Involvement of Serum Amyloid A1 in the Pathogenesis of Insulin Resistance in Ovarian Granulosa Cells in Polycystic Ovary Syndrome

Background: Insulin resistance (IR) contributes to ovarian dysfunctions in PCOS patients. Serum amyloid A1 (SAA1) is an acute phase protein produced primarily by the liver in response to inammation. In addition to its role in inammation, SAA1 may participate in IR development in peripheral tissues. Yet, expressional regulation of SAA1 in the ovary and its role in the pathogenesis of ovarian IR in PCOS remain elusive. Methods: Follicular uid and granulosa cells were collected from PCOS and non-PCOS patients with and without IR to measure SAA1 abundance for analysis of its correlation with IR status. The effects of SAA1 on its own expression and insulin signaling pathway were investigated in cultured primary granulosa cells. Results: Ovarian granulosa cells were capable of producing SAA1, which could be induced by SAA1 per se. Moreover, the abundance of SAA1 signicantly increased in granulosa cells and follicular uid in PCOS patients with IR. SAA1 treatment signicantly attenuated insulin-stimulated membrane translocation of glucose transporter 4 and glucose uptake in granulosa cells through induction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression with subsequent inhibition of Akt phosphorylation. These effects of SAA1 could be blocked by inhibitors for toll-like receptors 2/4 (TLR 2/4) and nuclear factor kappa light chain enhancer of activated B (NF-κB). Conclusions: Human granulosa cells are capable of feedforward production of SAA1, which signicantly increased in PCOS patients with IR. Excessive SAA1 reduces insulin sensitivity in granulosa cells via induction of PTEN and subsequent inhibition of Akt phosphorylation upon activation of TLR2/4 and NF-κB pathway. These ndings highlight that local excessive synthesis of SAA1 is associated with IR development in granulosa cells of PCOS patients.

Methods: Follicular uid and granulosa cells were collected from PCOS and non-PCOS patients with and without IR to measure SAA1 abundance for analysis of its correlation with IR status. The effects of SAA1 on its own expression and insulin signaling pathway were investigated in cultured primary granulosa cells.
Results: Ovarian granulosa cells were capable of producing SAA1, which could be induced by SAA1 per se. Moreover, the abundance of SAA1 signi cantly increased in granulosa cells and follicular uid in PCOS patients with IR. SAA1 treatment signi cantly attenuated insulin-stimulated membrane translocation of glucose transporter 4 and glucose uptake in granulosa cells through induction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression with subsequent inhibition of Akt phosphorylation. These effects of SAA1 could be blocked by inhibitors for toll-like receptors 2/4 (TLR 2/4) and nuclear factor kappa light chain enhancer of activated B (NF-κB).
Conclusions: Human granulosa cells are capable of feedforward production of SAA1, which signi cantly increased in PCOS patients with IR. Excessive SAA1 reduces insulin sensitivity in granulosa cells via induction of PTEN and subsequent inhibition of Akt phosphorylation upon activation of TLR2/4 and NF-κB pathway. These ndings highlight that local excessive synthesis of SAA1 is associated with IR development in granulosa cells of PCOS patients.

Background
Polycystic ovarian syndrome (PCOS) is a complex endocrine and metabolic disease, which affects up to 7-12% reproductive-aged women [1]. The key characteristics of PCOS include anovulation, hyperandrogenism, polycystic ovarian morphology and insulin resistance (IR) [2,3]. The prevalence of IR is up to 50-70% in PCOS patients [4,5], which is found in a number of tissues and cells including ovarian granulosa cells [6,7]. Given the essential roles of granulosa cells in folliculogenesis, IR of granulosa cells is a contributor to reproductive disorders in PCOS patients. Thus, elucidation of the mechanism underlying IR development in granulosa cells may help better understanding the pathogenesis of PCOS.
Accumulating evidence has indicated a strong association of low-grade chronic in ammation with the development of IR in peripheral tissues [8][9][10]. Serum amyloid A (SAA), an acute phase protein, is produced mainly by the liver in response to in ammation and trauma. Although the exact role of SAA in the acute phase response is not fully understood, SAA is at least known to participate in in ammatory reactions through activation of toll-like receptor 2 and 4 (TLR2/4) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway [11,12]. There are four SAA genes, SAA1-4. While SAA1 and SAA2 are inducible in in ammation, SAA4 is constitutively expressed and SAA3 is a pseudogene in humans [13]. Of interest, expression of SAA has been detected in non-hepatic tissues and its over-expression has been implicated in chronic in ammation and IR development in peripheral tissues in obesity and type 2 diabetes [14][15][16][17]. SAA1, SAA2 and SAA4 transcripts have also been detected in human ovarian granulosa cells with SAA1 being the most abundant isoform [18]. However, very little is known about the expression and production regulation of SAA1 in ovarian granulosa cells. It is neither known whether its over-production is associated with the development of IR through activation of the in ammatory pathway in granulosa cells in PCOS.
It is well known that insulin stimulates glucose uptake via induction of glucose transporter (GLUT4) translocation from cytoplasm to cell membrane upon activation of Akt phosphorylation [19,20]. This signaling pathway can be attenuated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) [21,22] and upregulation of PTEN expression has been shown to be responsible for insulin resistance in a number of tissues [23][24][25]. Thus, we hypothesize that increased SAA1 production may inhibit insulin-induced Akt phosphorylation via stimulation of PTEN expression upon activation of TLR2/4 and NF-κB pathway, resulting in the attenuation of insulin-induced GLUT4 translocation, glucose uptake in granulosa cells and IR development in granulosa cells in PCOS. Herein, we examined the hypothesis by using human ovarian granulosa cells obtained from PCOS and non-PCOS patients with or without IR.

Methods
Recruitment of patients and collection of blood, follicular uid and ovarian granulosa cells Blood, follicular uid and ovarian granulosa cells were collected from patients who underwent in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) at Center for Reproductive Medicine, Renji Hospital of Shanghai Jiao Tong University under a protocol approved by the Ethics Committee of Renji Hospital of Shanghai Jiao Tong University with informed consent. PCOS was diagnosed according to the revised Rotterdam consensus [26]. Women with tubal factor-related infertility were enrolled into the non-PCOS group. GnRH antagonist protocol was adopted for ovarian stimulation in both PCOS and non-PCOS patients. On the day of oocytes retrieval, peripheral venous blood and follicular uid were collected.
Hormonal pro les of recruited patients were determined by measuring follicular stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E 2 ), testosterone (T) and anti-mullerian hormone (AMH) in the serum with chemiluminescence (Beckman Access Health Company, Chaska, MN) or ELISA (Kangrun, Guangzhou, China) assay kits. Enrolled PCOS and non-PCOS patients were sub-divided into groups with and without IR according to HOMA-IR (homeostasis model of assessment for insulin resistance index) or blood fasting insulin level. Blood fasting insulin ≥ 15 μIU/ml or homeostasis model assessment of insulin resistance (HOMA-IR) ≥ 2.68 was diagnosed as IR [27]. Insulin and glucose in the blood were measured with a chemiluminescence assay kit (Beckman Acess Health Co) and a standard glucose oxidase method (Roche, Mannheim, Germany) respectively. Follicular uid from the same patient was pooled and centrifuged. After centrifugation, the supernatant follicular uid without contaminating cells was collected, and the cell pellet was resuspended in PBS for further dispersing in 0.1% hyaluronidase (Sigma Chemical Co, St-Louis, Mo). Dispersed cells were then centrifuged on Ficoll gradient to remove contaminating red blood cells. Puri ed granulosa cells were either frozen for later RNA extraction or cultured in DMEM/Hams F12 containing 10% FBS (Gibco, Grand Island, NY).

Determination of insulin sensitivity in granulosa cells from PCOS and non-PCOS patients
Six patients from each group (PCOS and non-PCOS patients with or without IR) were randomly chosen for this study. Granulosa cells from these patients were cultured overnight, and Akt phosphorylation and glucose uptake were then determined following insulin (100 nM, sigma) treatment for 15 min and 30 min. Study of SAA1 expression in granulosa cells SAA1 expression was examined in cultured granulosa cells prepared from non-PCOS patients with immuno uorescent staining, qRT-PCR and immunoassay. For immuno uorescent staining, cultured granulosa cells were xed with 4% paraformaldehyde and permeabilized with 0.2% trixonX-100. After blocking the non-speci c binding sites, the cells were incubated with primary antibodies against SAA1 (1:100) (Abcam, Cambrige, UK) overnight at 4°C. Following washing with PBS, the cells were incubated with Alexa Fluor 488-labeled (green color) secondary antibody in darkness for 2 h. Nuclei were counterstained with DAPI (blue color). The staining was examined using a uorescence microscope (Zeiss, DB, Germany).

Extraction of RNA and analysis with qRT-PCR
Total RNA was extracted from frozen and cultured granulosa cells using a total RNA kit (OMEGA Bio-Tek, Norcross, GA). After determination of RNA quality, mRNA was reverse-transcribed to cDNA using PrimeScript ® RT Master Mix (TaKaRa, Dalian, China). Amount of SAA1 mRNA was determined with qRT-PCR using the above reverse-transcribed cDNA and power SYBR Premix Ex Taq™ (TaKaRa) following a previously described protocol [29]. Abundance of mRNA in each sample was calculated using the 2 -ΔΔCT method, and the ratio of SAA1 transcript over ACTB transcript, the house-keeping gene, was obtained to indicate relative SAA1 mRNA abundance. Primer sequences for PCR were as follows: SAA1, forward 5'-TTTCTGCTCCTTGGTCCTGG-3' and reverse 5'-CTCTGGCATCGCTGATCACT-3'; ACTB, forward 5'-GGGAAATCGTGCGTGACATTAAG-3'and reverse 5'-TGTGTTGGCGTACAGGTCTTTG-3'.

Extraction of protein and analysis with Western blotting
Total cellular protein was extracted from treated granulosa cells in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Active Motif, Carlsbad, CA) containing protease inhibitor cocktail (Sigma) and phosphatase inhibitor (Active Motif). For GLUT4 translocation assay, cytoplasmic and membrane protein fractions were separated using a membrane and cytoplasmic extraction kit (Sheng gong, Shanghai, China). Protein sample (30 μg) was electrophoresed in 10% SDS-polyacrylamide gel and transferred to a nitrocellulose blot. After blocking the non-speci c binding sites with non-fat milk, the blot was incubated (1:1000) (Cell Signaling) at 4℃ overnight. After washing, the blot was further incubated with corresponding secondary antibodies conjugated with horseradish peroxidase (Sigma) for 1 h. Enhanced chemiluminescent detection system (Millipore, Billerica, MA) was then applied for band detection. To control sampling error, the same blot was probed with an antibody against the house keeping protein GAPDH (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). For GLUT4 translocation study, the blot was incubated with antibodies against GAPDH and Na + -K + -ATPase (1:10000) (Cell Signaling) as cytoplasm and cell membrane protein markers respectively. The ratio of target protein over GAPDH or Na + -K + -ATPase was obtained to indicate relative abundance of the target protein.

Statistical analysis
All the data were presented as mean ± SEM. The Kolmogorov-Smirnov test was used to determine whether the continuous variables were of normal distribution. Paired or unpaired Student T-test or Oneway ANOVA test followed by Student-Newman-Keuls test were used to assess the differences in normally distributed variables. All data analysis was performed with the Statistical Package for Social Science (SPSS, version 18.0 for windows). P < 0.05 was considered as statistically signi cant.

Results
Clinical characteristics and cycle outcomes of recruited PCOS and non-PCOS patients Table 1 illustrates clinical characteristics and cycle outcomes of recruited patients, who were classi ed into four groups: non-PCOS without IR (n=22) non-PCOS with IR (n=22), PCOS without IR (n=22) and PCOS with IR (n=22). There were no signi cant differences in the age, basal FSH and fasting glucose among these four groups, but higher serum levels of AMH, LH, LH/FSH, T and more retrieved oocytes were observed in PCOS patients than in non-PCOS patients regardless of IR status. BMI, blood fasting insulin level and HOMA-IR were higher in IR groups than in non-IR groups regardless of PCOS status. The rates of fertilization, cleavage and high-quality embryo were signi cantly lower in PCOS with IR group than non-PCOS without IR. However, these rates were comparable among PCOS without IR and non-PCOS with or without IR.

Impaired insulin signaling and sensitivity in granulosa cells from PCOS patients with IR
Insulin (100 nM, 15 min for Akt phosphorylation and 30 min for glucose uptake) increased Akt phosphorylation and glucose uptake signi cantly in granulosa cells from all other three groups but not from PCOS with IR group ( Figure.1A-C), although non-PCOS with IR group also presented signi cantly less Akt phosphorylation and glucose uptake than non-PCOS without IR group upon insulin stimulation ( Figure.1A -C). These data indicate that impaired insulin signaling and sensitivity are present in granulosa cells from both IR groups regardless of PCOS status, but appear to be more severe in PCOS with IR group.

SAA1 abundance in serum, follicular uid and granulosa cells in PCOS patients
Serum concentration of SAA1 was signi cantly higher in PCOS patients than in non-PCOS patients regardless of IR status with the highest concentration observed in PCOS with IR group (Figure 2A). Similarly, SAA1 concentration in follicular uid was also highest in PCOS with IR group but there was no difference among other three groups ( Figure 2B). Noteworthy, SAA1 concentration in the follicular uid was 10-fold higher than that in the serum, suggesting that granulosa cells may be an alternative source of SAA1 in the follicular uid (Figure 2A, B). Consistently, there was increased SAA1 mRNA abundance in granulosa cells from PCOS patients with IR ( Figure.2C).

Expression and production regulation of SAA1 in granulosa cells
We further examined expression and production regulation of SAA1 in cultured granulosa cells.

Discussion
The present study has demonstrated that human ovarian granulosa cells are capable of de novo SAA1 synthesis, which is inducible by proin ammatory factors including IL-1b and SAA1 itself. SAA1 overproduction may contribute to IR development in granulosa cells in PCOS patients via stimulation of TLR2/4 and NF-κB pathway.
Although SAA1 mRNA and protein have been revealed to be present in the ovary with in situ hybridization and immunohistochemical staining in a previous study [18], little is known about the expression and production regulation of SAA1 in the ovary. Our study demonstrated for the rst time that human granulosa cells are not only capable of SAA1 production but are also inducible by proin ammatory factors. Of interest, SAA1 per se can induce SAA1 expression in granulosa cells as well, which is consistent with the nding in amnion broblast cells [30]. As such, a feedforward expression of SAA1 may develop under chronic in ammatory conditions. Chronic low-grade in ammation is believed to be present in PCOS ovary with increased abundance of a number of proin ammatory factors including IL-1β [31][32][33]. Since TLR2/4 and NF-κB are involved in the induction of SAA1 expression by SAA1, it is likely that SAA1 synthesis can be induced by other proin ammatory factors which utilize similar signaling pathway. In this study, IL-1β was also found to induce SAA1 production in granulosa cells. This feedforward pattern SAA1 expression under chronic in ammatory conditions may explain why there is SAA1 accumulation in granulosa cells and follicular uid in PCOS patients with IR since patients with IR may experience severer chronic in ammation.
In this study, we found that SAA1 concentration in the follicular uid was about 10-fold higher than its concentration in the peripheral blood, suggesting that there may be alternative sources of SAA1 for the follicular uid in addition to the blood. Given the abundant SAA1 expression in granulosa cells, we believe that granulosa cells are an important source of SAA1 for the follicular uid. Since SAA1 is a small molecule which can cross the blood-follicle barrier [34], serum SAA1 may also be a source for SAA1 in the follicular uid. Since our present study demonstrated that there was increased SAA abundance in both granulosa cells and blood in PCOS with IR[35, 36], we believe that the increases in SAA1 in the follicular uid in PCOS with IR may derive from both granulosa cells and blood.
Till now, the biological functions of SAA1 in the acute phase are still not fully understood. SAA1 has been shown to be involved in the in ammatory process by promoting immune cells migration and cytokine/chemokine production [37,38]. SAA1 has also been linked to the development of IR in adipose tissue in obesity and type 2 diabetes [14,15]. Our study has demonstrated that SAA1 is a contributor to IR development in the ovarian granulosa cells in PCOS as well by disrupting insulin signaling via stimulating PTEN expression and inhibiting Akt phosphorylation and GLUT4 translocation. Given the important roles of granulose cells in steroidogenesis and oocytes development [39,40], IR in granulosa cells may distort these functions in PCOS. The observation of lower rates of fertilization and cleavage and low-quality embryo in this study may thus be linked, at least in part, to SAA1-induced IR in granulosa cells. A previous study has also demonstrated that elevated follicular SAA level is associated with decreased pregnancy rate [18].
Of interest, we found that serum SAA1 concentration increased in PCOS patients without IR as well, although not as much as PCOS patients with IR. This nding suggests that SAA1 may be involved in pathogenesis of other PCOS dysfunctions in addition to IR. SAA have been identi ed to play a role in extracellular matrix remodeling in the fetal membranes thereby participating in membrane rupture [29].
Since extracellular matrix plays important roles in the normal development of the follicle and ovulation [33,41,42], it would be of interest to examine whether SAA1 is also involved in extracellular matrix remodeling in the ovary and its abnormal function is linked with anovulation in PCOS in the future.

Conclusions
In conclusion, we report a novel local feedforward production of SAA1 in ovarian granulosa cells in this study. Excessive SAA1 production under chronic low-grade in ammation may contribute to IR development in granulosa cells through induction of PTEN expression and consequent inhibition of Akt phosphorylation, GLUT4 translocation and glucose uptake in PCOS patients. These effects of SAA1 were mediated by TLR2/4 with subsequent activation of NF-κB signaling. These observations support the hypothesis that SAA1 plays a crucial role in the pathogenesis of PCOS by altering insulin sensitivity and that elevated SAA1 may be an early biomarker and therapeutic target for PCOS Abbreviations AMH, anti-mullerian hormone; E 2 , estradiol; FSH, follicular stimulating hormone; GLUT4, glucose transporter 4; HOMA-IR, homeostasis model assessment of insulin resistance; IVF, in vitro fertilization; IL-1β, interleukin-1β; ICSI, intracytoplasmic sperm injection; IR, insulin resistance; NF-κB, nuclear factor kappa light chain enhancer of activated B; PCOS, polycystic ovary syndrome; PTEN, phosphatase and tensin homolog deleted on chromosome 10; qRT-PCR, quantitative real time PCR; PCOS, polycystic ovary syndrome; SAA1, Serum amyloid A1; TLR2/4, toll-like receptor 2 and 4; T, testosterone.

Declarations Ethics statement
The studies involving human participants were reviewed and approved by The Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. The patients provided their written informed consent to participate in this study.

Consent for publication
Formal written consent was obtained from each patient.