (the same Ct values without statistically significant differences) and was stable during the oestrous cycle. Both of the housekeeping genes were indicated as suitable reference genes (internal controls) in the independent studies (Zhong and Simons 1999). Quantitative real-time PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 59° for 1 min. Negative controls were performed in which water was substituted for cDNA, or reverse transcription was not performed prior to PCR. All samples were amplified in duplicate. The specificity of amplification was tested at the end of the PCR by melting-curve analysis. Product purity was confirmed by electrophoresis. Relative expression levels of adiponectin, AdipoR1 and AdipoR2 were calculated based on the comparative cycle threshold method (DDCt) (Livak and Schmittgen 2001) and normalized using the geometrical mean of reference genes expression levels: GAPDH and cyclophilin A. Western blotting Western blotting analysis was performed as described by Smolinska et al. (2007). Briefly, equal amounts of porcine hypothalamus lysates (MBH, POA and SME parts separately, 10 lg of total proteins) were resolved by SDS-PAGE (12.5% gel) for separating adiponectin, AdipoR1, AdipoR2 and actin, and then transferred to
Table 1. The sequence of oligonucleotide primer pairs Gene symbol
Primers pairs
Accession number
Adiponectin
F: 5′-ATGATGTCACCACTGGCAAATTC-3′ R: 5′-GACCGTGACGTGGAAGGAGA-3′ F: 5′-GCCATGGAGAAGATGGAGGA-3′ R: 5′-AGCACGTCGTACGGGATGA-3′ F: 5′-TGTTCGCCACCCCTCAGTAT-3′ R: 5′-AATGATTCCACTCAGGCCCA-3′ F: 5′-GCACTGGTGGCAAGTCCAT-3′ R: 5′-AGGACCCGTATGCTTCAGGA-3′ F: 5′ CCTTCATTGACCTCCACTACATGGT-3′ R: 5′-CCACAACATACGTAGCACCAGCATC-3′
AY135647
Lord et al. (2005)
AY452710
Lord et al. (2005)
AY452711
Lord et al. (2005)
AY266299
Lord et al. (2005)
AdipoR1 AdipoR2 Cyclophilin A GAPDH
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U48832
Reference
Nitkiewicz et al. (2010)
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nitrocellulose membranes (Whatman, Pittsburgh, PA, USA). Blots were blocked for 5 h at 4°C in Tris-buffered saline Tween-20 containing 5% skimmed milk powder, then incubated overnight at 4°C with the rabbit polyclonal adiponectin antibodies at the dilution of 1 : 150 (Santa Cruz Biotechnology, Dallas, TX, USA), the rabbit polyclonal AdipoR1 antibodies at a dilution of 1 : 150 (Phoenix Pharmaceuticals, Burlingame, CA, USA), rabbit polyclonal AdipoR2 antibodies at a dilution of 1 : 200 (Phoenix Pharmaceuticals) or rabbit polyclonal actin antibodies diluted 1 : 200 (Sigma, Sant Louis, MO, USA), which were used as a control for equal loading as well as to quantify porcine adiponectin, AdipoR1 and AdipoR2 proteins. To identify immunoreactive bands, membranes were incubated for 1.5 h at room temperature with mouse anti-rabbit IgG for adiponectin and AdipoR1 (Sigma; diluted 1 : 2000), goat anti-rabbit IgG for AdipoR2 (diluted 1 : 500) or for actin (diluted 1 : 5000) conjugated with alkaline phosphatase (Santa Cruz Biotechnology). Non-specific foetal calf serum (MP Biomedicals, Santa Ana, CA, USA) was used instead of primary antibodies to produce negative control blots. The immunocomplexes were visualized using 4-nitroblue tetrazolium chloride (NBT) and 5bromo-4-chloro-3-indolyl phosphate (BCIP), according to the manufacturer’s protocol (Promega, Madison, WI, USA). The results of Western blotting were quantified by
densitometric scanning of immunoblots with GELSCAN for Windows ver. 1.45 software (Kucharczyk, Poland). Data were expressed as a ratio of adiponectin, AdipoR1 or AdipoR2 protein relative to actin protein in arbitrary optical density units. Data Analysis Data were analysed by one-way ANOVA followed by least significant difference (LSD) post hoc test and are reported as the means SEM from five independent observations. Statistical analysis was performed using the STATISTICA program (StatSoft Inc., Tulsa, OK, USA). Values for p < 0.05 were considered statistically significant.
Results Adiponectin expression Adiponectin mRNA expression in MBH was significantly lower (p < 0.001) on days 14–16 of the oestrous cycle compared with the remaining studied phases of the cycle. In SME, the most pronounced gene expression was found on days 2–3 (p < 0.001), whereas in POA, the differences in the transcript content were not significant. Comparison of the transcript content in MBH, POA
Fig. 1. Comparison of adiponectin mRNA expression determined by quantitative real-time PCR in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Results are means SEM (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
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Adiponectin and its Receptors in Pig Hypothalamus
and SME revealed the highest expression of adiponectin mRNA on days 10–12 and 17–19 in MBH (p < 0.001 and p < 0.05, respectively), and on days 2–3 in SME (p < 0.001). On days 14–16, differences in the gene expression in these three hypothalamic structures were negligible (Fig. 1). Adiponectin protein in MBH was most abundant on days 17–19 (p < 0.001), and it reached the lowest level on days 2–3 and 14–16 of the cycle (p < 0.05). In POA, the enhanced content of the protein was observed on days 2–3, while the lowest on days 10–12 and 14–16 (p < 0.001). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.001) on days 14–16. Comparison of the protein content among MBH, POA and SME was differentiated depending on phase of the cycle: on days 2–3 and 14–16, the highest protein concentration was noted in POA (p < 0.001) on days 10–12 in SME (p < 0.001) and on days 17–19 in MBH (p < 0.05) (Fig. 2). AdipoR1 expression AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.001). In SME, the highest mRNA expression was noted on days
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2–3 and the lowest on days 10–12 and 17–19 of the cycle (p < 0.01). Variations in the gene expression in MBH were negligible. During three of four phases of the cycle, the highest AdipoR1 mRNA level was found in SME relative to MBH and POA (p < 0.01). The exception were days 10–12, where the transcript content was highest in POA (p < 0.01) (Fig. 3). AdipoR1 protein content in POA was similar during the luteal phase (days 2–14 of the cycle), and it reached a decrease on days 17–19 (p < 0.05). In SME, the protein expression peak occurred on days 2–3, whereas the lowest level was observed on days 14–16 (p < 0.001). Similarly to AdipoR1 gene expression in MBH, differences in the protein expression during the cycle were negligible. Variations in AdipoR1 protein content among MBH, POA and SME revealed the highest receptor protein expression in SME throughout the whole cycle (p < 0.001), with exception of days 14–16, where differences were insignificant (Fig. 4). AdipoR2 expression The expression pattern of AdipoR2 gene in MBH, POA and SME was to a high degree similar: the highest level of the mRNA in each of these tissues was found on days 2–3 of the cycle (p < 0.01). The transcript content
Fig. 2. Comparison of adiponectin protein expression determined by Western blotting analysis in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM – molecular marker, AT – adipose tissue used as a positive control); lower panels: densitometric analysis of adiponectin protein relative to actin protein. Values are expressed as means SEM in arbitrary optical density units (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
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Fig. 3. Comparison of adiponectin receptor 1 (AdipoR1) mRNA expression determined by quantitative real-time PCR in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Results are means SEM (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
during the rest of the cycle was constant; only in the case of POA, it was observed higher AdipoR2 gene expression on days 10–12 compared with days 14–16 and 17–19 (p < 0.01). Comparison of AdipoR2 mRNA content between the three studied hypothalamic structures indicated the highest gene expression in POA and SME on days 2–3 and in POA on days 10–12 (p < 0.01). In the remaining phases, the expression level was similar (Fig. 5). AdipoR2 protein content in MBH was the highest on days 2–3 (p < 0.001) and the lowest on days 14–16 of the cycle (p < 0.05). In POA, the protein concentration was stable on days 2–16 and statistically elevated on days 17–19 (p < 0.001). The presence of AdipoR2 protein in SME was more pronounced on days 10–12 and 14–16 and reached the lowest value on days 17–19 (p < 0.001). Unlike AdipoR2 gene expression, comparison of the receptor protein content between MBH, POA and SME revealed the highest protein expression in SME during the four studied stages of the cycle (p < 0.001) (Fig. 6).
Discussion The goal of the present study was to compare the expression levels of genes and proteins of adiponectin and its receptors in porcine hypothalamic structures responsible for GnRH production and secretion during
the oestrous cycle. The concentrations of adiponectin, AdipoR1 and AdipoR2 proteins generally do not reflect the content of specific mRNAs. The above can be attributed to transcriptional and post-transcriptional regulation, translational regulation and, consequently, variations in mRNA and protein stability (Gry et al. 2009; Vogel and Marcotte 2012). The polymorphism of genes encoding porcine adiponectin and its receptors is probably one of the factors that affect mRNA stability (Houde et al. 2008). In previous studies, AdipoR1 and AdipoR2 expression was observed in human and mouse brains (Yamauchi et al. 2003; Neumeier et al. 2007), including the hypothalamus (Kos et al. 2007; Guillod-Maximin et al. 2009). In the hypothalamus, adiponectin receptors were expressed in the pre-optic area, area postrema, arcuate and paraventricular nuclei and the lateral hypothalamus (Yamauchi et al. 2003; Kos et al. 2007; Guillod-Maximin et al. 2009; Psilopanagioti et al. 2009). In immunohistochemical analyses, adiponectin receptors were found in astrocytes and hypothalamic neurons, in particular in pro-opiomelanocortin and neuropeptide Y neurons (Guillod-Maximin et al. 2009). Their expression was also noted in GT1-7 hypothalamic GnRH neurons (Wen et al. 2008) and LbT2 pituitary gonadotroph cells (Lu et al. 2008). This study indicates that locally produced adiponectin can be a source of the ligand for AdipoR1 and © 2014 Blackwell Verlag GmbH
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Fig. 4. Comparison of adiponectin receptor 1 (AdipoR1) protein expression determined by Western blotting analysis in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM – molecular marker, SK – skeletal muscle used as a positive control); lower panels: densitometric analysis of AdipoR1 protein relative to actin protein. Values are expressed as means SEM in arbitrary optical density units (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
AdipoR2. The hypothesis of local adiponectin synthesis is supported by papers describing adiponectin expression in chicken and murine brains (Maddineni et al. 2005; Wilkinson et al. 2007) and in the pituitary glands of rats and humans (Rodriguez-Pacheco et al. 2007). Adiponectin is also supplied to the central nervous system with blood. It was detected in the cerebrospinal fluid of mice (Qi et al. 2004), humans (Kos et al. 2007; Kusminski et al. 2007; Neumeier et al. 2007) and rats (Caja et al. 2005). In every analysed model, adiponectin concentrations in the cerebrospinal fluid were many times lower than in blood plasma. Trimeric adiponectin was the predominant form of adiponectin in the cerebrospinal fluid (Kusminski et al. 2007), which suggests that multimer form is unable to cross the blood– brain barrier. Multiple hormones associated with reproductive processes, including the oestrous cycle, could be responsible for complex regulation of the adiponectin system. However, the obtained results did not fully explain the role of ovarian steroids in control of expression of adiponectin system components. On one hand, expression of AdipoR1 and AdipoR2 mRNAs in most hypothalamic structures was suppressed during the follicular phase, which seems to suggest the downregulating effect of oestrogens. On the other hand, expression © 2014 Blackwell Verlag GmbH
of adiponectin receptors genes was also diminished on days 10–12 of the cycle, when plasma level of oestrogens is very low. Interestingly, content of AdipoR1 and AdipoR2 transcripts in all examined structures was the highest on days 2–3. During this period, both oestrogens and progesterone plasma concentrations are at moderate levels. The role of estrogens in the regulation of adiponectin system is unclear also in women: the inhibitory effect of oestradiol on plasma adiponectin concentrations (Liu et al. 2006) and the lack of any action (Chalvatzas et al. 2009) were both noted. One cannot ruled out that other hormones controlling the oestrous cycle, such as pituitary follicle stimulating hormone (FSH), may be involved in the regulation of adiponectin receptors expression. This hypothesis is in good agreement with reports indicating that the highest plasma level of FSH takes place on day 3 of the pig oestrous cycle (Rayford et al. 1974; Van de Wiel et al. 1981). Nevertheless, this hypothesis should be verified in further studies. Fluctuations in adiponectin and its receptors expression throughout the oestrous cycle were also noticed in other porcine tissues (Kiezun et al. 2013) and in other species. In a study of rat pituitaries (Kim et al. 2013), changes were observed in the levels of adiponectin and AdipoR2 mRNAs, but not in the content of AdipoR1 genes during the oestrous
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Fig. 5. Comparison of adiponectin receptor 2 (AdipoR2) mRNA expression determined by quantitative real-time PCR in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Results are means SEM (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
cycle. Similar findings were reported by Takemura et al. (2006) who noted increased levels of AdipoR1 and AdipoR2 mRNAs in the human endometrium during the mid-secretory phase of the menstrual cycle in comparison with other stages of the cycle. The hypothesis that gonadal steroids control adiponectin synthesis is confirmed by sexual dimorphism in plasma adiponectin concentrations. Adiponectin levels are lower in men than in women (Arita et al. 1999), and they continue to decrease in both sexes throughout puberty (B€ ottner et al. 2004). The observed gender differences in adiponectin levels can probably be attributed to fluctuations in androgen levels because negative correlations were reported between the concentrations of plasma testosterone and adiponectin in humans and mice (Nishizawa et al. 2002; B€ ottner et al. 2004; Lanfranco et al. 2004; Page et al. 2005). An increase in the levels of circulating adiponectin in women receiving hCG and a drop in adiponectin levels in patients administered FSH were also observed (Liu et al. 2006). There is a general scarcity of research data describing local regulation of adiponectin and its receptors expression in nervous and pituitary tissues. Such studies were performed on immortalized LbT2 pituitary gonadotroph cells and primary pituitary cells of male rats (Kim et al. 2013). In both culture models, GnRH inhibited the expression of adiponectin mRNA and had no effect on adiponectin receptors. The expression of adiponectin
and adiponectin receptors in hypothalamic structures responsible for GnRH synthesis and secretion seems to suggest that the discussed hormone controls GnRH generation. This hypothesis is validated by the inhibition of GnRH release from GT1-7 hypothalamic neurons under the influence of adiponectin (Wen et al. 2008, 2012). The inhibitory effect of adiponectin was at least partly achieved by reducing Kiss1 gene transcription in AMPK-dependent manner (Wen et al. 2012). Adiponectin was also found to lower GnRH secretions by activating the AMPK pathway and inhibiting the ERK pathway in an in vivo study (Cheng et al. 2011). The drop in plasma LH concentration in male rats administered intracerebroventricular infusions of adiponectin confirms the above observations (Cheng et al. 2011). In conclusion, the expression of adiponectin and its receptors in porcine hypothalamic structures responsible for GnRH production and secretion suggests that the analysed hormone exerts autocrine/paracrine effects on GnRH synthesis and/or secretion. The variations in the expression levels of adiponectin and its receptor in the hypothalamus observed during the oestrous cycle could be attributed to the influence of ovarian steroids and other hormones controlling reproductive processes. The results of this study confirm the potential role of adiponectin as a key neuromodulator of reproductive functions. © 2014 Blackwell Verlag GmbH
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Fig. 6. Comparison of adiponectin receptor 2 (AdipoR2) protein expression determined by Western blotting analysis in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM – molecular marker, L – liver used as a positive control); lower panels: densitometric analysis of AdipoR2 protein relative to actin protein. Values are expressed as means SEM in arbitrary optical density units (n = 5). Bars with different superscripts within panels a, b, c and within days 2– 3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
Acknowledgement
Author contributions
This research was supported by National Science Centre (Project No. 2011/01/B/NZ4/01596).
T. Kaminski designed and coordinated the study, drafted the manuscript; N. Smolinska, A. Maleszka, M. Kiezun, K. Dobrzyn, J. Czerwinska, K. Szeszko and A. Nitkiewicz participated in mRNA and protein isolation, and carried out real-time PCR and Western blot analyses. All authors have read and approved the final version of the manuscript.
Conflict of interest None of the authors have any conflict of interest to declare.
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Submitted: 4 Oct 2013; Accepted: 30 Dec 2013 Author’s address (for correspondence): T Kaminski, Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn-Kortowo, Poland. E-mail: [email protected]
© 2014 Blackwell Verlag GmbH
Expression of Adiponectin and its Receptors in the Porcine Hypothalamus During the Oestrous Cycle T Kaminski, N Smolinska, A Maleszka, M Kiezun, K Dobrzyn, J Czerwinska, K Szeszko and A Nitkiewicz Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Contents Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14–16, whereas in SME, the most pronounced gene expression was found on days 2–3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17–19 and in POA on days 2–3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14–16. AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2–3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2–14 of the cycle), and they decreased on days 17–19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2–3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2–3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2–3 (p < 0.05), while in POA on days 17–19 and in SME on days 10–12 and 14–16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig’s endocrine status related to the oestrous cycle.
Introduction Adiponectin is one of the adipocytokines highly expressed in the adipose tissue and is one of the most abundant plasma proteins (2–25 lg/ml) in mammals (Nishizawa et al. 2002; B€ ottner et al. 2004). Adiponectin circulates in the serum in different multimeric forms, including trimers, hexamers and high-molecular-weight multimers, and as the globular fraction (globular adiponectin) generated by the proteolytic cleavage of multimers. Adiponectin controls energy homeostasis and insulin sensitivity, and it affects lipid synthesis, vasodilatation and atherogenic activity (Yamauchi et al. 2002; Trujillo and Scherer 2005). Plasma adiponectin concentrations are inversely correlated with the adipose tissue reservoir (Arita et al. 1999). Adiponectin is also identified in cerebrospinal fluid (Kos et al. 2007), and the hormone levels increase after its intravenous injection (Qi et al. 2004).
Adiponectin actions are mediated by two types of receptors: AdipoR1 and AdipoR2. Unlike G-coupled protein receptors, AdipoR1 and AdipoR2 are seventransmembrane domain receptors with an extracellular carboxyl terminus and an intracellular amino terminus. AdipoR1 shows high affinity for the globular form of adiponectin, and AdipoR2 has an intermediate binding affinity for both full-length and globular species. The receptors are highly related and share 66.7% sequence identity in mice (Yamauchi et al. 2003). AdipoR1 and AdipoR2 are widely expressed in humans and rodents, which suggests that adiponectin has pleiotropic effects. The highest levels of AdipoR1 expression are observed in skeletal muscles, whereas AdipoR2 is most abundantly expressed in the liver (Yamauchi et al. 2003; Lord et al. 2005). The presence of the adiponectin receptors was observed in human and rodent hypothalami, including in the paraventricular nucleus (Kos et al. 2007; Guillod-Maximin et al. 2009), in periventricular areas and the pituitary (Yamauchi et al. 2003; RodriguezPacheco et al. 2007; Psilopanagioti et al. 2009). Both types of receptors were also expressed in GT1-7 hypothalamic GnRH neuron cells (Wen et al. 2008). The expression of adiponectin and its receptors in species other than humans and rodents remains largely unexplored. Adiponectin seems to be a hormonal link between obesity and reproduction. It was described as a factor affecting ovarian steroidogenesis, oocyte maturation, embryo implantation and development (Palin et al. 2012). Several lines of evidence also suggest that adiponectin influences the reproductive system by exerting central effects on the highest branch of the hypothalamic–pituitary–ovarian axis, inhibiting GnRH (Wen et al. 2008) and GnRH-induced LH secretion (Rodriguez-Pacheco et al. 2007; Lu et al. 2008). To date, there has been no research investigating the gene and protein expression patterns of adiponectin and its receptors in porcine hypothalamic structures responsible for GnRH production and secretion, including the mediobasal hypothalamus (MBH), pre-optic area (POA) and stalk median eminence (SME), or the effect of the oestrous cycle on transcript and protein levels in animals. The objective of this study was to compare the expression levels of adiponectin, AdipoR1 and AdipoR2 mRNAs and proteins in selected areas of the porcine hypothalamus during the oestrous cycle.
Materials and Methods Animals and tissue collection This study was carried out in accordance with ethical standards of the Animal Ethics Committee at the © 2014 Blackwell Verlag GmbH
Adiponectin and its Receptors in Pig Hypothalamus
University of Warmia and Mazury in Olsztyn. Mature gilts (Large White 9 Polish Landrace), 7–8 months old, weighing 120–130 kg, descended from private breeding were used. Diets were balanced (crude protein, metabolisable energy, exogenous amino acids and minerals) in accordance with the nutrient requirements of domestic pigs. Twenty gilts were divided into four experimental groups (n = 5 per group) as follows: days 2–3 (the early luteal phase), 10–12 (the mid-luteal phase), 14–16 (the late luteal phase) and 17–19 (the follicular phase) of the oestrous cycle. The analysed days are representative of the stages of the oestrous cycle characterized by major hormonal and physiological changes observed throughout the cycle (Akins and Morrissette 1968; Henricks et al. 1972). Females were monitored daily for oestrous behaviour in the presence of an intact boar. The day of the second oestrous onset was designated as day 0 of the oestrous cycle. The phase of the oestrous cycle was also confirmed on the basis of ovarian morphology, and highly characteristic ovarian morphology was noted in each of the analysed periods with the presence of corpora haemorrhagica on days 2–3, fully active corpora lutea on days 10–12, corpora lutea with clear signs of luteolysis on days 14–16 and only corpora albicantia with pre-ovulatory follicles on days 17–19 (Akins and Morrissette 1968). Within a few minutes after the slaughter, the hypothalamus, muscle, liver and subcutaneous adipose tissue were removed. The hypothalamus has been divided into the pre-optic area, medial basal hypothalamus (the structures responsible for GnRH production) and stalk median eminence (the structure responsible for GnRH release) according to method by Sesti and Britt (1993). All the samples were frozen in liquid nitrogen and stored at 80°C until processing for RNA and protein analysis. Total RNA isolation and cDNA synthesis Total RNA was extracted from hypothalamic tissues using the Absolutety RNA Miniprep Kit (Stratagene, La Jolla, CA, USA). RNA concentration and quality were determined spectrophotometrically (NanoDrop ND-1000, NanoDrop Technologies Inc., Wilmington, DE, USA). Approximately 1 lg of RNA was reverse transcribed into cDNA in a total volume of 20 ll with 0.5 lg oligo(dt)15 primer (Roche, Berlin, Germany) using the Omniscript RT Kit (Qiagen, Valencia, CA, USA) at 37°C for 1 h and was terminated by incubation at 93°C for 5 min.
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Quantitative real-time PCR Quantitative real-time PCR analysis was performed using a PCR System 7300 (Applied Biosystems, Grand Island, NY, USA). The PCR included 20 ng cDNA, one of five primer pairs: adiponectin forward – 900 nM and adiponectin reverse – 300 nM, AdipoR1 forward – 300 nM and AdipoR1 reverse – 50 nM, AdipoR2 forward and reverse – 50 nM, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward and reverse – 500 nM, cyclophilin A forward and reverse – 300 nM as well as 12,5 ll SYBR Green PCR Master Mix (Applied Biosystems) and RNase free water in a final volume of 25 ll. The details concerning primers used in the study are described in Table 1. The constitutively expressed genes, cyclophilin A and GAPDH, were used as the internal control to verify the quantitative real-time PCR. During the preliminary experiments, it was found that expression of cyclophilin A and GAPDH was very similar in MBH, POA and SME (the same Ct values without statistically significant differences) and was stable during the oestrous cycle. Both of the housekeeping genes were indicated as suitable reference genes (internal controls) in the independent studies (Zhong and Simons 1999). Quantitative real-time PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 59° for 1 min. Negative controls were performed in which water was substituted for cDNA, or reverse transcription was not performed prior to PCR. All samples were amplified in duplicate. The specificity of amplification was tested at the end of the PCR by melting-curve analysis. Product purity was confirmed by electrophoresis. Relative expression levels of adiponectin, AdipoR1 and AdipoR2 were calculated based on the comparative cycle threshold method (DDCt) (Livak and Schmittgen 2001) and normalized using the geometrical mean of reference genes expression levels: GAPDH and cyclophilin A. Western blotting Western blotting analysis was performed as described by Smolinska et al. (2007). Briefly, equal amounts of porcine hypothalamus lysates (MBH, POA and SME parts separately, 10 lg of total proteins) were resolved by SDS-PAGE (12.5% gel) for separating adiponectin, AdipoR1, AdipoR2 and actin, and then transferred to
Table 1. The sequence of oligonucleotide primer pairs Gene symbol
Primers pairs
Accession number
Adiponectin
F: 5′-ATGATGTCACCACTGGCAAATTC-3′ R: 5′-GACCGTGACGTGGAAGGAGA-3′ F: 5′-GCCATGGAGAAGATGGAGGA-3′ R: 5′-AGCACGTCGTACGGGATGA-3′ F: 5′-TGTTCGCCACCCCTCAGTAT-3′ R: 5′-AATGATTCCACTCAGGCCCA-3′ F: 5′-GCACTGGTGGCAAGTCCAT-3′ R: 5′-AGGACCCGTATGCTTCAGGA-3′ F: 5′ CCTTCATTGACCTCCACTACATGGT-3′ R: 5′-CCACAACATACGTAGCACCAGCATC-3′
AY135647
Lord et al. (2005)
AY452710
Lord et al. (2005)
AY452711
Lord et al. (2005)
AY266299
Lord et al. (2005)
AdipoR1 AdipoR2 Cyclophilin A GAPDH
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U48832
Reference
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nitrocellulose membranes (Whatman, Pittsburgh, PA, USA). Blots were blocked for 5 h at 4°C in Tris-buffered saline Tween-20 containing 5% skimmed milk powder, then incubated overnight at 4°C with the rabbit polyclonal adiponectin antibodies at the dilution of 1 : 150 (Santa Cruz Biotechnology, Dallas, TX, USA), the rabbit polyclonal AdipoR1 antibodies at a dilution of 1 : 150 (Phoenix Pharmaceuticals, Burlingame, CA, USA), rabbit polyclonal AdipoR2 antibodies at a dilution of 1 : 200 (Phoenix Pharmaceuticals) or rabbit polyclonal actin antibodies diluted 1 : 200 (Sigma, Sant Louis, MO, USA), which were used as a control for equal loading as well as to quantify porcine adiponectin, AdipoR1 and AdipoR2 proteins. To identify immunoreactive bands, membranes were incubated for 1.5 h at room temperature with mouse anti-rabbit IgG for adiponectin and AdipoR1 (Sigma; diluted 1 : 2000), goat anti-rabbit IgG for AdipoR2 (diluted 1 : 500) or for actin (diluted 1 : 5000) conjugated with alkaline phosphatase (Santa Cruz Biotechnology). Non-specific foetal calf serum (MP Biomedicals, Santa Ana, CA, USA) was used instead of primary antibodies to produce negative control blots. The immunocomplexes were visualized using 4-nitroblue tetrazolium chloride (NBT) and 5bromo-4-chloro-3-indolyl phosphate (BCIP), according to the manufacturer’s protocol (Promega, Madison, WI, USA). The results of Western blotting were quantified by
densitometric scanning of immunoblots with GELSCAN for Windows ver. 1.45 software (Kucharczyk, Poland). Data were expressed as a ratio of adiponectin, AdipoR1 or AdipoR2 protein relative to actin protein in arbitrary optical density units. Data Analysis Data were analysed by one-way ANOVA followed by least significant difference (LSD) post hoc test and are reported as the means SEM from five independent observations. Statistical analysis was performed using the STATISTICA program (StatSoft Inc., Tulsa, OK, USA). Values for p < 0.05 were considered statistically significant.
Results Adiponectin expression Adiponectin mRNA expression in MBH was significantly lower (p < 0.001) on days 14–16 of the oestrous cycle compared with the remaining studied phases of the cycle. In SME, the most pronounced gene expression was found on days 2–3 (p < 0.001), whereas in POA, the differences in the transcript content were not significant. Comparison of the transcript content in MBH, POA
Fig. 1. Comparison of adiponectin mRNA expression determined by quantitative real-time PCR in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Results are means SEM (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
© 2014 Blackwell Verlag GmbH
Adiponectin and its Receptors in Pig Hypothalamus
and SME revealed the highest expression of adiponectin mRNA on days 10–12 and 17–19 in MBH (p < 0.001 and p < 0.05, respectively), and on days 2–3 in SME (p < 0.001). On days 14–16, differences in the gene expression in these three hypothalamic structures were negligible (Fig. 1). Adiponectin protein in MBH was most abundant on days 17–19 (p < 0.001), and it reached the lowest level on days 2–3 and 14–16 of the cycle (p < 0.05). In POA, the enhanced content of the protein was observed on days 2–3, while the lowest on days 10–12 and 14–16 (p < 0.001). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.001) on days 14–16. Comparison of the protein content among MBH, POA and SME was differentiated depending on phase of the cycle: on days 2–3 and 14–16, the highest protein concentration was noted in POA (p < 0.001) on days 10–12 in SME (p < 0.001) and on days 17–19 in MBH (p < 0.05) (Fig. 2). AdipoR1 expression AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.001). In SME, the highest mRNA expression was noted on days
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2–3 and the lowest on days 10–12 and 17–19 of the cycle (p < 0.01). Variations in the gene expression in MBH were negligible. During three of four phases of the cycle, the highest AdipoR1 mRNA level was found in SME relative to MBH and POA (p < 0.01). The exception were days 10–12, where the transcript content was highest in POA (p < 0.01) (Fig. 3). AdipoR1 protein content in POA was similar during the luteal phase (days 2–14 of the cycle), and it reached a decrease on days 17–19 (p < 0.05). In SME, the protein expression peak occurred on days 2–3, whereas the lowest level was observed on days 14–16 (p < 0.001). Similarly to AdipoR1 gene expression in MBH, differences in the protein expression during the cycle were negligible. Variations in AdipoR1 protein content among MBH, POA and SME revealed the highest receptor protein expression in SME throughout the whole cycle (p < 0.001), with exception of days 14–16, where differences were insignificant (Fig. 4). AdipoR2 expression The expression pattern of AdipoR2 gene in MBH, POA and SME was to a high degree similar: the highest level of the mRNA in each of these tissues was found on days 2–3 of the cycle (p < 0.01). The transcript content
Fig. 2. Comparison of adiponectin protein expression determined by Western blotting analysis in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM – molecular marker, AT – adipose tissue used as a positive control); lower panels: densitometric analysis of adiponectin protein relative to actin protein. Values are expressed as means SEM in arbitrary optical density units (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
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Fig. 3. Comparison of adiponectin receptor 1 (AdipoR1) mRNA expression determined by quantitative real-time PCR in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Results are means SEM (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
during the rest of the cycle was constant; only in the case of POA, it was observed higher AdipoR2 gene expression on days 10–12 compared with days 14–16 and 17–19 (p < 0.01). Comparison of AdipoR2 mRNA content between the three studied hypothalamic structures indicated the highest gene expression in POA and SME on days 2–3 and in POA on days 10–12 (p < 0.01). In the remaining phases, the expression level was similar (Fig. 5). AdipoR2 protein content in MBH was the highest on days 2–3 (p < 0.001) and the lowest on days 14–16 of the cycle (p < 0.05). In POA, the protein concentration was stable on days 2–16 and statistically elevated on days 17–19 (p < 0.001). The presence of AdipoR2 protein in SME was more pronounced on days 10–12 and 14–16 and reached the lowest value on days 17–19 (p < 0.001). Unlike AdipoR2 gene expression, comparison of the receptor protein content between MBH, POA and SME revealed the highest protein expression in SME during the four studied stages of the cycle (p < 0.001) (Fig. 6).
Discussion The goal of the present study was to compare the expression levels of genes and proteins of adiponectin and its receptors in porcine hypothalamic structures responsible for GnRH production and secretion during
the oestrous cycle. The concentrations of adiponectin, AdipoR1 and AdipoR2 proteins generally do not reflect the content of specific mRNAs. The above can be attributed to transcriptional and post-transcriptional regulation, translational regulation and, consequently, variations in mRNA and protein stability (Gry et al. 2009; Vogel and Marcotte 2012). The polymorphism of genes encoding porcine adiponectin and its receptors is probably one of the factors that affect mRNA stability (Houde et al. 2008). In previous studies, AdipoR1 and AdipoR2 expression was observed in human and mouse brains (Yamauchi et al. 2003; Neumeier et al. 2007), including the hypothalamus (Kos et al. 2007; Guillod-Maximin et al. 2009). In the hypothalamus, adiponectin receptors were expressed in the pre-optic area, area postrema, arcuate and paraventricular nuclei and the lateral hypothalamus (Yamauchi et al. 2003; Kos et al. 2007; Guillod-Maximin et al. 2009; Psilopanagioti et al. 2009). In immunohistochemical analyses, adiponectin receptors were found in astrocytes and hypothalamic neurons, in particular in pro-opiomelanocortin and neuropeptide Y neurons (Guillod-Maximin et al. 2009). Their expression was also noted in GT1-7 hypothalamic GnRH neurons (Wen et al. 2008) and LbT2 pituitary gonadotroph cells (Lu et al. 2008). This study indicates that locally produced adiponectin can be a source of the ligand for AdipoR1 and © 2014 Blackwell Verlag GmbH
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Fig. 4. Comparison of adiponectin receptor 1 (AdipoR1) protein expression determined by Western blotting analysis in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM – molecular marker, SK – skeletal muscle used as a positive control); lower panels: densitometric analysis of AdipoR1 protein relative to actin protein. Values are expressed as means SEM in arbitrary optical density units (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
AdipoR2. The hypothesis of local adiponectin synthesis is supported by papers describing adiponectin expression in chicken and murine brains (Maddineni et al. 2005; Wilkinson et al. 2007) and in the pituitary glands of rats and humans (Rodriguez-Pacheco et al. 2007). Adiponectin is also supplied to the central nervous system with blood. It was detected in the cerebrospinal fluid of mice (Qi et al. 2004), humans (Kos et al. 2007; Kusminski et al. 2007; Neumeier et al. 2007) and rats (Caja et al. 2005). In every analysed model, adiponectin concentrations in the cerebrospinal fluid were many times lower than in blood plasma. Trimeric adiponectin was the predominant form of adiponectin in the cerebrospinal fluid (Kusminski et al. 2007), which suggests that multimer form is unable to cross the blood– brain barrier. Multiple hormones associated with reproductive processes, including the oestrous cycle, could be responsible for complex regulation of the adiponectin system. However, the obtained results did not fully explain the role of ovarian steroids in control of expression of adiponectin system components. On one hand, expression of AdipoR1 and AdipoR2 mRNAs in most hypothalamic structures was suppressed during the follicular phase, which seems to suggest the downregulating effect of oestrogens. On the other hand, expression © 2014 Blackwell Verlag GmbH
of adiponectin receptors genes was also diminished on days 10–12 of the cycle, when plasma level of oestrogens is very low. Interestingly, content of AdipoR1 and AdipoR2 transcripts in all examined structures was the highest on days 2–3. During this period, both oestrogens and progesterone plasma concentrations are at moderate levels. The role of estrogens in the regulation of adiponectin system is unclear also in women: the inhibitory effect of oestradiol on plasma adiponectin concentrations (Liu et al. 2006) and the lack of any action (Chalvatzas et al. 2009) were both noted. One cannot ruled out that other hormones controlling the oestrous cycle, such as pituitary follicle stimulating hormone (FSH), may be involved in the regulation of adiponectin receptors expression. This hypothesis is in good agreement with reports indicating that the highest plasma level of FSH takes place on day 3 of the pig oestrous cycle (Rayford et al. 1974; Van de Wiel et al. 1981). Nevertheless, this hypothesis should be verified in further studies. Fluctuations in adiponectin and its receptors expression throughout the oestrous cycle were also noticed in other porcine tissues (Kiezun et al. 2013) and in other species. In a study of rat pituitaries (Kim et al. 2013), changes were observed in the levels of adiponectin and AdipoR2 mRNAs, but not in the content of AdipoR1 genes during the oestrous
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Fig. 5. Comparison of adiponectin receptor 2 (AdipoR2) mRNA expression determined by quantitative real-time PCR in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Results are means SEM (n = 5). Bars with different superscripts within panels a, b, c and within days 2–3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
cycle. Similar findings were reported by Takemura et al. (2006) who noted increased levels of AdipoR1 and AdipoR2 mRNAs in the human endometrium during the mid-secretory phase of the menstrual cycle in comparison with other stages of the cycle. The hypothesis that gonadal steroids control adiponectin synthesis is confirmed by sexual dimorphism in plasma adiponectin concentrations. Adiponectin levels are lower in men than in women (Arita et al. 1999), and they continue to decrease in both sexes throughout puberty (B€ ottner et al. 2004). The observed gender differences in adiponectin levels can probably be attributed to fluctuations in androgen levels because negative correlations were reported between the concentrations of plasma testosterone and adiponectin in humans and mice (Nishizawa et al. 2002; B€ ottner et al. 2004; Lanfranco et al. 2004; Page et al. 2005). An increase in the levels of circulating adiponectin in women receiving hCG and a drop in adiponectin levels in patients administered FSH were also observed (Liu et al. 2006). There is a general scarcity of research data describing local regulation of adiponectin and its receptors expression in nervous and pituitary tissues. Such studies were performed on immortalized LbT2 pituitary gonadotroph cells and primary pituitary cells of male rats (Kim et al. 2013). In both culture models, GnRH inhibited the expression of adiponectin mRNA and had no effect on adiponectin receptors. The expression of adiponectin
and adiponectin receptors in hypothalamic structures responsible for GnRH synthesis and secretion seems to suggest that the discussed hormone controls GnRH generation. This hypothesis is validated by the inhibition of GnRH release from GT1-7 hypothalamic neurons under the influence of adiponectin (Wen et al. 2008, 2012). The inhibitory effect of adiponectin was at least partly achieved by reducing Kiss1 gene transcription in AMPK-dependent manner (Wen et al. 2012). Adiponectin was also found to lower GnRH secretions by activating the AMPK pathway and inhibiting the ERK pathway in an in vivo study (Cheng et al. 2011). The drop in plasma LH concentration in male rats administered intracerebroventricular infusions of adiponectin confirms the above observations (Cheng et al. 2011). In conclusion, the expression of adiponectin and its receptors in porcine hypothalamic structures responsible for GnRH production and secretion suggests that the analysed hormone exerts autocrine/paracrine effects on GnRH synthesis and/or secretion. The variations in the expression levels of adiponectin and its receptor in the hypothalamus observed during the oestrous cycle could be attributed to the influence of ovarian steroids and other hormones controlling reproductive processes. The results of this study confirm the potential role of adiponectin as a key neuromodulator of reproductive functions. © 2014 Blackwell Verlag GmbH
Adiponectin and its Receptors in Pig Hypothalamus
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Fig. 6. Comparison of adiponectin receptor 2 (AdipoR2) protein expression determined by Western blotting analysis in porcine medial basal hypothalamus – MBH (a), pre-optic area – POA (b), stalk median eminence – SME (c) between days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle and (d) between MBH, POA and SME on days 2–3, 10–12, 14–16 and 17–19 of the cycle. Upper panels: representative immunoblots (MM – molecular marker, L – liver used as a positive control); lower panels: densitometric analysis of AdipoR2 protein relative to actin protein. Values are expressed as means SEM in arbitrary optical density units (n = 5). Bars with different superscripts within panels a, b, c and within days 2– 3, 10–12, 14–16 and 17–19 in panel d are significantly different (p < 0.05)
Acknowledgement
Author contributions
This research was supported by National Science Centre (Project No. 2011/01/B/NZ4/01596).
T. Kaminski designed and coordinated the study, drafted the manuscript; N. Smolinska, A. Maleszka, M. Kiezun, K. Dobrzyn, J. Czerwinska, K. Szeszko and A. Nitkiewicz participated in mRNA and protein isolation, and carried out real-time PCR and Western blot analyses. All authors have read and approved the final version of the manuscript.
Conflict of interest None of the authors have any conflict of interest to declare.
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Submitted: 4 Oct 2013; Accepted: 30 Dec 2013 Author’s address (for correspondence): T Kaminski, Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, Oczapowskiego 1A, 10-719 Olsztyn-Kortowo, Poland. E-mail: [email protected]
© 2014 Blackwell Verlag GmbH
