accepted preprint first posted on 11 may 2012 as manuscript joe

1

Adiponectin inhibits KiSS-1 gene transcription through AMPK and specificity protein-1 in 1

the hypothalamic GT1-7 neurons 2

Jun-Ping Wen1,2, Chune Liu1, Wen-kai Bi1,Ya-Ting Hu1, Qingshi Chen1, Huibing Huang1, Ji-xing 3

Liang1, Lian-tao Li

1, Li-xiang Lin

1 and Gang Chen

1 4

1. Department of Endocrinology, Fujian Provincial Hospital, Fujian Medical University, 5

2. Department of Endocrinology, North Branch of Fujian Provincial Hospital, Fujian Provincial 6

Geriatric Hospital, Fuzhou 350001, China. 7

Corresponding author: Gang Chen, PhD,MD, 8

Department of Endocrinology, Fujian Provincial Hospital, Fujian Medical University, Fuzhou 9

350001, China. 10

Jun-Ping Wen, PhD, MD 11

Department of Endocrinology, Fujian Provincial Hospital, Fujian Medical University, 12

North Branch of Fujian Provincial Hospital, Fujian Provincial Geriatric Hospital, Fuzhou 350001, 13

China. 14

Tel: (86)591-87557768 ext. 3079; Fax: (86)591-87532356 15

Email: [email protected], [email protected] 16

The first four authors contributed to the study equally. 17

Reprint requests to corresponding author. 18

Short title: Adiponectin inhibits KiSS-1 gene transcription. 19

Keywords: Adiponectin; KiSS-1 gene; AMPK; SP1 20

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of 42 Accepted Preprint first posted on 11 May 2012 as Manuscript JOE-12-0054

Copyright © 2012 by the Society for Endocrinology.

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Abstract 1

Adiponectin secreted from adipose tissues plays a role in regulation of energy homeostasis, food 2

intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin 3

significantly inhibited GnRH secretion from GT1-7 hypothalamic GnRH neuron cells. In present 4

study, we further investigated the effect of adiponectin on the hypothalamic KiSS-1 gene 5

transcription, which is the upstream signal of GnRH. We found that globular adiponectin(gAd) or 6

AICAR, an artificial AMPK activator, decreased KiSS-1 mRNA transcription and promoter 7

activity. Conversely, inhibition of AMPK by Compound C or AMPKα1-SiRNA, augmented 8

KiSS-1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the 9

translocation of specificity protein-1(SP1) from cytoplasm to nucleus; however, Compound C 10

and AMPKα1-SiRNA played an inverse role. Our experiments in vivo demonstrated that the 11

expression of KiSS-1 mRNA was stimulated 2-fold in the Compound C treated rats and 12

decreased about 60-70% in gAd or AICAR treated rats compared with control group. The 13

numbers of kisspeptin immunopositive neurons in the arcuate nucleus (ARC) region of 14

Sprague-Dawley rats mimicked the same trend seen in KiSS-1 mRNA levels in animal 15

groups with different treatments. In conclusion, our results provide the first evidence that 16

adiponectin reduces KiSS-1 gene transcription in GT1-7 cells through activation of AMPK and 17

subsequently decreased translocation of SP1. 18

Keywords: Adiponectin; KiSS-1 gene; AMPK; SP1 19

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1. Introduction 1

Human puberty is a mystifying process involving a complex series of hormonal events. 2

From a general neuroendocrine perspective, puberty is defined by the full activation of the 3

hypothalamic -pituitary-gonadal (HPG) axis (Grumbach 2002; Ojeda, et al. 1986). The onset of 4

puberty is marked by an increase in the secretion of gonadotropin-releasing hormone (GnRH) 5

from the hypothalamus. A crucial role in puberty and reproduction has been recently assigned to 6

a new hypothalamic signaling pathway, KiSS-1/GPR54 system (Seminara, et al. 2003). KiSS-1 7

gene encodes a premature 145-amino-acid protein called kisspeptin, which is an active ligand of 8

GPR54 in human (Kotani, et al. 2001; Muir, et al. 2001; Ohtaki, et al. 2001). The master role of 9

the KiSS-1/GPR54 system in the central regulation of the HPG axis was first unraveled by the 10

finding that inactivating mutations of the GPR54 gene were associated with the lack of puberty 11

onset and hypogonadotropic hypogonadism both in human and in mice (de Roux, et al. 2003; 12

Seminara et al. 2003), which indicates that KiSS-1 and GPR54 are the new gatekeepers of 13

reproduction. 14

Interestingly, hypothalamic KiSS-1 has been proposed as key molecular conduit for 15

relaying a number of peripheral signals into the GnRH system in the HPG axis. These include not 16

only feedback actions of androgen and estrogen (Navarro, et al. 2004; Smith, et al. 2005) but also 17

metabolic cues (Castellano, et al. 2006). Castellano et al. (Castellano, et al. 2005) showed an 18

interaction between energy status and the hypothalamic KiSS-1 system, which may constitute a 19

target for disruption of pubertal development in conditions of negative energy balance. Several 20

neuropeptides and hormones, including leptin, insulin and kisspeptin, have been reviewed 21

involved in communicating energy status to the reproductive axis (Budak, et al. 2006; Hill, et al. 22

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2008). Besides, an increasing body of evidence indicates that adiponectin secreted from adipose 1

tissues could also directly regulate reproductive functions. For instance, our prior research had 2

demonstrated that adiponectin inhibited GnRH secretion in the hypothalamus (Wen, et al. 2008). 3

Adiponectin also inhibits both basal and GnRH-stimulated LH secretion in rat and mouse 4

pituitary cells (Lu, et al. 2008; Rodriguez-Pacheco, et al. 2007). Overexpression of adiponectin 5

impairs female fertility in mice (Campos, et al. 2008). And serum adiponectin levels in boys and 6

girls decrease throughout puberty (Bottner, et al. 2004). These findings draw immediately 7

attention to that the changes of the hypothalamic KiSS-1 gene may be mediated by adiponectin 8

according to metabolic status. 9

Adiponectin, also termed Acrp30 or AdipoQ, was identified in 1995-1996 as an adipocyte 10

specific secreted factor with an N-terminal collagenous domain and a C-terminal globular 11

domain (Maeda, et al. 1996). Adiponectin activates down-stream targets such as AMP-activated 12

protein kinase (AMPK), peroxisome proliferator activated receptor-a (PPAR-a), and mitogen 13

activated protein kinase (MAPK) through binding to its two receptors, AdipoR1 and AdipoR2 14

(Kadowaki and Yamauchi 2005; Yamauchi, et al. 2003). The roles of adiponectin plays in 15

antidiabetic (Yamauchi, et al. 2001), antiinflammatory, antiangiogenic, and antiatherosclerotic 16

effects in peripheral tissues (Brakenhielm, et al. 2004; Goldstein and Scalia 2004) have been 17

reported in the early studies. Recent studies showed adiponectin participates in the regulation of 18

energy homeostasis (Kubota, et al. 2007; Wen, et al. 2010) and GnRH secretion through 19

activation AMPK in the hypothalamus (Wen et al. 2008). However, it has not yet been studied 20

whether and how adiponectin participates in the KiSS-1 expression in the hypothalamus. 21

It was clear that KiSS-1 can be regulated at the transcriptional level by specificity protein-1 22

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(SP1) in different cells (Li, et al. 2007; Mitchell, et al. 2006; Mitchell, et al. 2007). SP1 is a 1

ubiquitously expressed, prototypic C2H2-type zincfinger-containing DNA binding protein that 2

can activate or repress transcription through GC-rich elements in response to physiologic and 3

pathological stimuli. SP1 can be phosphorylated by various kinases at different sites, and the 4

effects of these modifications can lead to its translocation from the cytoplasm to the nucleus, 5

followed by the regulation of gene expression (Majumdar, et al. 2006; Solomon, et al. 2008; Tan 6

and Khachigian 2009). Report(Chu and Ferro 2005) shows that AMPK downstream signaling 7

molecules, such as ERK, can mediate the phosphorytion of transcription factor SP1. 8

In the present study, we investigated the intracellular signaling mechanisms and AMPK 9

activation in adiponectin treated GT1-7 cells, an immortalized hypothalamic KiSS-1gene positive 10

neuron(Chu and Ferro 2005) (Li et al. 2007). We hypothesized that adiponectin dynamically 11

regulated KiSS-1 gene transcription through activation of AMPK and sequentially subcellular 12

compartmentalization of SP1 in GT1-7 cells. To test this hypothesis, we evaluated KiSS-1 gene 13

transcription levels in response to adiponectin, as well as AMPKα1-SiRNA, 14

5-Amino-imidazole-4-carboxamide riboside (AICAR), and Compound C which regulate the 15

activity of AMPK specifically or nonspecifically. In addition, we conducted mechanistic studies, 16

aiming to disclose the mechanism by which adiponectin regulates KiSS-1 gene transcription. 17

18

2. Materials and Methods 19

2.1. Culture of neurons 20

Immortalized hypothalamic neurons GT1-7 cells were provided gratis by Shanghai Clinical 21

Center for Endocrine and Metabolic Diseases, Shanghai Jiaotong University School of Medicine 22

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(Shanghai, China). GT1-7 cells were maintained in 4.5g/l glucose Dulbecco’s modified Eagle’s 1

medium (DMEM) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 2

IU/ml streptomycin (at 37°C in a humidified atmosphere of 5% CO2). Cells were deprived of 3

serum overnight by changing incubation to serum-free medium prior to pharmaceutical exposure. 4

And cells were later exposed to globular adiponectin at concentrations of 0.1, 0.2, 1.0 and 5.0 ug 5

/ml, respectively, for 2, 6, 9, 18 and 24 h,, AICAR (0.1 and 1.0mM) for 2 or 24 h, compound C 6

(20 and 40uM) for 2 or 24 h, and mithramycin A (100 and 200nM) for 24 h. Mouse globular 7

adiponectin (Gingras, et al.) was purchased from R&D systems (Minneapolis, MN, USA), 8

5-amino-imidazole-4-carboxamide riboside (AICAR) and Compoud C from Sigma (Fluka 9

Chemie, USA) and mithramycin A from AppliChem (Germany). 10

11

2.2 Animals 12

Female Sprague-Dawley rats (5-6 months) were obtained from slac laboratory animal Inc, 13

shanghai, China. All animals were housed under an environment (12 h light/dark cycles; 20-24°C; 14

humidity 55±10%) with free access to food and water. The female rats having shown two 15

consecutive estrous cycles were bilaterally ovariectomized (OVX), and allowed to recover for 14 16

days. Animals were anaesthetized under intraperitoneal injection of pentobarbital anesthesia and 17

given an intracerebroventricular injection (-0.8 mm posterior, 4.0mm ventral to the bregma and 18

1.5mm lateral from the midline) of colchicines at 75µg/15µl using a protocol adapted from 19

Adachi(Adachi, et al. 2007). Two days later, the rats were randomly assigned into 4 groups (n=5) 20

and given intracerebro -ventricular injection of PBS/DMSO, gAd, AICAR or Compound C, 21

respectively. The method was described in Table 1. Three hours later, animals were perfused 22

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200ml 0.1M PBS and 200ml cold 4% paraformaldehyde in 0.1M PBS (pH7.4) through left 1

atrium in succession. Brains were immersed in paraformaldehyde for 12 h followed 25% 2

sucrose/0.1M PBS for 2 days. Fixed brain tissue was sectioned coronally at 30um using a sliding 3

microtome. All procedures were approved by the Animal Care Committee of Fujian Medical 4

University in accordance with the National Institutes of Health Guide for Care and Use of 5

Laboratory Animals. 6

7

2.3. Isolation of RNA and cDNA synthesis 8

GT1-7 cells, as well as hypothalamic tissues from female Sprague-Dawley rats were used in 9

this process. For Sprague-Dawley rats, their brain tissues were immediately removed on ice after 10

decapitation, the tissue of the ARC area was then dissected out of the brain with a micro-knife. 11

RNA extraction was conducted according to manufacturer’s instructions using TRIZOL reagent 12

(Invitrogen, Carlsbad CA). All RNA samples went through DNAse enzymatic digestion (Qiagen, 13

Carlsbad CA) to eliminate DNA contamination. In a subsequent analysis, cDNA was synthesized 14

based on 1ug total RNA from each sample, using the TaqMan Reverse Transcription kit 15

(Promega, CA) following manufacturer’s condition (60 min at 42°C, 5 min at 95°C, 5 min at 16

4°C). 17

18

2.4. Real-time quantitative PCR 19

Real-time quantitative PCR was carried out to evaluate KiSS-1 gene expression levels in the 20

GT1-7 cells and the hypothalamic tissues of Sprague-Dawley rats posterior to pharmaceutical 21

exposures (globular adiponectin, AICAR and Compound C). The extraction of RNA and 22

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synthesis of first-strand cDNA were as mentioned above. The conditions of this real-time PCR 1

were described as follows: 20ul reactions containing 4ul cDNA, 400nM primer pairs, and 10ul 2

SYBR green PCR master were mixed in 96 well plates on an ABI PRISM7300 sequence 3

detection system (Applied Biosystems, Foster City, CA). To normalize the quantity of cDNA in 4

the reaction, a housekeeping gene β-actin was run in all reactions separately under the same 5

experimental conditions. The primers for the PCR are KiSS-1 (forward 6

5’-GCTGCTGCTTCTCCTCTGTG-3’and reverse 5’-CTGCATACCGCGATTCCTT-3’ ) and 7

β-actin (forward 5’-GGCTGTATTCCCCTCCATCG-3’ and reverse 5’-CCAGTTGGTAA 8

CAATGCCATGT-3’). The PCR cycling conditions were 95°C for 10 min, followed by 40 cycles 9

at 95 °C for 5 sec and 60°C for 30 sec. Dissociation curve analysis was also carried out for each 10

gene at the end of the PCR. The raw data from each experiment was used to determine the 11

relative levels of expression for the gene by the -△△CT method. 12

13

2.5. Transient transfection and luciferase assay 14

Cells were cultured on 24-well plates in DMEM supplemented with 10% FBS for at least 24 15

h. When cells approximately reached 60% confluence, transient transfection process started. 16

PGL3-KiSS-1 promoter (provided gratis by Shanghai Clinical Center for Endocrine and 17

Metabolic Diseases, and the human KiSS-1 promoter was cloned from BAC clone RP11-203F10 18

using primers consisting of XhoI and KpnI sites for ligation into the pGL3-basic vector, as 19

previously described ) (Mitchell et al. 2007) was transfected with the aid of Lipofectamine 2000 20

transfection reagent (LF2000; Invitrogen Life Technologies) according to the manufacturer’s 21

protocol. To internally control the transfection efficiency, cells were cotransfected with pRL-TK 22

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(Promega), an expression vector for Renilla luciferase under the control of a thymidine kinase 1

promoter. This TK promoter would nullify any effect of adiponectin on the expression of 2

luciferase. Premixed DNA and Lipofectamine 2000 were applied to the cells for an 18 h term of 3

transfection. Cells were then incubated with globular adiponectin, AICAR, compound C, 4

AMPKα1-SiRNA, SP1SiRNA and mithramycin A for a further 24 h term. Cell lysates were 5

prepared using reporter lysates buffer (Promega) and other routine techniques such as 6

centrifugation (18,000 rpm for 5 min). Aliquots of cell lysates (20ul) were used to mix with 7

luciferase substrate for luminescent measurement (Berthold Detection Systems). 8

9

2.6. Western blot analysis 10

GT1-7 cells were harvested and lysed in radioimmunoprecipitation (RIPA) buffer 11

containing protease inhibitors for total cell protein extract. Nuclear and cytoplasmic proteins 12

were extracted using a kit (Beyotime Institute of iotechnology, Jiangsu, China). In brief, 13

after the cells were treated as indicated, cytoplasmic proteins were prepared by 14

repeating cycles of freezing and thawing in 0.2 ml of cold uffer A. Nuclear protein was extracted 15

by using ice-cold buffer C. Total cellular extracts (40ug of protein) were separated in 10% 16

SDS-PAGE gels, and then electrophoretically transferred onto polyvinylidene difluoride 17

membranes (Millipore Bedford, MA, USA). The membranes were incubated first with blocking 18

buffer (Tris-buffered saline containing 0.05% Tween 20 and 10% nonfat milk) for 3h at room 19

temperature. Primary polyclonal antibodies against SP1, β-actin, β-tubulin (Santa Cruz 20

Biotechnology, CA, USA) and Lamin B1(Bioworld Technology, USA) were applied overnight at 21

4°C. After three washes, the membranes were subsequently incubated with secondary antibody 22

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conjugated with HRP (invitrogen) for 2h at room temperature. The blots were visualized by ECL 1

using Kodak X-OMATLS film (Eastman Kodak). Quantitative data were obtained using a 2

computing densitometer and ImageQuant software. 3

4

2.7. Fluorescence microscopy 5

Cells were cultivated on coverslips, washed in PBS three times (5 min each), and fixed in 6

10% paraformaldehyde (10 min). Cells were washed three times in PBS (5 min each), and made 7

permeable in 0.3% Triton X-100 for 15 min, then washed three times in PBS (5 min each). To 8

block nonspecific antibody-binding sites, cells were incubated in 10% goat serum albumin in 9

PBS for 1 h at room temperature. The cells were then incubated overnight at 4°C with rabbit 10

polyclonal antibodies to SP1, washed with PBS for three times (5 min each), and incubated for 11

1h at 37°C with fluorescein isothiocyanate-conjugated goat antibodies to rabbit immunoglobulin 12

G (ZSGB-BIO, China)(green, 1:250) in the dark. The cells were washed three times with PBS 13

again and then were stained by 1-Aminopyridinium iodide for 30 min in the dark, followed by 14

three washes in PBS. Coverslips were air-dried and mounted with anti-fading glycerinum, then 15

mounted on glass slides, finally observed with a fluorescence microsc. 16

17

2.8. Immunohistochemistry 18

Brain tissues of the sacrificed rats were immersed in paraformaldehyde for 12 h followed 19

25% sucrose/0.1M PBS for 2 days. Fixed brain tissue was sectioned coronally at 30um using a 20

sliding microtome. Then the sections through ARC was preserved in ethylene glycol 21

cryoprotectant at -20°C until use. The free-floating sections were rinsed for three times (10 min 22

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each) in 0.01 M PBS and incubated in 3% H2O2/PBS for 10 min. Before incubation in the 1

primary antiserum, the sections were rinsed and treated in the blocking solution (PBS with 0.3% 2

Triton X-100, 5% sheep serum, and 0.25% bovine serum albumin) at room temperature for 1 h. 3

Then they were incubated at 4°C for 48 h in anti-KiSS-1 rabbit polyclonal primary antibody 4

(1:200,000; antiserum 566, a generous gift from Dr. A. Caraty, NOUZILLY, France). After 5

incubation in primary antiserum, the sections were washed and incubated in secondary 6

biotinylated goat anti-rabbit IgG (1:500; Vector Laboratories, Burlingame, USA) for 2 h. Rinsed 7

again for three times (10 min each), the sections were incubated for 1 h in ABC Elite Kit (Vector 8

Laboratories) at room temperature. Then the free-floating brain sections were stained with DAB/ 9

chromogen solution for 15 min, and sequentially rinsed in PBS for three times (2 min each). At 10

last sections were mounted on gelatinized glass slides, dried, dehydrated in ascending alcohol 11

concentrations, cleared by xylenes, and cover slipped with neutral gum. 12

13

2.9. Cell counting 14

To quantify kisspeptin immunopositive neurons in the ARC, a Nikon microscope attached to a 15

digital camera (NIKON, ECLIPSE-TS100, Japan) was utilized. Cell counting was performed by 16

two researchers. Bilateral cell counts for each section were averaged between researchers. Total 17

cell counts of the ARC (eight sections/rat) were summed and analyzed. 18

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2.10. Statistical analysis 20

All experiments were repeated at least three times. All analyses were performed using the 21

Statistical Package for Social Sciences (SPSS for Windows, version 16.0; Chicago, IL). Data 22

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were log-transformed owing to skewed distributions, and were expressed as mean±SD for 1

quantitative values. Unless otherwise noted, data were analyzed using ANOVA followed by 2

Tukey post hoc tests. The significant differences of variate between two groups were assessed by 3

Student’s t tests. P＜0.05 was considered statistically significant. 4

5

3. Results 6

3.1. Globular adiponectin and AMPK inhibited KiSS-1 gene mRNA transcription in GT1-7 cells 7

GT1-7 cells exposed to 0.1, 0.2, 1.0 and 5.0ug/ml globular adiponectin for 2 h have lower 8

transcription of KiSS-1 mRNA determined by real-time PCR (shown in Fig.1A). The inhibition 9

of KiSS-1 mRNA by adiponectin is in a concentration-dependent manner with statistical 10

significance (P<0.05). 11

AMPK is believed to be a major cellular target of adiponectin and its activation has been 12

reported in GT1-7 cells. In order to discover whether AMPK plays a role in the modulation of 13

KiSS-1 mRNA transcription, we used artificial means to activate or inhibit AMPK on GT1-7 14

cells, and followed it with the quantification of mRNA transcription in our assay. As results 15

shown, AICAR (1mM), an AMPK activator, caused an outstanding decrease in KiSS-1 mRNA 16

transcription (Fig.1B) (P<0.001). Conversely, Compound C (20/40uM), a relatively specific 17

AMPK inhibitor, promoted the transcription of KiSS-1 mRNA with a statistical significance in a 18

concentration-dependent manner (P<0.05). 19

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3.2. Globular adiponectin inhibited KiSS-1 gene promoter activity 21

To examine the transcriptional regulation of KiSS-1 gene in hypothalamus by adiponectin, a 22

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2-kb full-length KiSS-1 gene promoter (PGL3-KiSS-1 promoter) was transiently transfected into 1

GT1-7 cells. Cells were treated with adiponectin at the concentration of 1.0, 5.0, 10.0ug/ml for 24 2

h, or at 1.0ug/ml for 6, 9, 18, 24 h respectively. The promoter activity was significantly decreased 3

in a time-dependent (Fig.2A) (P<0.05) and concentration-dependent manner (Fig.2B) 4

(P<0.05).The promoter activity was significantly suppressed by the administration of globular 5

adiponectin at the lowest concentration of 1.0ug/ml for a 6 h term of incubation, and the activity 6

was further reduced as time extended. 7

8

3.3. AMPK contributed to KiSS-1 gene promoter activity 9

Following to our discovery of AMPK’s involvement in KiSS-1 gene transcription, it is 10

interesting to know whether AMPK functions as usual under some known pathways, or unusually 11

at the transcriptional level. With these questions unaddressed, we transiently transfected the 12

GT1-7 cells with 2-kb PGL3-KiSS-1 promoter. Then pharmacological regulators of AMPK, 13

AICAR and Compound C as well as the sequence specific interfering RNA of AMPKα1subunit, 14

were applied to the GT1-7 cell culture. The results showed that activation of AMPK by AICAR 15

decreased the promoter activity of KiSS-1gene (Fig.2C) (P<0.05). However, the suppression of 16

AMPK by Compound C or AMPKα1-SiRNA enhanced the promoter activity of KiSS-1gene 17

(Fig.2C,D) (P<0.001). We also pre-treated the GT1-7 cells for 30 min with AICAR (1uM) or 18

Compound C (20uM), and then incubated with globular adiponectin (5ug/ml) for 24 h. As shown 19

in Fig.2E, globular adiponectin (5ug/ml) and AICAR (1mM) decrease KiSS-1gene promoter 20

activity, while Compound C (20uM) augmented promoter activity of KiSS-1gene (P<0.05). 21

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3.4. Globular adiponectin regulated subcellular redistribution of SP1 in GT1-7 cells 1

SP1 functions as an important transcriptional factor by binding to GC-rich elements in 2

response to physiologic and pathological stimuli, and it can either activate or repress gene 3

transcription. Previous studies have shown that the mechanism of its gene regulation partially lies 4

in the translocation of SP1 triggered by its posttranslational modifications. And this translocation, 5

from the cytoplasm to the nucleus, is more commonly described as subcellular 6

compartmentalization. 7

This experiment is to investigate whether the sequential subcellular compartmentalization of 8

SP1 is involved in the regulation of KiSS-1 gene in GT1-7 cells after globular adiponectin 9

administration. Western blotting technique was used to observe the changes of SP1 content 10

before and after subcellular compartmentalization. As shown in Fig.3, the nuclear fraction of SP1 11

decreased by 50% compared with the control group; however, the cytoplasmic fraction increased 12

1.2 fold after global adiponectin incubation (10.0ug/ml) (P<0.05), and this increase is 13

corresponding in a dependent manner to the concentration of globular adiponectin administration. 14

Total SP1 content in whole cells remain constant in GT1-7 cells (P=0.55). 15

16

3.5. AMPK regulated subcellular redistribution of SP1 in GT1-7 cells 17

From the above mentioned experiments, it is clear that both AMPK and SP1 are taking roles in 18

regulating KiSS-1 gene transcription. Then our curiosity leads us to question whether AMPK 19

regulates the procedure of subcellular compartmentalization of SP1 or vice versa. We treated the 20

GT1-7 cells with AICAR, Compound C and AMPKα1-SiRNA, then we ran western blot to 21

evaluate the changes of SP1 content upon subcellular compartmentalization. The results showed 22

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that AICAR decreased the nuclear fraction and increased the cytoplasmic fraction of SP1 content 1

(Fig.4A) (P<0.05). Compound C decreased the cytoplasmic SP1 content and increased the 2

nuclear fraction, so did AMPKα1-SiRNA (P<0.05). As shown in Fig.4, pharmacological 3

regulators of AMPK, AICAR and Compound C, as well as AMPKα1-SiRNA had no effect on the 4

total SP1 content in whole GT1-7 cells (P=0.46). 5

6

3.6. Immunofluorescence analysis supported the regulation of subcellular redistribution of SP1 by 7

AMPK 8

Other than western blot analysis, we also performed immunofluorescence analysis to directly 9

visualize the effect of AMPK on the subcellular redistribution of SP1 protein. After incubation 10

with AICAR or Compound C for 24 h, changes in SP1 content were assessed by using highly 11

specific primary antibodies, which was later couple with fluorescent tagged secondary antibodies 12

(Witte, et al.). As Fig.5 fluorescence demonstrates, in response to AICAR treatment, SP1 in the 13

cytoplasm increased greatly, but the SP1 in the nucleus reduced compared with the control group. 14

To the contrary, Compound C demonstrated a reverse effect on the subcellular localization of SP1. 15

It increased the amount of SP1 in the nucleus and decreased the amount of SP1 in the cytoplasm. 16

These findings were in accordance with the results from western blot. 17

18

3.7. SP1 contributed to KiSS-1 promoter activity 19

We performed luciferase assay to demonstrate the important role of SP1 in the KiSS-1 20

promoter activity. The 2-kb full-length KiSS-1 gene promoter (PGL3-KiSS-1 promoter) was 21

transiently transfected into GT1-7 cells. After one passage, we treated the cells with SP1SiRNA 22

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and mithramycin A (known as a pharmacological suppressor of SP1). Fig.6 illustrated that 1

suppression of SP1 resulted in a greatly decrease in the activity of KiSS-1 promoter (P<0.05). 2

Our western blot results demonstrated that the SP1SiRNA we used in the luciferase assay 3

reduced the expression of SP1 dramatically in the GT1-7 cells (P<0.05). 4

5

3.8. SP1 promoted KiSS-1 gene mRNA transcription in GT1-7 cells 6

In order to confirm SP1 has an definitely effect on the expression of KiSS-1 mRNA, real time 7

PCR was carried out to detect the KiSS-1 mRNA levels when SP1 SiRNA was transiently 8

transfected into in GT1-7 cells. SP1 protein was definitely knockdown by SP1SiRNA as shown 9

by western blot in Fig.6. As demonstrated in Fig.7, KiSS-1 mRNA transcription was decreased 10

obviously when the expression of transcription factor SP1 was knockdown by SP1SiRNA 11

(P<0.05). 12

13

3.9. Globular adiponectin and AMPK contributed to KiSS-1 gene mRNA transcription in vivo 14

Except for the in vitro experiments, we used real-time PCR to detect the expression levels of 15

KiSS-1 mRNA in the hypothalamic tissues of Sprague-Dawley rats given intracerebro 16

-ventricular injection of PBS/DMSO, globular adiponectin, AICAR or Compound C. As 17

shown in Fig.8, the expression of KiSS-1 mRNA was stimulated almost 2-fold in the Compound 18

C treated rats (P<0.05). Correspondently, the KiSS-1 mRNA level decreased about 60-70% in 19

globular adiponectin or AICAR treated rats compared with control group (P<0.05). 20

21

3.10. Globular adiponectin and AMPK had an effect on the expression of kisspeptin in the ARC 22

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area of Sprague-Dawley rats 1

We performed immunohistochemistry and counted the cell number of kisspeptin 2

immunopositive neurons to demonstrate globular adiponectin and AMPK play an important role 3

in the expression of kisspeptin in the ARC area of Sprague-Dawley rats. As Fig.9 shown, 4

injection of Compound C increased the number of kisspeptin immunopositive neurons in ARC 5

area of Sprague-Dawley rats (Fig.9 D), while injection of globular adiponectin or AICAR 6

decreased the number of kisspeptin immunopositive neurons (Fig.9 B,C) compared with control 7

group treated with PBS/DMSO with significant difference (P<0.05). 8

9

4. Discussion 10

Puberty is a complicated process that has not been comprehensively understood by human 11

beings ourselves. Lots of factors are believed to play important regulatory roles in this process, 12

which include genetic heritages, environmental effects, neuroendocrinological encounters, 13

metabolic status and other growth related factors. Reproductive function is highly sensitive to 14

changes in the metabolic status and energy reserves of an organism, and adverse metabolic 15

conditions are commonly associated with defective reproductive capability involving the 16

alteration in the hypothalamic KiSS-1 system (Castellano et al. 2006). Kisspeptin, ligand of G 17

protein-coupled receptor 54 (GPR54), encoded by the KiSS-1gene, have recently emerged as 18

major gatekeepers of the gonadotropic axis. Shahab et al. (Shahab, et al. 2005) shows that KiSS-1 19

mRNA levels increases with puberty in both male and female monkeys. In intact females, GPR54 20

mRNA levels in the hypothalamus increased 3-fold from the juvenile to midpubertal stage. 21

Hybridization histochemistry indicated robust KiSS-1 and GPR54 mRNA transcription in the 22

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region of the arcuate nucleus. Dhillo (Dhillo, et al. 2005) reports that systemic administration of 1

kisspeptin to male volunteers results in a significant increase in plasma LH, FSH, and 2

testosterone. Converging evidence (Kauffman 2009; Messager, et al. 2005; Navarro et al. 2004) 3

demonstrates that a key action of KiSS-1/GPR54 system on the hypothalamo-pituitary-gonadal 4

axis occurs directly at the level of GnRH release. It is well established that GPR54-deficient mice 5

and human display phenocopy syndromes characterized as isolated hypogonadotropic 6

hypogonadism (de Roux et al. 2003; Seminara et al. 2003). So KiSS-1/GPR54 represents the 7

molecular ‘‘gatekeepers’’ underlying the onset of pubertal development. 8

Adiponectin is exclusively expressed and secreted by adipose tissue (Kadowaki and 9

Yamauchi 2005). Except for its regulation effect in energy metabolism and insulin sensitivity, 10

adiponectin has also been approved to be involved in antiinflammatory, antiangiogenic, and 11

antiatherosclerotic processes (Brakenhielm et al. 2004; Goldstein and Scalia 2004; Yamauchi et 12

al. 2001). An increasing body of evidence indicates that adiponectin could also directly regulate 13

reproductive functions. Adiponectin also inhibits both basal and GnRH-stimulated LH secretion 14

in rat and mouse pituitary cells (Lu et al. 2008; Rodriguez-Pacheco et al. 2007). Study by 15

Campos et al. sugests that overexpression of adiponectin impairs female fertility in mice 16

(Campos et al. 2008). Bottner’s research shows that serum adiponectin levels in boys and girls 17

decrease throughout puberty (Bottner et al. 2004). 18

In this study, we used globular domain of adiponectin (gAd) to treat GT1-7 cells and found 19

that both transcription levels and promoter activity of KiSS-1gene were significantly inhibited. 20

According to the literature, along with the initiation of puberty, adiponectin levels in men 21

decrease dramatically, while the KiSS-1 gene transcription levels increase rapidly, which also 22

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19

shows the intrinsic relationship may exist between adiponectin and KiSS-1 gene before puberty. 1

AMPK plays as an “energy sensor” in vivo as well as an important signaling molecule mediating 2

the physiological role of adiponectin. Adiponectin facilitates glucose uptake and fatty acid 3

oxidation through activation of AMPK in peripheral tissues (Ceddia, et al. 2005; Wu, et al. 2003; 4

Yamauchi, et al. 2002). Lu et al. (Lu et al. 2008) reported that adiponectin could reduce LH 5

secretion in the pituitary gonadotrophic cells in an AMPK-dependent manner. Additionally, our 6

prior research had demonstrated that adiponectin inhibited GnRH secretion via AMPK in GT1-7 7

cells (Wen et al. 2008). Therefore, AMPK may play as a conjunction between energy metabolism 8

and central reproduction, as well as a mediator for the regulation of metabolism and reproduction 9

by adiponectin. In order to investigate whether the AMPK signaling pathway is involved in the 10

regulation of KiSS-1 gene transcription by adiponectin, we used AMPK agonist AICAR as 11

control to observe their impact on the KiSS-1 gene transcription. The results showed that AICAR 12

also inhibited the KiSS-1 gene transcription and their promoter activity obviously, and the 13

AMPK inhibitor Compound C could increase the KiSS-1 gene transcription and enhance the 14

promoter activity. AMPKα1-SiRNA by RNA interference reduced AMPK α catalytic subunit 15

protein level, significantly enhanced the KiSS-1 gene transcription and promoter activity as 16

Compound C did. 17

Report (Chu and Ferro 2005) shows that AMPK downstream signaling molecules such as 18

ERK can mediate the phosphorytion of transcription factor SP1. SP1 has been proved to be a 19

vital regulator of gene transcription of KiSS-1gene (Li et al. 2007; Mitchell et al. 2006; Mitchell 20

et al. 2007). SP1 is a ubiquitously expressed, prototypic C2H2-type zincfinger-containing DNA 21

binding protein. The primary sequence and domain organization of SP1 protein is highly 22

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20

conserved. In addition to three zinc fingers, SP1 possesses two glutamine-rich modules that 1

together make up their transcriptional activation domains (AD). Adjacent to the two activation 2

domains is a serine/threonine (S/T)-rich module that may be targeted for posttranslational 3

modifications (e.g. phosphorylation or acetylation). Posttranslational modifications of SP1 not 4

only regulate its subcellular compartmentalization but also modulate its interactions with other 5

transcription factors to regulate gene expression. For example, phosphorylation of SP1 leads to 6

its enhanced nuclear migration and results in the decrease in SP1 DNA binding, and hence 7

suppression of transcription activation of EP4 gene in glioblastoma cells (Kambe, et al. 2008). 8

Therefore, we hypothesized that adiponectin regulates the gene transcription of KiSS-1 and the 9

onset of puberty through AMPK and the activity of its downstream signaling molecules. 10

Through the combination of GC/GT box and the interaction with other proteins, SP1 plays an 11

important role in the enhancement or inhibition of gene transcription and has been implicated in 12

the control of a diverse array of cellular processes exemplified by not limited to cell growth, 13

differentiation, apoptosis, angiogenesis, and immune response (Tan and Khachigian 2009). Study 14

by Mitchell et al. (Mitchell et al. 2007) indicate that co-expression of SP1 and DRIP-130, which 15

is encoded by human chromosome 6q16.3-q23 and has been proved to be SP1-coactivator protein, 16

not only rescues KiSS-1 expression, but also induces an inhibition of the invasive and migratory 17

behavior in highly metastatic melanoma cells. Mitchell et al. (Mitchell et al. 2006) has also 18

demonstrated that SP1 directly interacts with activator protein 2α（AP2）to form transcription 19

complexes at two tandem SP1-binding sites of the promoter of KiSS-1 gene which leads to 20

activation of KiSS-1 transcription in breast cancer cells. Dali Li (Li et al. 2007) has found that 21

there are four proximal GC-rich SP1 binding sites at -188 to -87 of the KiSS-1 promoter and the 22

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21

differential regulation of KiSS-1 expression by E2 in different brain regions can be achieved by 1

the different ratio of SP1 to SP3 in the protein complexes. It can be explained by the regulatory 2

mechanism of SP1: 1) SP1 combines to different sites of the KiSS-1 gene promoter, resulting in 3

changes in binding affinity; 2) The role of SP1 in regulation of gene expression depends on its 4

expression level which varies with cell type during different developmental stages; 3) Other 5

factors (as mentioned above involving in DRIP-130, AP-2α, SP3, etc.) can be involved in 6

regulating the transcriptional activity of SP1; 4) as a transcription factor, posttranslational 7

modifications of SP1 play a crucial role for its transcriptional regulatory function. Growing 8

evidence indicates that phosphorylation, acetylation, sumoylation, ubiquitylation, and 9

glycosylation are among the posttranslational modifications that influence the transcriptional 10

activity and stability of SP1. Phosphorylation of SP1 occurs in glutamate-rich region usually 11

enhances transcriptional activation of SP1 in the corresponding pathway, and phosphorylation 12

within the zinc finger always reduces affinity of SP1 to the DNA, thereby reducing 13

transcriptional activity. 14

In our study, the nuclear fraction of SP1 decreased in content while the cytoplasmic fraction 15

increased in a concentration-dependent manner after administration of globular adiponectin in 16

hypothalamic GT1-7 cells. Meanwhile, we didn’t find significant changes in the total cell extracts 17

of SP1 after globular adiponectin treatment in GT1-7 cells. Also in our study, we discovered that 18

AICAR mimicked globular adiponectin in enhancing the cytoplasmic fraction, while Compound 19

C and AMPKα1-SiRNA functioned inversely in the subcellular compartmentalization of SP1 in 20

hypothalamic GT1-7 cells. So we conclude that the changes of subcellular compartmentalization 21

of SP1, instead of the changes of total cell extracts of SP1, take part in the regulations of KiSS-1 22

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22

gene transcription by globular adiponectin and AMPK. Our results showed knockdown of SP1 1

expression by SP1SiRNA induced the inhibitory expression of KiSS-1 mRNA in GT1-7 cells. 2

Besides, the luciferase assay demonstrated that SP1 contributed to promote the promoter activity 3

of KiSS-1 gene, since both SP1 SiRNA and mithramycin A, the pharmacological suppressor of 4

SP1, dramatically suppressed the promoter activity of KiSS-1gene. Collectively, these results 5

demonstrated SP1 could activate KiSS-1 promoter activity and promote KiSS-1 mRNA 6

transcription, knockdown of SP1 induced lower promoter activity of KiSS-1 and reduced 7

expression of KiSS-1 mRNA. The suppressive effect of globular adiponectin and AMPK activitor 8

on KiSS-1 promoter activity and the mRNA transcription could be partly explained by the 9

detained transcriptional factor SP1 in the cytoplasm induced by globular adiponectin and AMPK 10

activitor which mimicked the effect on expression of KiSS-1 mRNA and promoter activity when 11

expression of SP1 was knockdown. While, Compound C or AMPKα1-SiRNA, which inhibited 12

the activity of AMPK nonspecifically or specifically, played an inverse role as globular 13

adiponectin and AICAR did. 14

Except for the in vitro studies, our results from in vivo experiments demonstrated injection 15

of globular adiponectin and AICAR could inhibit the expression of KiSS-1 mRNA and decrease 16

the number of kisspeptin immunopositive neurons in the ARC area of Sprague-Dawley rats, 17

while Compound C performed an inverse role as globular adiponectin and AICAR did, the 18

transcription of KiSS mRNA almost doubled and the number of kisspeptin immunopositive 19

neurons in the ARC area also increased significantly in Sprague-Dawley rats treated with 20

Compound C compared with control group. These results further confirmed the results 21

found in vitro studies. 22

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In summary, our study demonstrated that adiponectin caused the activation of AMPK in the 1

hypothalamic GT1-7 neurons and whereby decreased the translocation of SP1 from cytoplasm to 2

nucleus; as a result, the promoter activity and transcription levels of KiSS-1gene are repressed. 3

This may further influence the hypothalamic release of GnRH and the onset of puberty. To our 4

best knowledge, the data presented here is the first experimental evidence for the characterization 5

of reproductive regulation by adiponectin, as well as the involvement of AMPK and SP1 6

pathways in such a process. However, the limitation of the present study is that only one kind of 7

cell line was utilized, and hence the conclusions of the paper seem a little bit thin. Although we have 8

performed some in vivo studies in Sprague-Dawley rats, further investigations about the 9

molecular mechanism in vivo will be conducted more completely in our future studies. 10

Declaration of interest 11

The authors of this manuscript have nothing to declare. 12

Funding 13

This study was supported by the grants from Natural Science Foundation of China (No.30900510 14

and No.81170774) and Natural Science Foundation of Fujian Province (No. 2011J01127 and 15

No.2011J06012). 16

Acknowledgments 17

Immortalized Gonadotropin-Releasing Hormone Neurons (GT1-7 Cells), the 2-kb 18

full-length KiSS-1 gene promoter (PGL3-KiSS-1 promoter) were provided gratis by Shanghai 19

Clinical Center for Endocrine and Metabolic Diseases, Shanghai Jiaotong University School of 20

Medicine. This manuscript was reviewed for grammatical accuracies by G. Alexander Wen at 21

Lowell General Hospital, Lowell MA. We appreciate all the supports. 22

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Figure legends 1

Fig.1. Regulation of KiSS-1 mRNA expression by different concentrations of gAd or AMPK. 2

GT1-7 cells were serum starved, and then treated with gAd for 2 h in different concentrations 3

(from 0-5 ug/ml) (A) or treated with 1mM AICAR or 20/40uM Compound C for 2 h (B). KiSS-1 4

mRNA levels were determined by realtime-PCR analysis. Results were representative of at least 5

three replicate determinations for each treatment group. *P<0.05, compared with the control 6

group. 7

8

Fig.2. Regulation of KiSS-1 promoter activity by gAd or AMPK. GT1-7 cells were serum starved, 9

and then transiently transfected with 2-kb full-length KiSS-1 pormoter (PGL3-2k), along with a 10

Renilla luciferase to control for transcription efficiency. After 18 h of transfection, cells were 11

treated with gAd (1.0 ug/ml) for different times (A) or at different concentrations for 24 h (B), or 12

treated with 1mM AICAR or 20uM Compound C (C) or AMPKα1-SiRNA (D), or pretreated with 13

AICAR (1mM), Compound C (20uM) for 30 min before incubation with gAd (5.0 ug/ml) for 24 14

h (E). Luciferase expression level driven by KiSS-1 promoter was measured and normalized to 15

Renilla luciferase activity. Results were representative of at least three replicate determinations 16

for each treatment group. *P<0.05, compared with the control group. 17

18

Fig.3 gAd induces subcellular redistribution of SP1 protein. GT1-7 cells were treated with gAd in 19

different concentrations (from1-10ug/ml), after which cell lysates were separated into nuclear and 20

cytoplasmic fractions. The nuclear and cytoplasmic fractions as well as total cell extracts were 21

subjected to western blot analysis with antibodies to SP1 protein , Lamin B1 (nuclear marker), 22

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30

β-tubulin (cytoplasmic marker) or β-actin (total protein marker), as indicated. Results are 1

representative of at least three replicate determinations for each treatment group. *P<0.05, 2

compared with the control group. 3

4

Fig.4 AMPK induces subcellular redistribution of SP1 protein. GT1-7 cells were treated with 5

AICAR (0.1, 1.0mM), Compound C (40uM) (A) or AMPK SiRNA (B), after which cell lysates 6

were separated into nuclear and cytoplasmic fractions. The nuclear and cytoplasmic fractions as 7

well as total protein extracts were subjected to western blot analysis with antibodies to SP1 8

protein, Lamin B1 (nuclear marker), β-tubulin (cytoplasmic marker) or β-actin (total protein 9

marker), as indicated. Results were representative of at least three replicate determinations for 10

each treatment group. *P<0.05, compared with the control group. 11

12

Fig.5. AMPK induces subcellular redistribution of SP1 protein by immunofluorescence analysis 13

(200×). GT1-7 cells were treated with AICAR (1mM) or Compound C (20uM) for 24 h, fixed, 14

immunostained with antibodies to SP1 which was visualized by a secondary antibody coupled to 15

FITC. Nucleus was stained by 1-Aminopyridinium iodide (red), and then examined with a 16

fluorescence microscope. SP1 is distributed in cytoplasm and nucleus of untreated GT1-7 cells 17

(Witte et al.) (a). In response to AICAR treatment, SP1 in the cytoplasm increased greatly (Witte 18

et al.) (b). SP1 in the nucleus increased greatly after treated with Compound C (Witte et al.) (c). 19

Nucleus was stained by 1-Aminopyridinium iodide (red) (d, e, and f). Merged figures with graph 20

a and d, b and e, c and f were respectively shown as graph g, h, and i. Color of SP1 localized in 21

nucleus merged with nucleus stain shows yellow. The yellow color was more deep in Compound 22

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31

C group compared with the control group and AICAR group demonstrated the subcellular 1

localization of SP1(g, h, and i). 2

3

Fig.6. Regulation of KiSS-1 promoter activity by SP1 SiRNA and mithramycin A. Cells were 4

transfected with full-length KiSS-1 promoter (PGL3-2k), along with a Renilla luciferase to 5

control for transcription efficiency, then passaged and transiently transfected with SP1 SiRNA or 6

treated with mithramycin A (100nM, 200nM). Luciferase activity was then assayed. Regulation 7

of SP1 protein levels by SP1 SiRNA from western blot analysis. Results were representative of at 8

least three independent experiments. *P<0.05, compared with the control group. 9

10

11

Fig.7. Regulation of KiSS-1 mRNA expression by SP1 SiRNA. GT1-7 cells were transfected 12

with SP1 SiRNA (SiRNA 1669, SiRNA 965, SiRNA 1867). KiSS-1 mRNA levels were 13

determined by realtime-PCR analysis. Results were representative of at least three replicate 14

determinations for each treatment group. *P<0.05, compared with the control group. 15

16

17

Fig.8. Regulation of KiSS-1 mRNA expression by gAd, AICAR or Compound C in 18

Sprague-Dawley rats. The rats were given intracerebroventricular injection of 19

PBS/DMSO, gAd, AICAR or Compound C, their heads were immediately removed on the 20

ice, then the ARC area was dissected out of the brains, followed by gene expression 21

assays as described in method part. KiSS-1 mRNA levels were determined by realtime-PCR 22

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32

analysis. Results were representative of at least three replicate determinations for each treatment 1

group. *P<0.05, compared with the control group. 2

3

4

Fig.9. Regulation of kisspeptin expression in the ARC area of Sprague-Dawley rats by gAd, 5

AICAR or Compound C. The Sprague-Dawley rats were anaesthetized under intraperitoneal 6

injection of pentobarbital anesthesia and given an intracerebroventricular injection of colchicines at 7

75µg/15µl and followed by intracerebroventricular injection of PBS/DMSO, gAd, AICAR and 8

Compound C, then kisspeptin levels were determined by immunohistochemistry as described in 9

method part. Graph A, B, C and D stands for treatment of PBS/DMSO, gAd, AICAR and 10

Compound C respectively. To quantify kisspeptin immunopositive neurons in the ARC, cell counting 11

was performed by two researchers. Bilateral cell counts for each section were averaged between 12

researchers. Total cell counts of the ARC (eight sections/rat) were summed and analyzed. Results 13

were representative of at least three replicate determinations for each treatment group. *P<0.05, 14

compared with the control group. 15

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254x190mm (96 x 96 DPI)

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529x533mm (96 x 96 DPI)

of 42

254x190mm (96 x 96 DPI)

of 42

159x91mm (96 x 96 DPI)

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146x105mm (96 x 96 DPI)

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254x190mm (96 x 96 DPI)

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Table1. The methods of i.c.v for the four groups

i.c.v

Control Adiponectin

(gAd)

AICAR CompoundC

(Comp C)

Drug(ug)

SolutionⅠ(ul)

----

PBS(10)

gAd (7)

PBS (10)

AICAR(10)

PBS(10)

Comp C (40)

DMSO (10)

SolutionⅡ(ul) DMSO(10) DMSO(10) DMSO(10) PBS(10)

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accepted preprint first posted on 11 may 2012 as manuscript joe

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