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Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression
Biochemical and Biophysical Research Communications 408 (2011) 422–426
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression Pawel K. Olszewski a,b,⇑, Robert Fredriksson a, Jenny D. Eriksson a, Anaya Mitra c, Katarzyna J. Radomska a, Blake A. Gosnell c, Maria N. Solvang a, Allen S. Levine b,c, Helgi B. Schiöth a a b c
Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala, Sweden Minnesota Obesity Center, Saint Paul, MN 55108, USA Department of Food Science and Nutrition, Saint Paul, MN 55108, USA
a r t i c l e
i n f o
Article history: Received 31 March 2011 Available online 13 April 2011 Keywords: Obesity Feeding Brain
a b s t r a c t Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance through a functional relationship with specific genes encoding feedingrelated peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction There is a strong link between the fat mass and obesity-associated (FTO) gene and the control of energy balance. Single nucleotide polymorphisms in this gene are associated with human obesity [1– 6]. Animal experiments indicate that the relationship between Fto and body weight is complex and it involves mechanisms regulating energy metabolism and food intake. Fto knockout mice have a lower body weight yet they eat more food [7], whereas animals with a dominant point mutation in the Fto gene display reduced fat mass and increased energy expenditure [8]. Mice carrying additional copies of the Fto gene are heavier and eat significantly more than controls [9]. In wild-type mice and rats, food restriction leads to changes in hypothalamic FTO mRNA levels; and so does the treatment of hypothalamic explants with the anorexigenic amino acid, leucine [10]. It is not understood what physiological mechanisms underlie Fto’s involvement in energy homeostasis. Fto is a demethylase Abbreviations: AgRP, Agouti-related protein; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular hypothalamic nucleus; SON, supraoptic nucleus. ⇑ Corresponding author at: Department of Neuroscience, Functional Pharmacology, Uppsala University, BMC, 75124 Uppsala, Sweden. Fax: + 46 18511540. E-mail address: [email protected] (P.K. Olszewski). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.037
[11,12], but it may also serve as a transcriptional co-activator [13]. In vitro expression of Fto results in localization of the recombinant protein to the nucleus [14] and, in the brain – where this gene is most abundantly expressed – Fto immunoreactivity is predominantly present in neuronal nuclei [15,16]. It has been suggested that the Fto protein affects energy homeostasis by regulating expression of other genes involved in this process [12]. In fact, Fto mRNA has been found in neurons positive for transcripts/peptides known to regulate feeding, including oxytocin and proopiomelanocortin (POMC) [7,10,15]. Fto deficiency reduces expression of genes encoding neuropeptide Y (NPY) and POMC [7]. The link with oxytocin appears particularly interesting in the functional context: hypothalamic Fto mRNA-positive neurons display an increased level of colocalization with an immediate-early gene product, c-Fos, at meal termination compared to initiation [10]. The distribution of c-Fos immunoreactive Fto cells in the paraventricular and supraoptic nuclei at the end of a meal closely resembles the topography of neuronal populations synthesizing a satiety mediator, oxytocin [10,17,18]. The current project was aimed at exploring the presumed functional relationship between Fto and oxytocin. We used double immunohistochemistry to examine the percentage of oxytocin neurons containing nuclear profiles positive for the Fto protein in the male and female murine brain. We examined the level of
P.K. Olszewski et al. / Biochemical and Biophysical Research Communications 408 (2011) 422–426
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colocalization in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, the two main sites encompassing oxytocin neuronal populations. We then overexpressed Fto in a mouse hypothalamic cell line and employed real-time PCR to study whether overexpression of this transcription co-activator affects oxytocin mRNA levels. Relative expression of other feeding-related candidate interactor genes: POMC, NPY and Agouti-related protein (AgRP), was also determined. Finally, we evaluated whether addition of oxytocin to the culture medium serves as negative feedback for the expression of Fto in hypothalamic cells.
control (mock) vector to reveal a more pronounced signal in Ftotransfected cells. Since our Fto + oxytocin staining was done in male and female brains and our published Fto data had defined only Fto distribution in males, we performed an additional control single staining for Fto in females to exclude sex-related neuroanatomical differences in localization of this protein. Through a visual comparison of sections stained with the same antibody we did not detect any obvious differences in central Fto topography between males and females (see Supplement).
2. Materials and methods
2.2. Expression of oxytocin and other feeding-related genes in the hypothalamic cell line transfected with Fto
2.1. Fto -oxytocin colocalization assessment 2.1.1. Animals and perfusions Adult C57BL/6 J male (n = 4) and female (n = 4) mice (Scanbur, Sweden) weighing 32 g were housed individually in macrolon cages with LD 12:12 (lights on at 06:00; T = 21 °C). Water and standard chow (Lactamin, Sweden) were available ad libitum. All procedures were approved by the Uppsala Animal Welfare Committee. Mice were anesthetized with sodium pentobarbital (90 mg/kg IP) and intracardially perfused with 10 ml saline followed by 50 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The animals were sacrificed between 11:00 and 12:00. Brains were excised, postfixed for 24 h in the same fixative, sectioned and processed to detect oxytocin and Fto. 2.1.2. Immunohistochemistry Coronal sections (50 lm) were cut with a Vibratome. Every fourth section encompassing the PVN and SON, the sites where oxytocin is synthesized, was processed using standard double immunohistochemistry. Sections were treated for 10 min in 3% H2O2 and 10% methanol (in TBS, pH 7.4) and incubated for 24 h at 4 °C in the rabbit Fto antibody (1:2000; Inovagen, Sweden). Subsequently, the tissue was incubated for 1 h in the goat anti-rabbit antibody (1:400; Vector, Burlingame, CA) and then in the avidin– biotin complex (1:800; ABC Elite; Vector, Burlingame, CA). Peroxidase was visualized with 0.05% DAB, 0.35% nickel sulfate and 0.01% H2O2. Following the completion of the Fto staining, we applied the same protocol to detect oxytocin; however, the primary oxytocin antibody (Millipore, Temecula, CA; 1:15,000) and the DAB solution without nickel sulfate were used. The vehicle for incubations was a mixture of 0.25% gelatin and 0.5% Triton X-100 in TBS. Rinsing was done in TBS alone. Sections were mounted, dehydrated in alcohol, soaked in xylene, and embedded in DPX (BDH, UK). Evaluation of staining was based on the visual inspection of sections containing neuronal cell bodies positive for oxytocin. A percentage of oxytocin neurons co-expressing Fto was established for the PVN and SON in each animal and the results were averaged for males and females separately. Percentages of Fto immunoreactive oxytocin neurons are presented as means ± SEM. 2.1.3. Controls for specificity of the Fto antibody Additional test data for the specificity of this commercially available antibody (aside from those performed routinely by the manufacturer) have been published [16]. They included: (1) staining sections in the primary antibody solution preincubated for 3 h at 4 °C with the protein that the antibody had been raised against (Ab:Ag 1:5) resulting in no positive signal; (2) performing the BLAST search of the peptide sequence to confirm the uniqueness of the epitope binding site and exclude cross binding; and (3) performing the Western blot of Fto in mouse cells transfected with a pcDNA3 vector encoding N-terminal flag-tagged Fto versus the
2.2.1. Fto overexpression in hypothalamic cells in vitro We have used a previously described protocol for overexpressing the Fto gene in cell lines [16]. In short, we transfected the embryonal hypothalamic neuronal line (Cedarlane, Canada) with a pcDNA3 expression vector coding for N-terminal flag-tagged Fto and compared the results with those obtained in cells transfected with the control (mock) vector. Cells were cultured in the complete Dulbecco’s Modified Eagle Medium (high glucose, Gibco) supplemented with 1% Pen/Strep (50 U/ml penicillin and 50 lg/ ml streptomycin) and 10% FBS (Gibco). They were passaged onto new plates after reaching 70–80% confluency using trypsin (0.5 mg/ml, Invitrogen)/EDTA (0.2 mg/ml, Invitrogen). The cells were transfected at 70–80% confluency using Attractene according to the manufacturer’s protocol (Qiagen). Transfection efficiency was at least 65% as determined by immunostaining using the anti-flag antibody with the GFP-conjugated secondary antibody (as described in [16]). An increase in Fto protein level in transfected cells was confirmed also with Western blotting. 225 lg (15 lg/ll) of protein was loaded into wells as determined using DC protein assay (Bio-Rad). Proteins were transferred from the gel (Criterion precast 10% polyacrylamide gel, BioRad) to membranes (0.45 lm PVDF Immobilon-P, Millipore) using electroblot. Membranes were blocked with 1% non-fat dry milk in TTBS (TBS with 0.5% Tween 20) and incubated with the primary Fto or anti-flag antibody (1:2500) in TTBS overnight. After incubation, the filters were washed 3 10 min with TTBS. The filters were then incubated in the secondary antibody (ECL-Anti-Rabbit IgG, horseradish peroxidase-linked, Amersham) diluted 1:10,000 in TTBS for 60 min and washed in TTBS. Membranes were developed with the Immun-Star HRP Chemiluminescent Kit (Bio-Rad) and ECL hyperfilm (Amersham). The visual analysis revealed a much stronger signal in Fto-transfected cells in which the gene was overexpressed compared to control cells; anti-Fto and anti-flag antibodies produced comparable bands (an example of the Fto signal detected with Western blotting is shown in Fig. 2). 2.2.2. Real-time PCR analysis of expression of genes encoding oxytocin and other feeding-related genes, POMC, NPY and AgRP, in Ftotransfected cells Six separate hypothalamic cell cultures were transfected with the Fto containing vector, whereas the other six were transfected with the mock vector. RNA was extracted 48 h later and cDNA was synthesized as described before [19]. Cells were immersed in TRIzol (Invitrogen, Sweden). RNA was extracted with chloroform and precipitated with isopropanol. The samples were centrifuged and the pellet was washed, air dried, and dissolved in the 1 deoxyribonuclease buffer. The samples were incubated with ribonuclease-free deoxyribonuclease I (37 °C, 90 min; Roche, Sweden). The absence of genomic DNA was established by PCR: 0.5 ll template was mixed in a final volume of 10 ll 1 PCR mix [1 ll MgCl2-free buffer 10, 0.3 ll 50 mM MgCl2, 0.25 ll 1% Tween, 0.1 ll 20 mM dNTP, 1 ll primer mix (forward and backward at
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10 pmol/ll), 0.1 ll Taq polymerase, 5 U/ll (Biotools, Spain), and 6.75 ll MilliQ H2O]. 0.5 ll 100 ng/ll genomic DNA served as a positive control; 0.5 ll MilliQ H2O was added in a negative control. The product was analyzed with electrophoresis. Total RNA concentration was measured with Nanodrop ND-1000 spectrophotometer. For cDNA synthesis, 5-lg RNA samples were diluted in MilliQ H2O to 12 ll. RNA was reverse-transcribed in 20 ll 1 Mastermix (4 ll 5 first-strand buffer, 2 ll 0.1 M dithiothreitol, 0.5 ll 20 mM dNTP, 0.5 ll N6 1/6.25 and 1 ll MMLV reverse transcriptase). Samples were incubated at 37 °C for 1 h, followed by PCR (as above) to confirm cDNA synthesis. 25 ng cDNA template from each sample was used per primer (primers for oxytocin, POMC, NPY and AgRP). Each reaction (20 ll) utilized 2 ll MgCl2-free buffer 10, 0.2 ll 20 mM dNTP, 1.6 ll 50 mM MgCl2, 0.05 ll of a primer at 100 pmol/ll (forward and reverse), 1 ll dimethyl sulfoxide, 0.5 ll Sybr Green (1:50,000), 0.08 ll Taq polymerase (5 U/ll) (Biotools), and 9.52 ll MilliQ water. All PCRs were done in duplicates with negative controls included on each plate. Amplification was performed: denaturation at 95 °C (3 min), 50 cycles of denaturing at 95 °C (15 s), annealing at temperatures established for the primers (15 s), and extension at 72 °C (30 s). Seven housekeeping genes (HKGs) were analyzed (glyceraldehyde-3-phosphate-dehydrogenase, b-tubulin, ribosomal protein 19, histone H3, cyclophilin, b-actin, and succinate dehydrogenase complex subunit B). Data were analyzed with MyiQ 1.04 software (Bio-Rad, Sweden) (30). Primer efficiencies were calculated with LinRegPCR (31), and samples were corrected for differences in primer efficiencies. The GeNorm protocol by Vandesompele et al. [20] was employed to calculate normalization factors based on HKG expression. Grubb’s test was used to identify outliers. Differences between groups were analyzed with Student’s t-test. Values were considered different when P < 0.05.
2.3. Expression of Fto in the hypothalamic cell line treated with oxytocin To investigate whether Fto mRNA level is affected by oxytocin, mouse embryonic hypothalamic neurons (Cedarlane, Canada) were cultured in the DMEM media (2 ml) containing 0 (control; n = 4), 10 (n = 4) or 100 nM/l (n = 4) oxytocin (Sigma, Sweden) for 48 h. These two concentrations of the peptide have been shown to affect activity of cells in vitro [21–23]. Expression of Fto was analyzed with real-time PCR as described above. 3. Results Double immunohistochemical staining revealed that the majority of oxytocin neurons in the PVN and SON colocalized with Fto (Fig. 1). In both males and females the level of colocalization was similar. In the PVN, approximately 75% of oxytocin cells were positive for Fto, whereas just over 50% of SON oxytocin cells were Ftopositive. We did not observe any PVN subdivision-specific variability in colocalization. Real-time PCR analysis of the hypothalamic cell cultures treated with the Fto-containing or ‘‘mock’’ vector showed that Fto overexpression leads to an increase in oxytocin mRNA levels by ca. 50% (P = 0.019; Fig. 2). Fto transfection caused an increase in POMC expression suggesting a trend (P = 0.110), whereas AgRP and NPY mRNA levels were unaffected (Fig. 3). Oxytocin had no effect on Fto gene expression: Hypothalamic cells incubated for 48 h in the medium containing 10 or 100 nmol oxytocin had the same Fto mRNA levels as cells grown in the control medium (Fig. 4). 4. Discussion The role of Fto in the regulation of energy balance seems to stem to a large extent from its activity within central circuits.
Fig. 1. Fto-oxytocin colocalization. The majority of oxytocin (OT) neurons in the SON (A) and PVN (B) colocalize with Fto. Double immunohistochemistry was used to detect oxytocin (brown cytoplasmic staining) and Fto (black nuclear profiles) in the brain of male (n = 4) and female (n = 4) mice. Data are shown as means ± SEM.
P.K. Olszewski et al. / Biochemical and Biophysical Research Communications 408 (2011) 422–426
Fig. 2. Oxytocin mRNA levels in neurons overexpressing Fto. Hypothalamic neurons transfected with the plasmid containing the Fto cDNA (Fto++) express higher levels of oxytocin mRNA than cells transfected with the control vector (control) in vitro, as determined with real-time PCR (Panel A). They also show higher levels of the Fto signal assessed through Western blotting (Panel B). Data are shown as means ± SEM; – P < 0.05.
Fig. 3. Effect of Fto overexpression on mRNA levels of select feeding-related genes. Neuronal expression levels of proopiomelanocortin (POMC), Agouti-related protein (AgRP) and neuropeptide Y (NPY) encoding genes are unaffected by transfection with the plasmid containing the Fto cDNA (Fto++) versus the control vector (control) in vitro, as determined with real-time PCR. A trend signifying upregulation of POMC mRNA was detected.
Fig. 4. Effect of oxytocin on Fto expression. Hypothalamic neuronal cells cultured in the medium containing 10 nmol (A) or 100 nmol (B) oxytocin (OT) compared to the control medium did not display changes in Fto mRNA levels determined with realtime PCR.
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Real-time PCR studies have shown responsiveness of hypothalamic Fto mRNA levels to organism’s energy status. Immunohistochemical and in situ hybridization analyses have identified brain areas involved in calorie-driven feeding as being particularly rich in Fto signal. The actual link between Fto and central mechanisms controlling energy balance is however unknown, though it has been speculated that this transcription co-activator affects expression of feeding-related genes [24]. The current project defines one such specific relationship, between Fto and a satiety mediator, oxytocin. The majority of oxytocin neurons in the PVN and SON contain Fto-positive nuclei. Since this colocalization is not restricted to any particular subdivision(s) of the PVN, but it rather appears to be uniformly distributed throughout the magno- and parvocellular parts, it is likely that Fto plays a role in transcriptional activity of oxytocin neurons that project to the pituitary as well as those that innervate the brain stem and other central areas. Neurotoxin, tracing and pharmacological experiments have identified the hypothalamus-brainstem oxytocin pathway as a key element of central mechanisms shaping a satiety response [17,18,25]. Here, we report that Fto is expressed in a large percentage of oxytocin neurons, including those that belong to feeding-related subpopulations projecting to the hindbrain. This is in concert with our previous observation that c-Fos-positive Fto cells in the PVN at the end of a deprivation-induced meal show a distribution pattern resembling localization of oxytocin neurons [10]. Importantly, we found that the level of Fto-oxytocin colocalization is identical in males and females. We have thus expanded on our previous findings that Fto immunoreactivity is widespread throughout the brain, by providing evidence that the protein is distributed very similarly in the male and female murine central nervous system (see also the Supplement where Fto immunoreactivity alone is mapped). Some gender differences that have been seen in the context of genetic variants of FTO on human metabolic phenotypes [26,27] seem thus unrelated to sex differences in the basic protein expression pattern in the brain. Since the high level of colocalization between Fto and oxytocin indicated that there are solid neuroanatomical bases for a possible interaction, we sought to obtain functional evidence whether this interaction indeed exists. Therefore, we increased Fto levels in cultured hypothalamic cells by transfecting them with a vector containing the Fto gene (versus a ‘‘mock’’ vector) and studied the relative expression of oxytocin mRNA. We found a ca. 50% upregulation of the oxytocin gene in these cells. Importantly, the link between Fto and oxytocin appears specific: among the three additional feeding-related transcripts analyzed alongside oxytocin, only POMC showed a trend suggesting upregulation (and this finding reflects previously published neuroanatomical descriptions [7]), whereas NPY and AgRP mRNA levels were unchanged in the transfected cells. Our data imply that Fto does not act as a promiscuous co-activator of gene expression, but that there is at least some selectivity pertaining to which genes are affected. It remains to be elucidated whether the effect of Fto on expression of oxytocin (and likely, other mRNAs, considering that Fto was present in many other populations of cells, not just those synthesizing oxytocin) is direct or indirect. Nonetheless, theoretical modeling (through databases such as Coexprsdb and FunCoup) of networks of Fto’s functional interactions with other genes strongly suggests high complexity of such networks and, hence, may involve multiple mechanisms/pathways (data not shown). This may serve as an explanation of why treating cultured hypothalamic cells with oxytocin at concentrations known to induce secretory activity was not sufficient to cause a detectable negative feedback response in the Fto gene expression level. It should also be noted that although we discuss here the link between Fto and oxytocin only from the standpoint of a feeding control-related interaction, this relationship may affect other known roles of oxytocin that have not yet
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been studied in conjunction with Fto. Therefore, our data are of potential importance to understand oxytocin gene expression regulation in a much broader context than food intake alone. In sum, we show that the majority of oxytocin neurons in the male and female mouse brain coexpress Fto. This transcription co-activator up-regulates oxytocin mRNA levels. The relationship between Fto and oxytocin could thus be an important mechanism through which Fto exerts its effect on energy balance. Acknowledgments The studies were supported by the Swedish Research Council (Medicine), Swedish Brain Research Foundation, Novo Nordisk Foundation, National Institute of Drug Abuse, and National Institute of Diabetes and Ingestive and Kidney Diseases. We thank Kedar Ghimire for technical help with Western blotting. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.04.037. References [1] T.M. Frayling, N.J. Timpson, M.N. Weedon, et al., A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity, Science 316 (2007) 889–894. [2] C. Dina, D. Meyre, S. Gallina, et al., Variation in FTO contributes to childhood obesity and severe adult obesity, Nat. Genet. 39 (2007) 724–726. [3] J.A. Jacobsson, J. Klovins, I. Kapa, et al., Novel genetic variant in FTO influences insulin levels and insulin resistance in severely obese children and adolescents, Int. J. Obes. (Lond.) 32 (2008) 1730–1735. [4] A. Hinney, T.T. Nguyen, A. Scherag, et al., Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants, PLoS One 2 (2007) e1361. [5] S.C. Hunt, S. Stone, Y. Xin, et al., Association of the FTO gene with BMI, Obesity 16 (2008) 902–904 (Silver Spring). [6] A. Scuteri, S. Sanna, W.M. Chen, et al., Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits, PLoS Genet. 3 (2007) e115. [7] J. Fischer, L. Koch, C. Emmerling, et al., Inactivation of the Fto gene protects from obesity, Nature 458 (2009) 894–898. [8] C. Church, S. Lee, E.A. Bagg, et al., A mouse model for the metabolic effects of the human fat mass and obesity associated FTO gene, PLoS Genet. 5 (2009) e1000599. [9] C. Church, L. Moir, F. McMurray, et al., Overexpression of Fto leads to increased food intake and results in obesity, Nat. Genet. 42 (2010) 1086–1092.
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Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Fto colocalizes with a satiety mediator oxytocin in the brain and upregulates oxytocin gene expression Pawel K. Olszewski a,b,⇑, Robert Fredriksson a, Jenny D. Eriksson a, Anaya Mitra c, Katarzyna J. Radomska a, Blake A. Gosnell c, Maria N. Solvang a, Allen S. Levine b,c, Helgi B. Schiöth a a b c
Department of Neuroscience, Functional Pharmacology, Uppsala University, 75124 Uppsala, Sweden Minnesota Obesity Center, Saint Paul, MN 55108, USA Department of Food Science and Nutrition, Saint Paul, MN 55108, USA
a r t i c l e
i n f o
Article history: Received 31 March 2011 Available online 13 April 2011 Keywords: Obesity Feeding Brain
a b s t r a c t Single nucleotide polymorphisms in the fat mass and obesity-associated (FTO) gene have been associated with obesity in humans. Alterations in Fto expression in transgenic animals affect body weight, energy expenditure and food intake. Fto, a nuclear protein and proposed transcription co-factor, has been speculated to affect energy balance through a functional relationship with specific genes encoding feedingrelated peptides. Herein, we employed double immunohistochemistry and showed that the majority of neurons synthesizing a satiety mediator, oxytocin, coexpress Fto in the brain of male and female mice. We then overexpressed Fto in a murine hypothalamic cell line and, using qPCR, detected a 50% increase in the level of oxytocin mRNA. Expression levels of several other feeding-related genes, including neuropeptide Y (NPY) and Agouti-related protein (AgRP), were unaffected by the FTO transfection. Addition of 10 and 100 nmol oxytocin to the cell culture medium did not affect Fto expression in hypothalamic cells. We conclude that Fto, a proposed transcription co-factor, influences expression of the gene encoding a satiety mediator, oxytocin. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction There is a strong link between the fat mass and obesity-associated (FTO) gene and the control of energy balance. Single nucleotide polymorphisms in this gene are associated with human obesity [1– 6]. Animal experiments indicate that the relationship between Fto and body weight is complex and it involves mechanisms regulating energy metabolism and food intake. Fto knockout mice have a lower body weight yet they eat more food [7], whereas animals with a dominant point mutation in the Fto gene display reduced fat mass and increased energy expenditure [8]. Mice carrying additional copies of the Fto gene are heavier and eat significantly more than controls [9]. In wild-type mice and rats, food restriction leads to changes in hypothalamic FTO mRNA levels; and so does the treatment of hypothalamic explants with the anorexigenic amino acid, leucine [10]. It is not understood what physiological mechanisms underlie Fto’s involvement in energy homeostasis. Fto is a demethylase Abbreviations: AgRP, Agouti-related protein; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular hypothalamic nucleus; SON, supraoptic nucleus. ⇑ Corresponding author at: Department of Neuroscience, Functional Pharmacology, Uppsala University, BMC, 75124 Uppsala, Sweden. Fax: + 46 18511540. E-mail address: [email protected] (P.K. Olszewski). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.037
[11,12], but it may also serve as a transcriptional co-activator [13]. In vitro expression of Fto results in localization of the recombinant protein to the nucleus [14] and, in the brain – where this gene is most abundantly expressed – Fto immunoreactivity is predominantly present in neuronal nuclei [15,16]. It has been suggested that the Fto protein affects energy homeostasis by regulating expression of other genes involved in this process [12]. In fact, Fto mRNA has been found in neurons positive for transcripts/peptides known to regulate feeding, including oxytocin and proopiomelanocortin (POMC) [7,10,15]. Fto deficiency reduces expression of genes encoding neuropeptide Y (NPY) and POMC [7]. The link with oxytocin appears particularly interesting in the functional context: hypothalamic Fto mRNA-positive neurons display an increased level of colocalization with an immediate-early gene product, c-Fos, at meal termination compared to initiation [10]. The distribution of c-Fos immunoreactive Fto cells in the paraventricular and supraoptic nuclei at the end of a meal closely resembles the topography of neuronal populations synthesizing a satiety mediator, oxytocin [10,17,18]. The current project was aimed at exploring the presumed functional relationship between Fto and oxytocin. We used double immunohistochemistry to examine the percentage of oxytocin neurons containing nuclear profiles positive for the Fto protein in the male and female murine brain. We examined the level of
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colocalization in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, the two main sites encompassing oxytocin neuronal populations. We then overexpressed Fto in a mouse hypothalamic cell line and employed real-time PCR to study whether overexpression of this transcription co-activator affects oxytocin mRNA levels. Relative expression of other feeding-related candidate interactor genes: POMC, NPY and Agouti-related protein (AgRP), was also determined. Finally, we evaluated whether addition of oxytocin to the culture medium serves as negative feedback for the expression of Fto in hypothalamic cells.
control (mock) vector to reveal a more pronounced signal in Ftotransfected cells. Since our Fto + oxytocin staining was done in male and female brains and our published Fto data had defined only Fto distribution in males, we performed an additional control single staining for Fto in females to exclude sex-related neuroanatomical differences in localization of this protein. Through a visual comparison of sections stained with the same antibody we did not detect any obvious differences in central Fto topography between males and females (see Supplement).
2. Materials and methods
2.2. Expression of oxytocin and other feeding-related genes in the hypothalamic cell line transfected with Fto
2.1. Fto -oxytocin colocalization assessment 2.1.1. Animals and perfusions Adult C57BL/6 J male (n = 4) and female (n = 4) mice (Scanbur, Sweden) weighing 32 g were housed individually in macrolon cages with LD 12:12 (lights on at 06:00; T = 21 °C). Water and standard chow (Lactamin, Sweden) were available ad libitum. All procedures were approved by the Uppsala Animal Welfare Committee. Mice were anesthetized with sodium pentobarbital (90 mg/kg IP) and intracardially perfused with 10 ml saline followed by 50 ml 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The animals were sacrificed between 11:00 and 12:00. Brains were excised, postfixed for 24 h in the same fixative, sectioned and processed to detect oxytocin and Fto. 2.1.2. Immunohistochemistry Coronal sections (50 lm) were cut with a Vibratome. Every fourth section encompassing the PVN and SON, the sites where oxytocin is synthesized, was processed using standard double immunohistochemistry. Sections were treated for 10 min in 3% H2O2 and 10% methanol (in TBS, pH 7.4) and incubated for 24 h at 4 °C in the rabbit Fto antibody (1:2000; Inovagen, Sweden). Subsequently, the tissue was incubated for 1 h in the goat anti-rabbit antibody (1:400; Vector, Burlingame, CA) and then in the avidin– biotin complex (1:800; ABC Elite; Vector, Burlingame, CA). Peroxidase was visualized with 0.05% DAB, 0.35% nickel sulfate and 0.01% H2O2. Following the completion of the Fto staining, we applied the same protocol to detect oxytocin; however, the primary oxytocin antibody (Millipore, Temecula, CA; 1:15,000) and the DAB solution without nickel sulfate were used. The vehicle for incubations was a mixture of 0.25% gelatin and 0.5% Triton X-100 in TBS. Rinsing was done in TBS alone. Sections were mounted, dehydrated in alcohol, soaked in xylene, and embedded in DPX (BDH, UK). Evaluation of staining was based on the visual inspection of sections containing neuronal cell bodies positive for oxytocin. A percentage of oxytocin neurons co-expressing Fto was established for the PVN and SON in each animal and the results were averaged for males and females separately. Percentages of Fto immunoreactive oxytocin neurons are presented as means ± SEM. 2.1.3. Controls for specificity of the Fto antibody Additional test data for the specificity of this commercially available antibody (aside from those performed routinely by the manufacturer) have been published [16]. They included: (1) staining sections in the primary antibody solution preincubated for 3 h at 4 °C with the protein that the antibody had been raised against (Ab:Ag 1:5) resulting in no positive signal; (2) performing the BLAST search of the peptide sequence to confirm the uniqueness of the epitope binding site and exclude cross binding; and (3) performing the Western blot of Fto in mouse cells transfected with a pcDNA3 vector encoding N-terminal flag-tagged Fto versus the
2.2.1. Fto overexpression in hypothalamic cells in vitro We have used a previously described protocol for overexpressing the Fto gene in cell lines [16]. In short, we transfected the embryonal hypothalamic neuronal line (Cedarlane, Canada) with a pcDNA3 expression vector coding for N-terminal flag-tagged Fto and compared the results with those obtained in cells transfected with the control (mock) vector. Cells were cultured in the complete Dulbecco’s Modified Eagle Medium (high glucose, Gibco) supplemented with 1% Pen/Strep (50 U/ml penicillin and 50 lg/ ml streptomycin) and 10% FBS (Gibco). They were passaged onto new plates after reaching 70–80% confluency using trypsin (0.5 mg/ml, Invitrogen)/EDTA (0.2 mg/ml, Invitrogen). The cells were transfected at 70–80% confluency using Attractene according to the manufacturer’s protocol (Qiagen). Transfection efficiency was at least 65% as determined by immunostaining using the anti-flag antibody with the GFP-conjugated secondary antibody (as described in [16]). An increase in Fto protein level in transfected cells was confirmed also with Western blotting. 225 lg (15 lg/ll) of protein was loaded into wells as determined using DC protein assay (Bio-Rad). Proteins were transferred from the gel (Criterion precast 10% polyacrylamide gel, BioRad) to membranes (0.45 lm PVDF Immobilon-P, Millipore) using electroblot. Membranes were blocked with 1% non-fat dry milk in TTBS (TBS with 0.5% Tween 20) and incubated with the primary Fto or anti-flag antibody (1:2500) in TTBS overnight. After incubation, the filters were washed 3 10 min with TTBS. The filters were then incubated in the secondary antibody (ECL-Anti-Rabbit IgG, horseradish peroxidase-linked, Amersham) diluted 1:10,000 in TTBS for 60 min and washed in TTBS. Membranes were developed with the Immun-Star HRP Chemiluminescent Kit (Bio-Rad) and ECL hyperfilm (Amersham). The visual analysis revealed a much stronger signal in Fto-transfected cells in which the gene was overexpressed compared to control cells; anti-Fto and anti-flag antibodies produced comparable bands (an example of the Fto signal detected with Western blotting is shown in Fig. 2). 2.2.2. Real-time PCR analysis of expression of genes encoding oxytocin and other feeding-related genes, POMC, NPY and AgRP, in Ftotransfected cells Six separate hypothalamic cell cultures were transfected with the Fto containing vector, whereas the other six were transfected with the mock vector. RNA was extracted 48 h later and cDNA was synthesized as described before [19]. Cells were immersed in TRIzol (Invitrogen, Sweden). RNA was extracted with chloroform and precipitated with isopropanol. The samples were centrifuged and the pellet was washed, air dried, and dissolved in the 1 deoxyribonuclease buffer. The samples were incubated with ribonuclease-free deoxyribonuclease I (37 °C, 90 min; Roche, Sweden). The absence of genomic DNA was established by PCR: 0.5 ll template was mixed in a final volume of 10 ll 1 PCR mix [1 ll MgCl2-free buffer 10, 0.3 ll 50 mM MgCl2, 0.25 ll 1% Tween, 0.1 ll 20 mM dNTP, 1 ll primer mix (forward and backward at
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10 pmol/ll), 0.1 ll Taq polymerase, 5 U/ll (Biotools, Spain), and 6.75 ll MilliQ H2O]. 0.5 ll 100 ng/ll genomic DNA served as a positive control; 0.5 ll MilliQ H2O was added in a negative control. The product was analyzed with electrophoresis. Total RNA concentration was measured with Nanodrop ND-1000 spectrophotometer. For cDNA synthesis, 5-lg RNA samples were diluted in MilliQ H2O to 12 ll. RNA was reverse-transcribed in 20 ll 1 Mastermix (4 ll 5 first-strand buffer, 2 ll 0.1 M dithiothreitol, 0.5 ll 20 mM dNTP, 0.5 ll N6 1/6.25 and 1 ll MMLV reverse transcriptase). Samples were incubated at 37 °C for 1 h, followed by PCR (as above) to confirm cDNA synthesis. 25 ng cDNA template from each sample was used per primer (primers for oxytocin, POMC, NPY and AgRP). Each reaction (20 ll) utilized 2 ll MgCl2-free buffer 10, 0.2 ll 20 mM dNTP, 1.6 ll 50 mM MgCl2, 0.05 ll of a primer at 100 pmol/ll (forward and reverse), 1 ll dimethyl sulfoxide, 0.5 ll Sybr Green (1:50,000), 0.08 ll Taq polymerase (5 U/ll) (Biotools), and 9.52 ll MilliQ water. All PCRs were done in duplicates with negative controls included on each plate. Amplification was performed: denaturation at 95 °C (3 min), 50 cycles of denaturing at 95 °C (15 s), annealing at temperatures established for the primers (15 s), and extension at 72 °C (30 s). Seven housekeeping genes (HKGs) were analyzed (glyceraldehyde-3-phosphate-dehydrogenase, b-tubulin, ribosomal protein 19, histone H3, cyclophilin, b-actin, and succinate dehydrogenase complex subunit B). Data were analyzed with MyiQ 1.04 software (Bio-Rad, Sweden) (30). Primer efficiencies were calculated with LinRegPCR (31), and samples were corrected for differences in primer efficiencies. The GeNorm protocol by Vandesompele et al. [20] was employed to calculate normalization factors based on HKG expression. Grubb’s test was used to identify outliers. Differences between groups were analyzed with Student’s t-test. Values were considered different when P < 0.05.
2.3. Expression of Fto in the hypothalamic cell line treated with oxytocin To investigate whether Fto mRNA level is affected by oxytocin, mouse embryonic hypothalamic neurons (Cedarlane, Canada) were cultured in the DMEM media (2 ml) containing 0 (control; n = 4), 10 (n = 4) or 100 nM/l (n = 4) oxytocin (Sigma, Sweden) for 48 h. These two concentrations of the peptide have been shown to affect activity of cells in vitro [21–23]. Expression of Fto was analyzed with real-time PCR as described above. 3. Results Double immunohistochemical staining revealed that the majority of oxytocin neurons in the PVN and SON colocalized with Fto (Fig. 1). In both males and females the level of colocalization was similar. In the PVN, approximately 75% of oxytocin cells were positive for Fto, whereas just over 50% of SON oxytocin cells were Ftopositive. We did not observe any PVN subdivision-specific variability in colocalization. Real-time PCR analysis of the hypothalamic cell cultures treated with the Fto-containing or ‘‘mock’’ vector showed that Fto overexpression leads to an increase in oxytocin mRNA levels by ca. 50% (P = 0.019; Fig. 2). Fto transfection caused an increase in POMC expression suggesting a trend (P = 0.110), whereas AgRP and NPY mRNA levels were unaffected (Fig. 3). Oxytocin had no effect on Fto gene expression: Hypothalamic cells incubated for 48 h in the medium containing 10 or 100 nmol oxytocin had the same Fto mRNA levels as cells grown in the control medium (Fig. 4). 4. Discussion The role of Fto in the regulation of energy balance seems to stem to a large extent from its activity within central circuits.
Fig. 1. Fto-oxytocin colocalization. The majority of oxytocin (OT) neurons in the SON (A) and PVN (B) colocalize with Fto. Double immunohistochemistry was used to detect oxytocin (brown cytoplasmic staining) and Fto (black nuclear profiles) in the brain of male (n = 4) and female (n = 4) mice. Data are shown as means ± SEM.
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Fig. 2. Oxytocin mRNA levels in neurons overexpressing Fto. Hypothalamic neurons transfected with the plasmid containing the Fto cDNA (Fto++) express higher levels of oxytocin mRNA than cells transfected with the control vector (control) in vitro, as determined with real-time PCR (Panel A). They also show higher levels of the Fto signal assessed through Western blotting (Panel B). Data are shown as means ± SEM; – P < 0.05.
Fig. 3. Effect of Fto overexpression on mRNA levels of select feeding-related genes. Neuronal expression levels of proopiomelanocortin (POMC), Agouti-related protein (AgRP) and neuropeptide Y (NPY) encoding genes are unaffected by transfection with the plasmid containing the Fto cDNA (Fto++) versus the control vector (control) in vitro, as determined with real-time PCR. A trend signifying upregulation of POMC mRNA was detected.
Fig. 4. Effect of oxytocin on Fto expression. Hypothalamic neuronal cells cultured in the medium containing 10 nmol (A) or 100 nmol (B) oxytocin (OT) compared to the control medium did not display changes in Fto mRNA levels determined with realtime PCR.
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Real-time PCR studies have shown responsiveness of hypothalamic Fto mRNA levels to organism’s energy status. Immunohistochemical and in situ hybridization analyses have identified brain areas involved in calorie-driven feeding as being particularly rich in Fto signal. The actual link between Fto and central mechanisms controlling energy balance is however unknown, though it has been speculated that this transcription co-activator affects expression of feeding-related genes [24]. The current project defines one such specific relationship, between Fto and a satiety mediator, oxytocin. The majority of oxytocin neurons in the PVN and SON contain Fto-positive nuclei. Since this colocalization is not restricted to any particular subdivision(s) of the PVN, but it rather appears to be uniformly distributed throughout the magno- and parvocellular parts, it is likely that Fto plays a role in transcriptional activity of oxytocin neurons that project to the pituitary as well as those that innervate the brain stem and other central areas. Neurotoxin, tracing and pharmacological experiments have identified the hypothalamus-brainstem oxytocin pathway as a key element of central mechanisms shaping a satiety response [17,18,25]. Here, we report that Fto is expressed in a large percentage of oxytocin neurons, including those that belong to feeding-related subpopulations projecting to the hindbrain. This is in concert with our previous observation that c-Fos-positive Fto cells in the PVN at the end of a deprivation-induced meal show a distribution pattern resembling localization of oxytocin neurons [10]. Importantly, we found that the level of Fto-oxytocin colocalization is identical in males and females. We have thus expanded on our previous findings that Fto immunoreactivity is widespread throughout the brain, by providing evidence that the protein is distributed very similarly in the male and female murine central nervous system (see also the Supplement where Fto immunoreactivity alone is mapped). Some gender differences that have been seen in the context of genetic variants of FTO on human metabolic phenotypes [26,27] seem thus unrelated to sex differences in the basic protein expression pattern in the brain. Since the high level of colocalization between Fto and oxytocin indicated that there are solid neuroanatomical bases for a possible interaction, we sought to obtain functional evidence whether this interaction indeed exists. Therefore, we increased Fto levels in cultured hypothalamic cells by transfecting them with a vector containing the Fto gene (versus a ‘‘mock’’ vector) and studied the relative expression of oxytocin mRNA. We found a ca. 50% upregulation of the oxytocin gene in these cells. Importantly, the link between Fto and oxytocin appears specific: among the three additional feeding-related transcripts analyzed alongside oxytocin, only POMC showed a trend suggesting upregulation (and this finding reflects previously published neuroanatomical descriptions [7]), whereas NPY and AgRP mRNA levels were unchanged in the transfected cells. Our data imply that Fto does not act as a promiscuous co-activator of gene expression, but that there is at least some selectivity pertaining to which genes are affected. It remains to be elucidated whether the effect of Fto on expression of oxytocin (and likely, other mRNAs, considering that Fto was present in many other populations of cells, not just those synthesizing oxytocin) is direct or indirect. Nonetheless, theoretical modeling (through databases such as Coexprsdb and FunCoup) of networks of Fto’s functional interactions with other genes strongly suggests high complexity of such networks and, hence, may involve multiple mechanisms/pathways (data not shown). This may serve as an explanation of why treating cultured hypothalamic cells with oxytocin at concentrations known to induce secretory activity was not sufficient to cause a detectable negative feedback response in the Fto gene expression level. It should also be noted that although we discuss here the link between Fto and oxytocin only from the standpoint of a feeding control-related interaction, this relationship may affect other known roles of oxytocin that have not yet
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been studied in conjunction with Fto. Therefore, our data are of potential importance to understand oxytocin gene expression regulation in a much broader context than food intake alone. In sum, we show that the majority of oxytocin neurons in the male and female mouse brain coexpress Fto. This transcription co-activator up-regulates oxytocin mRNA levels. The relationship between Fto and oxytocin could thus be an important mechanism through which Fto exerts its effect on energy balance. Acknowledgments The studies were supported by the Swedish Research Council (Medicine), Swedish Brain Research Foundation, Novo Nordisk Foundation, National Institute of Drug Abuse, and National Institute of Diabetes and Ingestive and Kidney Diseases. We thank Kedar Ghimire for technical help with Western blotting. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2011.04.037. References [1] T.M. Frayling, N.J. Timpson, M.N. Weedon, et al., A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity, Science 316 (2007) 889–894. [2] C. Dina, D. Meyre, S. Gallina, et al., Variation in FTO contributes to childhood obesity and severe adult obesity, Nat. Genet. 39 (2007) 724–726. [3] J.A. Jacobsson, J. Klovins, I. Kapa, et al., Novel genetic variant in FTO influences insulin levels and insulin resistance in severely obese children and adolescents, Int. J. Obes. (Lond.) 32 (2008) 1730–1735. [4] A. Hinney, T.T. Nguyen, A. Scherag, et al., Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants, PLoS One 2 (2007) e1361. [5] S.C. Hunt, S. Stone, Y. Xin, et al., Association of the FTO gene with BMI, Obesity 16 (2008) 902–904 (Silver Spring). [6] A. Scuteri, S. Sanna, W.M. Chen, et al., Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits, PLoS Genet. 3 (2007) e115. [7] J. Fischer, L. Koch, C. Emmerling, et al., Inactivation of the Fto gene protects from obesity, Nature 458 (2009) 894–898. [8] C. Church, S. Lee, E.A. Bagg, et al., A mouse model for the metabolic effects of the human fat mass and obesity associated FTO gene, PLoS Genet. 5 (2009) e1000599. [9] C. Church, L. Moir, F. McMurray, et al., Overexpression of Fto leads to increased food intake and results in obesity, Nat. Genet. 42 (2010) 1086–1092.
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