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Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk
ARTICLE IN PRESS The Veterinary Journal ■■ (2015) ■■–■■
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The Veterinary Journal j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t v j l
Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk Cristina Lecchi a,b,*, Chiara Giudice a, Martina Uggè a, Alessio Scarafoni b,c, Antonella Baldi b,d, Paola Sartorelli a,b a
Department of Veterinary Sciences and Public Health, Università degli Studi di Milano, 20133 Milan, Italy Centro Interdipartimentale per lo Studio sulla Ghiandola Mammaria (CISMA), Università degli Studi di Milano, 20133 Milan, Italy c Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, 20133 Milan, Italy d Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, 20133 Milan, Italy b
A R T I C L E
I N F O
Article history: Accepted 10 January 2015 Keywords: Adipokine Adiponectin Cattle Mammary gland Milk
A B S T R A C T
Adiponectin is an adipocyte-derived hormone, which circulates in the form of homo-multimers. The individual oligomers have a distinct profile of activity, playing crucial roles in several biological processes, including metabolism and inflammation. Adiponectin exerts many of its effects by interacting with the receptors, AdipoR1 and AdipoR2. In the present study, mRNA expression of adiponectin, AdipoR1 and AdipoR2 was evaluated by quantitative PCR in different areas of the mammary gland in healthy lactating cows. The adiponectin isoforms in milk and blood were investigated by Western blotting and 2D-electrophoresis, and the presence of adiponectin protein was determined by immunohistochemistry. Low level expression of adiponectin mRNA was found in all areas of bovine mammary gland tissues examined. AdipoR1 and AdipoR2 mRNAs were also detected in mammary tissues and their expression was particularly prominent in the parenchyma and cistern. Western blotting revealed a heterogeneous electrophoretic pattern, indicating that different adiponectin isoforms exist in milk, compared with blood. In particular, milk shows a low molecular weight isoform of adiponectin, corresponding to the globular domain. Adiponectin in milk is characterised by a more complex 2D electrophoretic pattern, compared with blood, as illustrated by the presence of proteins of different molecular weights and isoelectric points. Adiponectin protein was detected by immunohistochemistry in epithelial cells lining the secretory alveoli, in secretum within the alveolar lumen and in small peripheral nerves. The study findings support a role for adiponectin in regulating metabolism and immunity of the bovine mammary gland and potentially the calf intestine, following ingestion of milk. © 2015 Elsevier Ltd. All rights reserved.
Introduction Adiponectin (AdipoQ) is a hormone, produced almost exclusively by adipocytes, which is present in serum at concentrations that are inversely related to the degree of hypertrophy of adipose tissue. In addition, circulating AdipoQ usually shows an inverse relationship with insulin, triglycerides, very low density lipoproteins (VLDL) and pro-inflammatory cytokines (Matsubara et al., 2002). AdipoQ has anti-inflammatory effects and demonstrates other properties, including insulin-sensitisation of tissues and vascular protection (Yamauchi et al., 2002; Fantuzzi, 2013). AdipoQ belongs to the complement 1q (C1q) family of proteins (Scherer et al., 1995). Its primary structure is highly conserved, with over 80% amino acid sequence identity comparing mammalian species (Wang et al., 2002). Bovine AdipoQ is a 240 amino acid
* Corresponding author. Tel.: +39 025 031 8100. E-mail address: [email protected] (C. Lecchi).
protein of approximately 28–30 kDa and circulates as homomultimers, which vary from low molecular weight trimers to high molecular weight (12- to 18-mer) oligomers (Wang et al., 2004; Tsao, 2014). The protein undergoes extensive post-translational modification, such as hydroxylation and hydroxyl-glycosylation (Wang et al., 2002; Richards et al., 2006, 2010). AdipoQ exerts many of its biological effects by binding to the receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2), which have distinct distribution patterns in different tissues. AdipoR1 is abundantly expressed in skeletal muscle and is linked to activation of cAMP-mediated kinase pathways, whereas AdipoR2 is abundantly expressed in the liver and is associated with activation of PPARα pathways (Kadowaki and Yamauchi, 2005). Both AdipoR1 and AdipoR2 are involved in regulating energy metabolism in these tissues. Expression of AdipoQ has been studied in the adipose tissue of cattle during the transition period (Lemor et al., 2009; Ohtani et al., 2012), particularly in the mammary gland (Ohtani et al., 2011) and ovaries, including follicles, oocytes (Tabandeh et al., 2012) and
http://dx.doi.org/10.1016/j.tvjl.2015.01.009 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cristina Lecchi, Chiara Giudice, Martina Uggè, Alessio Scarafoni, Antonella Baldi, Paola Sartorelli, Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.01.009
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granulosa cells (Maillard et al., 2010). AdipoQ has also been quantified in bovine plasma (mean ± SD of 32 ± 1.0 μg/mL) and in milk (0.61 ± 0.03 μg/mL) (Singh et al., 2014). The aim of the present study was to investigate the distribution pattern of AdipoQ and AdipoRs in the mammary gland of lactating cows to elucidate the possible role of adiponectin on mammary gland health.
10 mg/mL DTT for 15 min, then with 25 mg/mL iodoacetamide in the same buffer without DTT for 10 min. Second dimension separation was performed using a MiniProtean III cell (Bio-Rad) with a 12% polyacrylamide gel (acrylamide:bis-acrylamide 37.5:1) at 16 mA constant current for 90 min. Electrotransfer onto nitrocellulose membranes (Whatmann Protran BA85) was performed for 75 min at 50 mA with a semi-dry apparatus (GE Healthcare) using 39 mM glycine, 48 mM Tris-HCl (pH 9.0), 1.3 mM SDS and 12% methanol as transfer buffer.
Materials and methods
Immunohistochemistry
Sample collection
Formalin-fixed, paraffin-embedded mammary and adipose tissues were routinely processed for histology. Five micrometre sections were obtained from paraffin blocks and mounted on polylysine-coated slides. Immunolabelling was performed by the standard ABC method (Hsu et al., 1981). Primary anti-adiponectin antibody (diluted 1: 2000 in Tris buffer) was incubated overnight at 4 °C. Immunolabelling was revealed using 3-amino, 9 ethyl-carbazole (AEC; Vector Laboratories) as the chromogen and sections were counterstained with Mayer’s haematoxylin. As a negative control, primary antibody was replaced with Tris buffer. The presence of immunostaining (positive or negative) with anti-adiponectin antibody and its localisation within the mammary and adipose tissue were assessed by light microscopy.
Blood and milk samples were collected from clinically healthy multiparous Holstein-Friesian cows, aged between 3 and 8 years, obtained 4–10 weeks after calving as part of routine health monitoring. Samples were made available for research following completion of diagnostic testing. Whole blood was collected via coccygeal venepuncture using tubes coated with clot activator and silicone. Serum was obtained by centrifugation of clotted blood at 1200 g for 10 min at 4 °C, which was stored in aliquots at −80 °C. Milk whey was prepared from milk with a somatic cell count (SCC) < 250,000 cells/mL. Ten millilitres of milk were centrifuged at 1200 g for 15 min at 4 °C to remove fat and cells, then the whey was further centrifuged at 13,000 g at room temperature and stored in aliquots at −80 °C. Mammary tissue samples were obtained from six cows immediately after routine slaughtering procedures. Representative tissues of each area of the mammary gland were sampled and stored in RNAlater (Sigma-Aldrich) at −80 °C before RNA extraction. For the immunohistochemical study, samples of mammary tissues of approximately 1 cm × 1 cm were collected and fixed in 10% buffered formalin. Samples of subcutaneous adipose tissue, to be used as a positive control, were similarly collected and formalin fixed. Real-time quantitative PCR Total RNA was extracted from tissue lysates in TRIzol reagent (Invitrogen), according to the manufacturer’s recommendations. RNA concentrations were quantified by use of the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The purity of RNA (A260/A280) was ~2. Genomic DNA was eliminated using DNase I (Invitrogen) and reverse transcription was performed with 1 μg RNA as the template, using the iSCRIPT cDNA Synthesis Kit (BioRad). Quantitative PCR was performed in 12 μL reactions, using the Eco Real Time PCR detection System (Illumina). Each reaction contained Eva Green mix (BioRad) and 400 nM of primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), AdipoR1 and AdipoR2 or 300 nM primers specific for AdipoQ (see Appendix: Supplementary material). The thermocycler conditions consisted of 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 8 s and 60 °C for 18 s. For assessment of melting curves, PCR products were incubated at 55 °C for 60 s then the temperature was increased to 95 °C at 0.5 °C increments for 10 s. The MIQE guidelines (Bustin et al., 2009) were followed. The PCR efficiencies were determined using four-fold serial dilutions of cDNA, prepared from adipose tissue, performed in triplicate. cDNA samples from mammary tissue were assessed in duplicate. Non-reverse transcribed controls and no template controls were included in the assays. Three reference genes (GAPDH, ACTB, YWHAZ) (Lecchi et al., 2012) were selected and the geometric mean of reference gene abundance was used for normalisation purposes. Relative quantification of genes of interest was carried out with adipose tissue used as the reference sample for mammary gland tissues. Western blotting A rabbit anti-human AdipoQ polyclonal antibody (PAI-84881, Pierce Biotechnology), raised against a peptide located near the carboxy-terminal globular domain, was used for experiments. Cross-reactivity of this antibody with bovine AdipoQ was assessed by testing bovine serum and defatted milk in Western blotting. AdipoQ multimers were converted to dimers and monomers by reducing the samples with 200 mM DTT and denaturing for 5 min at 95 °C (Mielenz et al., 2013). Following SDSPAGE and transfer of protein to nitrocellulose membranes, these were incubated with the anti-adiponectin primary antibody for 60 min at room temperature. The optimal antibody concentration was determined to be 0.5 μg/mL (1:2000 dilution). Immunoreactive bands were visualised by enhanced chemiluminescence (Millipore), using human serum as the positive control. 2D-electrophoresis Defatted milk and serum were concentrated 10 and 5 times, respectively, by ultrafiltration using Amicon Ultra-15 centrifugal filter devices (Millipore), with Ultracel membranes (molecular weight cut off of 3000), spun at 3800 g. 2D isoelectric focusing (IEF) SDS-PAGE was performed as reported by Lecchi et al. (2013). Strips were focused in the first dimension at 8500 Vh, with a maximum of 2500 V, at 20 °C using a Multiphor II Electrophoresis unit (GE Healthcare). Strips were then incubated in equilibration buffer (375 mM Tris–HCl, pH 8.8, 6 M urea, 2% SDS, 20% glycerol) with
Statistical analysis All statistical analyses were performed using IBM SPSS 21.0 for Windows. Data were log10 transformed, to normalise their distribution, conformed by the Shapiro– Wilk test. Analysis of variance (ANOVA) was performed to compare the means of the values of gene expression, followed by post-hoc testing using the Bonferroni method. Statistical significance was accepted at P < 0.05.
Results Evaluation of AdipoQ and AdipoR mRNA expression Different levels of expression of AdipoQ, AdipoR1 and AdipoR2 mRNA were detected in the different areas of the mammary gland tissue evaluated (duct, parenchyma, Fürstenberg’s rosette and cistern; Fig. 1). AdipoQ was expressed in mammary gland parenchyma, cistern, ducts and in Fürstenberg’s rosette to a more limited degree, compared with adipose tissue (Fig. 1A). AdipoR1 mRNA was found to be expressed at a relatively high level in all the analysed areas of the mammary gland and particularly in parenchyma and cistern (Fig. 1B). Expression of AdipoR2 mRNA was similar comparing mammary gland parenchyma and adipose tissue and lower in Fürstenberg’s rosette, cistern and duct (Fig. 1C). Validation of anti-adiponectin polyclonal antibody and detection of AdipoQ in bovine milk Western blotting was performed to determine whether the selected anti-human adiponectin antibody cross-reacted with the corresponding bovine protein. The antibody reacted to a band of approximately 28–30 kDa (Fig. 2) that corresponds to the anticipated molecular weight of the AdipoQ monomer. Larger molecular weight bands of ~52 kDa were also present, corresponding to the expected size of dimers in both bovine and human serum samples with a similar electrophoresis pattern, but different stoichiometry. A weak band of ~28 kDa, corresponding to the AdipoQ monomer, was detected in bovine milk (Fig. 2). Although the AdipoQ dimer was not identified in milk, additional high molecular weight proteins, ranging from 60 to 100 kDa, corresponding to AdipoQ oligomers were detected. A band with a low molecular weight of 15 kDa, corresponding to the globular domain, was also detected in bovine milk. Several AdipoQ isoforms can be detected in bovine milk by 2D electrophoresis A number of different bovine AdipoQ isoforms were detected by 2D electrophoresis. In milk samples, the electrophoretic pattern was more complex than those obtained from serum (Fig. 3).
Please cite this article in press as: Cristina Lecchi, Chiara Giudice, Martina Uggè, Alessio Scarafoni, Antonella Baldi, Paola Sartorelli, Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.01.009
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Fig. 2. Cross-reactivity between anti-human adiponectin antibody and bovine AdipoQ in Western blotting. Human or bovine serum (0.2 μL) or bovine defatted milk (5 μL) was loaded onto separate lanes and subjected to SDS-PAGE and transferred to nitrocellulose membranes. Immunoreactivity was detected with anti-human adiponectin polyclonal antibody using ECL detection. The globular fragment is the carboxylterminal domain of AdipoQ with a molecular weight of 15–18 kDa.
Immunohistochemical localisation of bovine AdipoQ in mammary tissue Subcutaneous adipose tissue, used as positive control, and adipose tissue collected at the interstitium of mammary parenchyma, showed immunoreactivity for AdipoQ, primarily along adipocyte cell borders (Fig. 4A). In the mammary gland parenchyma, AdipoQ multifocally stained epithelial cells that were lining the secretory alveoli (Fig. 4B). Mammary secretum within scattered alveolar and cisternal lumina also stained positive for AdipoQ. AdipoQ immunoreactivity was occasionally observed associated with the endothelium lining small vessels (Fig. 4C). A positive signal for AdipoQ was also
Fig. 1. Relative expression of AdipoQ and AdipoR mRNA in different bovine mammary gland areas. QPCR results for the target genes, (A) AdipoQ, (B) AdipoR1 and (C) AdipoR2 were normalised using the geometric mean of reference genes (ACTB, YWHAZ and GAPDH). Adipose tissue was used as the reference sample for calculation of relative expression and data are shown as the mean ± SE of six animals. *P < 0.05; **P < 0.01.
The monomeric forms of Mr of approximately 28–29 kDa were present in both samples, but showed different pI values, ranging from 5.88 to 6.90 in milk and 6.20, 6.75 and 6.90 in serum. In serum, but not milk, another spot having a pI of 6.75 and a Mr compatible with that expected for the dimeric form of the protein was visible. Two groups of oligomeric AdipoQ isoforms were present in milk. The first group comprised the dimeric forms (Mr ~60 kDa) showing three spots, with pIs of 4.40, 6.75 and 6.90. The second group consisted of polypeptides with a molecular weight of approximately 76 kDa, found exclusively in the milk sample.
Fig. 3. Different bovine AdipoQ isoforms in (A) milk and (B) serum, separated by 2D electrophoresis (IEF/SDS-PAGE). Immunoreactivity was detected with antihuman adiponectin polyclonal antibody using ECL detection. Relative mass (Mr) values are expressed as kDa.
Please cite this article in press as: Cristina Lecchi, Chiara Giudice, Martina Uggè, Alessio Scarafoni, Antonella Baldi, Paola Sartorelli, Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.01.009
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Fig. 4. Immunohistochemistry for AdipoQ in bovine tissues. (A) Adipose tissue. Cytoplasmic borders of adipocytes showed strong immunoreactivity for adiponectin. Haematoxylin counterstain. Bar = 15 μm. (B) Mammary parenchyma. Strong intracytoplasmic immunolabelling for adiponectin was multifocally detected in epithelial cells lining the mammary alveoli. Haematoxylin counterstain. Bar = 35 μm. (C) Small arterial vessel. Scattered endothelial cells demonstrated immunoreactivity for adiponectin. Haematoxylin counterstain. Bar = 20 μm. (D) Section of a small peripheral nerve. Nerve fibres demonstrated intense immunoreactivity for adiponectin. Haematoxylin counterstain. Bar = 10 μm.
frequently observed in structures, morphologically consistent with small peripheral nerve fibres (Fig. 4D). In order to confirm the nature of this anatomical structure, serial sections of the same sample were immunostained with neuron specific enolase (NSE) (see Appendix: Supplementary material). Discussion The present study has provided an insight into the distribution of AdipoQ and its two receptors, AdipoR1 and AdipoR2, in various anatomical regions of the bovine mammary gland and characterisation of AdipoQ isoforms in milk. The immunohistochemical results identified AdipoQ in the cytoplasm of epithelial cells, lining the secretory alveoli and occasionally in the secretum. Since AdipoQ mRNA expression was negligible in the different mammary tissues investigated, it can be hypothesised that AdipoQ is primarily transferred from the blood to the mammary epithelial cells and into milk, with a relatively small contribution from local synthesis. This hypothesis is supported by the finding that the endothelial cells lining small blood vessels occasionally immunostained positively for AdipoQ. What is proposed is that the positive staining in the endothelium is due to the AdipoQ molecules that pass across the blood-mammary gland barrier to the mammary alveoli. AdipoR1 and AdipoR2 mRNA expression was detected throughout the mammary gland and predominantly in the parenchyma. These results confirmed a previous report (Ohtani et al., 2011) and expanded upon their findings, by identifying AdipoRs in distinct areas of the mammary gland. AdipoQ regulates a number of biological processes by diverse and complex pathways. Binding of AdipoQ to its cognate receptors activates an insulin-sensitising signal in liver
and muscle (Yamauchi et al., 2002), thus contributing to the fine tuning of energy metabolism during lactation. The results of Western blotting and 2D-electrophoresis demonstrated that bovine AdipoQ is not present exclusively as a monomer in milk or blood, with dimers and oligomers also present. The 2Delectrophoresis findings further emphasise the differences between milk and blood, revealing that bovine AdipoQ exists as multiple forms with different Mr and up to five isoforms in milk. These differences might be explained by post-translational modification (Wang et al., 2008; Richards et al., 2010), which plays a key role in AdipoQ oligomerisation and in mediating its biological effects. Moreover, the AdipoQ globular domain (Wang et al., 2002; Waki et al., 2005) was apparent in milk. Given that monomeric AdipoQ, the globular domain and the variants produced by post-translational modification perform different biological functions (Wang et al., 2008), these findings could have several consequences on maintenance of the healthy status of the mammary gland. The study findings suggest that AdipoQ might modulate a wider set of pathways in bovine mammary gland and milk as compared to the situation in humans, where only the high molecular weight isoforms of AdipoQ have been detected (Woo et al., 2009). The precise role of AdipoQ in cow’s milk remains to be elucidated. However, on the basis of research in other species (Newburg et al., 2010), several hypotheses can be proposed. AdipoQ is known to exert anti-inflammatory and immunomodulatory activities (Fantuzzi, 2013), which might be important in the mammary gland in preventing an excessive inflammatory responses. AdipoQ has been demonstrated to limit the detrimental effect of some pathogens on epithelial cells, by activating anti-inflammatory pathways (Kraus et al., 2012). Equally important is the role that AdipoQ might play
Please cite this article in press as: Cristina Lecchi, Chiara Giudice, Martina Uggè, Alessio Scarafoni, Antonella Baldi, Paola Sartorelli, Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.01.009
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in the calf intestine following ingestion of milk. The AdipoQ ingested by human infants is resistant to proteolysis and is absorbed through the enteric mucosal epithelium (Savino et al., 2008; Woo et al., 2009). AdipoQ multimers in bovine milk could contribute to modulating calf mucosal immunity or interacting with intestinal microflora by a direct binding to pathogen-associated molecular patterns (Peake et al., 2006). Such interaction might be important in establishing a commensal microbial population in the gut. AdipoQ was detected in small peripheral nerve fibres within the mammary gland, suggesting that there might be a degree of neuroendocrine input. AdipoQ has been reported to be present in the cerebrospinal fluid of rodents (Qi et al., 2004) and humans (Kusminski et al., 2007), suggesting a role in the central nervous system (Thundyil et al., 2012). Furthermore, AdipoRs are expressed by neurons and astrocytes (Guillod-Maximin et al., 2009). Nerve fibres in the mammary gland provide sympathetic innervation to blood vessels, thus regulating the blood supply and controlling the smooth muscle surrounding the milk collecting ducts and the sphincter muscles. AdipoQ has been shown to have a hypotensive action (Tanida et al., 2007), and its detection in mammary gland nerves suggests a potentially novel activity for AdipoQ, i.e. regulation of the blood supply in the udder during lactation. Conclusions Although AdipoQ was originally thought to be expressed exclusively by adipose tissue, the present study adds to a growing body of evidence, indicating that other cell types can express this adipokine. Adipocytes produce AdipoQ which circulates in the plasma, although recent evidence describes important physiological roles for locally produced AdipoQ. The findings support an emerging new paradigm that AdipoQ is an important local mediator of metabolism and immunity of bovine mammary gland. Moreover, the presence of different AdipoQ isoforms in milk reinforces the hypothesis that AdipoQ can potentially influence metabolic and immunological processes in the gut of milk-fed calves. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisation that could inappropriately influence or bias the content of the paper. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.tvjl.2015.01.009. References Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., et al., 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55, 611–622. Fantuzzi, G., 2013. Adiponectin in inflammatory and immune-mediated diseases. Cytokine 64, 1–10. doi:10.1016/j.cyto.2013.06.317. Guillod-Maximin, E., Roy, A.F., Vacher, C.M., Aubourg, A., Bailleux, V., Lorsignol, A., Pénicaud, L., Parquet, M., Taouis, M., 2009. Adiponectin receptors are expressed in hypothalamus and colocalized with proopiomelanocortin and neuropeptide Y in rodent arcuate neurons. Journal of Endocrinology 200, 93–105. Hsu, S.M., Raine, L., Fanger, H., 1981. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry 29, 577–580. Kadowaki, T., Yamauchi, T., 2005. Adiponectin and adiponectin receptors. Endocrine Reviews 26, 439–451.
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Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk Cristina Lecchi a,b,*, Chiara Giudice a, Martina Uggè a, Alessio Scarafoni b,c, Antonella Baldi b,d, Paola Sartorelli a,b a
Department of Veterinary Sciences and Public Health, Università degli Studi di Milano, 20133 Milan, Italy Centro Interdipartimentale per lo Studio sulla Ghiandola Mammaria (CISMA), Università degli Studi di Milano, 20133 Milan, Italy c Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, 20133 Milan, Italy d Department of Health, Animal Science and Food Safety, Università degli Studi di Milano, 20133 Milan, Italy b
A R T I C L E
I N F O
Article history: Accepted 10 January 2015 Keywords: Adipokine Adiponectin Cattle Mammary gland Milk
A B S T R A C T
Adiponectin is an adipocyte-derived hormone, which circulates in the form of homo-multimers. The individual oligomers have a distinct profile of activity, playing crucial roles in several biological processes, including metabolism and inflammation. Adiponectin exerts many of its effects by interacting with the receptors, AdipoR1 and AdipoR2. In the present study, mRNA expression of adiponectin, AdipoR1 and AdipoR2 was evaluated by quantitative PCR in different areas of the mammary gland in healthy lactating cows. The adiponectin isoforms in milk and blood were investigated by Western blotting and 2D-electrophoresis, and the presence of adiponectin protein was determined by immunohistochemistry. Low level expression of adiponectin mRNA was found in all areas of bovine mammary gland tissues examined. AdipoR1 and AdipoR2 mRNAs were also detected in mammary tissues and their expression was particularly prominent in the parenchyma and cistern. Western blotting revealed a heterogeneous electrophoretic pattern, indicating that different adiponectin isoforms exist in milk, compared with blood. In particular, milk shows a low molecular weight isoform of adiponectin, corresponding to the globular domain. Adiponectin in milk is characterised by a more complex 2D electrophoretic pattern, compared with blood, as illustrated by the presence of proteins of different molecular weights and isoelectric points. Adiponectin protein was detected by immunohistochemistry in epithelial cells lining the secretory alveoli, in secretum within the alveolar lumen and in small peripheral nerves. The study findings support a role for adiponectin in regulating metabolism and immunity of the bovine mammary gland and potentially the calf intestine, following ingestion of milk. © 2015 Elsevier Ltd. All rights reserved.
Introduction Adiponectin (AdipoQ) is a hormone, produced almost exclusively by adipocytes, which is present in serum at concentrations that are inversely related to the degree of hypertrophy of adipose tissue. In addition, circulating AdipoQ usually shows an inverse relationship with insulin, triglycerides, very low density lipoproteins (VLDL) and pro-inflammatory cytokines (Matsubara et al., 2002). AdipoQ has anti-inflammatory effects and demonstrates other properties, including insulin-sensitisation of tissues and vascular protection (Yamauchi et al., 2002; Fantuzzi, 2013). AdipoQ belongs to the complement 1q (C1q) family of proteins (Scherer et al., 1995). Its primary structure is highly conserved, with over 80% amino acid sequence identity comparing mammalian species (Wang et al., 2002). Bovine AdipoQ is a 240 amino acid
* Corresponding author. Tel.: +39 025 031 8100. E-mail address: [email protected] (C. Lecchi).
protein of approximately 28–30 kDa and circulates as homomultimers, which vary from low molecular weight trimers to high molecular weight (12- to 18-mer) oligomers (Wang et al., 2004; Tsao, 2014). The protein undergoes extensive post-translational modification, such as hydroxylation and hydroxyl-glycosylation (Wang et al., 2002; Richards et al., 2006, 2010). AdipoQ exerts many of its biological effects by binding to the receptors, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2), which have distinct distribution patterns in different tissues. AdipoR1 is abundantly expressed in skeletal muscle and is linked to activation of cAMP-mediated kinase pathways, whereas AdipoR2 is abundantly expressed in the liver and is associated with activation of PPARα pathways (Kadowaki and Yamauchi, 2005). Both AdipoR1 and AdipoR2 are involved in regulating energy metabolism in these tissues. Expression of AdipoQ has been studied in the adipose tissue of cattle during the transition period (Lemor et al., 2009; Ohtani et al., 2012), particularly in the mammary gland (Ohtani et al., 2011) and ovaries, including follicles, oocytes (Tabandeh et al., 2012) and
http://dx.doi.org/10.1016/j.tvjl.2015.01.009 1090-0233/© 2015 Elsevier Ltd. All rights reserved.
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granulosa cells (Maillard et al., 2010). AdipoQ has also been quantified in bovine plasma (mean ± SD of 32 ± 1.0 μg/mL) and in milk (0.61 ± 0.03 μg/mL) (Singh et al., 2014). The aim of the present study was to investigate the distribution pattern of AdipoQ and AdipoRs in the mammary gland of lactating cows to elucidate the possible role of adiponectin on mammary gland health.
10 mg/mL DTT for 15 min, then with 25 mg/mL iodoacetamide in the same buffer without DTT for 10 min. Second dimension separation was performed using a MiniProtean III cell (Bio-Rad) with a 12% polyacrylamide gel (acrylamide:bis-acrylamide 37.5:1) at 16 mA constant current for 90 min. Electrotransfer onto nitrocellulose membranes (Whatmann Protran BA85) was performed for 75 min at 50 mA with a semi-dry apparatus (GE Healthcare) using 39 mM glycine, 48 mM Tris-HCl (pH 9.0), 1.3 mM SDS and 12% methanol as transfer buffer.
Materials and methods
Immunohistochemistry
Sample collection
Formalin-fixed, paraffin-embedded mammary and adipose tissues were routinely processed for histology. Five micrometre sections were obtained from paraffin blocks and mounted on polylysine-coated slides. Immunolabelling was performed by the standard ABC method (Hsu et al., 1981). Primary anti-adiponectin antibody (diluted 1: 2000 in Tris buffer) was incubated overnight at 4 °C. Immunolabelling was revealed using 3-amino, 9 ethyl-carbazole (AEC; Vector Laboratories) as the chromogen and sections were counterstained with Mayer’s haematoxylin. As a negative control, primary antibody was replaced with Tris buffer. The presence of immunostaining (positive or negative) with anti-adiponectin antibody and its localisation within the mammary and adipose tissue were assessed by light microscopy.
Blood and milk samples were collected from clinically healthy multiparous Holstein-Friesian cows, aged between 3 and 8 years, obtained 4–10 weeks after calving as part of routine health monitoring. Samples were made available for research following completion of diagnostic testing. Whole blood was collected via coccygeal venepuncture using tubes coated with clot activator and silicone. Serum was obtained by centrifugation of clotted blood at 1200 g for 10 min at 4 °C, which was stored in aliquots at −80 °C. Milk whey was prepared from milk with a somatic cell count (SCC) < 250,000 cells/mL. Ten millilitres of milk were centrifuged at 1200 g for 15 min at 4 °C to remove fat and cells, then the whey was further centrifuged at 13,000 g at room temperature and stored in aliquots at −80 °C. Mammary tissue samples were obtained from six cows immediately after routine slaughtering procedures. Representative tissues of each area of the mammary gland were sampled and stored in RNAlater (Sigma-Aldrich) at −80 °C before RNA extraction. For the immunohistochemical study, samples of mammary tissues of approximately 1 cm × 1 cm were collected and fixed in 10% buffered formalin. Samples of subcutaneous adipose tissue, to be used as a positive control, were similarly collected and formalin fixed. Real-time quantitative PCR Total RNA was extracted from tissue lysates in TRIzol reagent (Invitrogen), according to the manufacturer’s recommendations. RNA concentrations were quantified by use of the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The purity of RNA (A260/A280) was ~2. Genomic DNA was eliminated using DNase I (Invitrogen) and reverse transcription was performed with 1 μg RNA as the template, using the iSCRIPT cDNA Synthesis Kit (BioRad). Quantitative PCR was performed in 12 μL reactions, using the Eco Real Time PCR detection System (Illumina). Each reaction contained Eva Green mix (BioRad) and 400 nM of primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), AdipoR1 and AdipoR2 or 300 nM primers specific for AdipoQ (see Appendix: Supplementary material). The thermocycler conditions consisted of 50 °C for 2 min, 95 °C for 10 min followed by 40 cycles of 95 °C for 8 s and 60 °C for 18 s. For assessment of melting curves, PCR products were incubated at 55 °C for 60 s then the temperature was increased to 95 °C at 0.5 °C increments for 10 s. The MIQE guidelines (Bustin et al., 2009) were followed. The PCR efficiencies were determined using four-fold serial dilutions of cDNA, prepared from adipose tissue, performed in triplicate. cDNA samples from mammary tissue were assessed in duplicate. Non-reverse transcribed controls and no template controls were included in the assays. Three reference genes (GAPDH, ACTB, YWHAZ) (Lecchi et al., 2012) were selected and the geometric mean of reference gene abundance was used for normalisation purposes. Relative quantification of genes of interest was carried out with adipose tissue used as the reference sample for mammary gland tissues. Western blotting A rabbit anti-human AdipoQ polyclonal antibody (PAI-84881, Pierce Biotechnology), raised against a peptide located near the carboxy-terminal globular domain, was used for experiments. Cross-reactivity of this antibody with bovine AdipoQ was assessed by testing bovine serum and defatted milk in Western blotting. AdipoQ multimers were converted to dimers and monomers by reducing the samples with 200 mM DTT and denaturing for 5 min at 95 °C (Mielenz et al., 2013). Following SDSPAGE and transfer of protein to nitrocellulose membranes, these were incubated with the anti-adiponectin primary antibody for 60 min at room temperature. The optimal antibody concentration was determined to be 0.5 μg/mL (1:2000 dilution). Immunoreactive bands were visualised by enhanced chemiluminescence (Millipore), using human serum as the positive control. 2D-electrophoresis Defatted milk and serum were concentrated 10 and 5 times, respectively, by ultrafiltration using Amicon Ultra-15 centrifugal filter devices (Millipore), with Ultracel membranes (molecular weight cut off of 3000), spun at 3800 g. 2D isoelectric focusing (IEF) SDS-PAGE was performed as reported by Lecchi et al. (2013). Strips were focused in the first dimension at 8500 Vh, with a maximum of 2500 V, at 20 °C using a Multiphor II Electrophoresis unit (GE Healthcare). Strips were then incubated in equilibration buffer (375 mM Tris–HCl, pH 8.8, 6 M urea, 2% SDS, 20% glycerol) with
Statistical analysis All statistical analyses were performed using IBM SPSS 21.0 for Windows. Data were log10 transformed, to normalise their distribution, conformed by the Shapiro– Wilk test. Analysis of variance (ANOVA) was performed to compare the means of the values of gene expression, followed by post-hoc testing using the Bonferroni method. Statistical significance was accepted at P < 0.05.
Results Evaluation of AdipoQ and AdipoR mRNA expression Different levels of expression of AdipoQ, AdipoR1 and AdipoR2 mRNA were detected in the different areas of the mammary gland tissue evaluated (duct, parenchyma, Fürstenberg’s rosette and cistern; Fig. 1). AdipoQ was expressed in mammary gland parenchyma, cistern, ducts and in Fürstenberg’s rosette to a more limited degree, compared with adipose tissue (Fig. 1A). AdipoR1 mRNA was found to be expressed at a relatively high level in all the analysed areas of the mammary gland and particularly in parenchyma and cistern (Fig. 1B). Expression of AdipoR2 mRNA was similar comparing mammary gland parenchyma and adipose tissue and lower in Fürstenberg’s rosette, cistern and duct (Fig. 1C). Validation of anti-adiponectin polyclonal antibody and detection of AdipoQ in bovine milk Western blotting was performed to determine whether the selected anti-human adiponectin antibody cross-reacted with the corresponding bovine protein. The antibody reacted to a band of approximately 28–30 kDa (Fig. 2) that corresponds to the anticipated molecular weight of the AdipoQ monomer. Larger molecular weight bands of ~52 kDa were also present, corresponding to the expected size of dimers in both bovine and human serum samples with a similar electrophoresis pattern, but different stoichiometry. A weak band of ~28 kDa, corresponding to the AdipoQ monomer, was detected in bovine milk (Fig. 2). Although the AdipoQ dimer was not identified in milk, additional high molecular weight proteins, ranging from 60 to 100 kDa, corresponding to AdipoQ oligomers were detected. A band with a low molecular weight of 15 kDa, corresponding to the globular domain, was also detected in bovine milk. Several AdipoQ isoforms can be detected in bovine milk by 2D electrophoresis A number of different bovine AdipoQ isoforms were detected by 2D electrophoresis. In milk samples, the electrophoretic pattern was more complex than those obtained from serum (Fig. 3).
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Fig. 2. Cross-reactivity between anti-human adiponectin antibody and bovine AdipoQ in Western blotting. Human or bovine serum (0.2 μL) or bovine defatted milk (5 μL) was loaded onto separate lanes and subjected to SDS-PAGE and transferred to nitrocellulose membranes. Immunoreactivity was detected with anti-human adiponectin polyclonal antibody using ECL detection. The globular fragment is the carboxylterminal domain of AdipoQ with a molecular weight of 15–18 kDa.
Immunohistochemical localisation of bovine AdipoQ in mammary tissue Subcutaneous adipose tissue, used as positive control, and adipose tissue collected at the interstitium of mammary parenchyma, showed immunoreactivity for AdipoQ, primarily along adipocyte cell borders (Fig. 4A). In the mammary gland parenchyma, AdipoQ multifocally stained epithelial cells that were lining the secretory alveoli (Fig. 4B). Mammary secretum within scattered alveolar and cisternal lumina also stained positive for AdipoQ. AdipoQ immunoreactivity was occasionally observed associated with the endothelium lining small vessels (Fig. 4C). A positive signal for AdipoQ was also
Fig. 1. Relative expression of AdipoQ and AdipoR mRNA in different bovine mammary gland areas. QPCR results for the target genes, (A) AdipoQ, (B) AdipoR1 and (C) AdipoR2 were normalised using the geometric mean of reference genes (ACTB, YWHAZ and GAPDH). Adipose tissue was used as the reference sample for calculation of relative expression and data are shown as the mean ± SE of six animals. *P < 0.05; **P < 0.01.
The monomeric forms of Mr of approximately 28–29 kDa were present in both samples, but showed different pI values, ranging from 5.88 to 6.90 in milk and 6.20, 6.75 and 6.90 in serum. In serum, but not milk, another spot having a pI of 6.75 and a Mr compatible with that expected for the dimeric form of the protein was visible. Two groups of oligomeric AdipoQ isoforms were present in milk. The first group comprised the dimeric forms (Mr ~60 kDa) showing three spots, with pIs of 4.40, 6.75 and 6.90. The second group consisted of polypeptides with a molecular weight of approximately 76 kDa, found exclusively in the milk sample.
Fig. 3. Different bovine AdipoQ isoforms in (A) milk and (B) serum, separated by 2D electrophoresis (IEF/SDS-PAGE). Immunoreactivity was detected with antihuman adiponectin polyclonal antibody using ECL detection. Relative mass (Mr) values are expressed as kDa.
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Fig. 4. Immunohistochemistry for AdipoQ in bovine tissues. (A) Adipose tissue. Cytoplasmic borders of adipocytes showed strong immunoreactivity for adiponectin. Haematoxylin counterstain. Bar = 15 μm. (B) Mammary parenchyma. Strong intracytoplasmic immunolabelling for adiponectin was multifocally detected in epithelial cells lining the mammary alveoli. Haematoxylin counterstain. Bar = 35 μm. (C) Small arterial vessel. Scattered endothelial cells demonstrated immunoreactivity for adiponectin. Haematoxylin counterstain. Bar = 20 μm. (D) Section of a small peripheral nerve. Nerve fibres demonstrated intense immunoreactivity for adiponectin. Haematoxylin counterstain. Bar = 10 μm.
frequently observed in structures, morphologically consistent with small peripheral nerve fibres (Fig. 4D). In order to confirm the nature of this anatomical structure, serial sections of the same sample were immunostained with neuron specific enolase (NSE) (see Appendix: Supplementary material). Discussion The present study has provided an insight into the distribution of AdipoQ and its two receptors, AdipoR1 and AdipoR2, in various anatomical regions of the bovine mammary gland and characterisation of AdipoQ isoforms in milk. The immunohistochemical results identified AdipoQ in the cytoplasm of epithelial cells, lining the secretory alveoli and occasionally in the secretum. Since AdipoQ mRNA expression was negligible in the different mammary tissues investigated, it can be hypothesised that AdipoQ is primarily transferred from the blood to the mammary epithelial cells and into milk, with a relatively small contribution from local synthesis. This hypothesis is supported by the finding that the endothelial cells lining small blood vessels occasionally immunostained positively for AdipoQ. What is proposed is that the positive staining in the endothelium is due to the AdipoQ molecules that pass across the blood-mammary gland barrier to the mammary alveoli. AdipoR1 and AdipoR2 mRNA expression was detected throughout the mammary gland and predominantly in the parenchyma. These results confirmed a previous report (Ohtani et al., 2011) and expanded upon their findings, by identifying AdipoRs in distinct areas of the mammary gland. AdipoQ regulates a number of biological processes by diverse and complex pathways. Binding of AdipoQ to its cognate receptors activates an insulin-sensitising signal in liver
and muscle (Yamauchi et al., 2002), thus contributing to the fine tuning of energy metabolism during lactation. The results of Western blotting and 2D-electrophoresis demonstrated that bovine AdipoQ is not present exclusively as a monomer in milk or blood, with dimers and oligomers also present. The 2Delectrophoresis findings further emphasise the differences between milk and blood, revealing that bovine AdipoQ exists as multiple forms with different Mr and up to five isoforms in milk. These differences might be explained by post-translational modification (Wang et al., 2008; Richards et al., 2010), which plays a key role in AdipoQ oligomerisation and in mediating its biological effects. Moreover, the AdipoQ globular domain (Wang et al., 2002; Waki et al., 2005) was apparent in milk. Given that monomeric AdipoQ, the globular domain and the variants produced by post-translational modification perform different biological functions (Wang et al., 2008), these findings could have several consequences on maintenance of the healthy status of the mammary gland. The study findings suggest that AdipoQ might modulate a wider set of pathways in bovine mammary gland and milk as compared to the situation in humans, where only the high molecular weight isoforms of AdipoQ have been detected (Woo et al., 2009). The precise role of AdipoQ in cow’s milk remains to be elucidated. However, on the basis of research in other species (Newburg et al., 2010), several hypotheses can be proposed. AdipoQ is known to exert anti-inflammatory and immunomodulatory activities (Fantuzzi, 2013), which might be important in the mammary gland in preventing an excessive inflammatory responses. AdipoQ has been demonstrated to limit the detrimental effect of some pathogens on epithelial cells, by activating anti-inflammatory pathways (Kraus et al., 2012). Equally important is the role that AdipoQ might play
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in the calf intestine following ingestion of milk. The AdipoQ ingested by human infants is resistant to proteolysis and is absorbed through the enteric mucosal epithelium (Savino et al., 2008; Woo et al., 2009). AdipoQ multimers in bovine milk could contribute to modulating calf mucosal immunity or interacting with intestinal microflora by a direct binding to pathogen-associated molecular patterns (Peake et al., 2006). Such interaction might be important in establishing a commensal microbial population in the gut. AdipoQ was detected in small peripheral nerve fibres within the mammary gland, suggesting that there might be a degree of neuroendocrine input. AdipoQ has been reported to be present in the cerebrospinal fluid of rodents (Qi et al., 2004) and humans (Kusminski et al., 2007), suggesting a role in the central nervous system (Thundyil et al., 2012). Furthermore, AdipoRs are expressed by neurons and astrocytes (Guillod-Maximin et al., 2009). Nerve fibres in the mammary gland provide sympathetic innervation to blood vessels, thus regulating the blood supply and controlling the smooth muscle surrounding the milk collecting ducts and the sphincter muscles. AdipoQ has been shown to have a hypotensive action (Tanida et al., 2007), and its detection in mammary gland nerves suggests a potentially novel activity for AdipoQ, i.e. regulation of the blood supply in the udder during lactation. Conclusions Although AdipoQ was originally thought to be expressed exclusively by adipose tissue, the present study adds to a growing body of evidence, indicating that other cell types can express this adipokine. Adipocytes produce AdipoQ which circulates in the plasma, although recent evidence describes important physiological roles for locally produced AdipoQ. The findings support an emerging new paradigm that AdipoQ is an important local mediator of metabolism and immunity of bovine mammary gland. Moreover, the presence of different AdipoQ isoforms in milk reinforces the hypothesis that AdipoQ can potentially influence metabolic and immunological processes in the gut of milk-fed calves. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisation that could inappropriately influence or bias the content of the paper. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.tvjl.2015.01.009. References Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., et al., 2009. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 55, 611–622. Fantuzzi, G., 2013. Adiponectin in inflammatory and immune-mediated diseases. Cytokine 64, 1–10. doi:10.1016/j.cyto.2013.06.317. Guillod-Maximin, E., Roy, A.F., Vacher, C.M., Aubourg, A., Bailleux, V., Lorsignol, A., Pénicaud, L., Parquet, M., Taouis, M., 2009. Adiponectin receptors are expressed in hypothalamus and colocalized with proopiomelanocortin and neuropeptide Y in rodent arcuate neurons. Journal of Endocrinology 200, 93–105. Hsu, S.M., Raine, L., Fanger, H., 1981. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry 29, 577–580. Kadowaki, T., Yamauchi, T., 2005. Adiponectin and adiponectin receptors. Endocrine Reviews 26, 439–451.
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Please cite this article in press as: Cristina Lecchi, Chiara Giudice, Martina Uggè, Alessio Scarafoni, Antonella Baldi, Paola Sartorelli, Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.01.009
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Please cite this article in press as: Cristina Lecchi, Chiara Giudice, Martina Uggè, Alessio Scarafoni, Antonella Baldi, Paola Sartorelli, Characterisation of adiponectin and its receptors in the bovine mammary gland and in milk, The Veterinary Journal (2015), doi: 10.1016/j.tvjl.2015.01.009