cGMP/PKG-I Pathway–Mediated GLUT1/4 Regulation by NO in Female Rat Granulosa Cells

cGMP/PKG-I Pathway–Mediated GLUT1/4 Regulation by NO in Female Rat Granulosa Cells Abstract Nitric oxide (NO) is a multifunctional gaseous molecule that plays important roles in mammalian reproductive functions, including follicular growth and development. Although our previous study showed that NO mediated 3,5,3′-triiodothyronine and follicle-stimulating hormone–induced granulosa cell development via upregulation of glucose transporter protein (GLUT)1 and GLUT4 in granulosa cells, little is known about the precise mechanisms regulating ovarian development via glucose. The objective of the present study was to determine the cellular and molecular mechanism by which NO regulates GLUT expression and glucose uptake in granulosa cells. Our results indicated that NO increased GLUT1/GLUT4 expression and translocation in cells, as well as glucose uptake. These changes were accompanied by upregulation of cyclic guanosine monophosphate (cGMP) level and cGMP-dependent protein kinase (PKG)-I protein content. The results of small interfering RNA (siRNA) analysis showed that knockdown of PKG-I significantly attenuated gene expression, translocation, and glucose uptake. Moreover, the PKG-I inhibitor also blocked the above processes. Furthermore, NO induced cyclic adenosine monophosphate response element binding factor (CREB) phosphorylation, and CREB siRNA attenuated NO-induced GLUT expression, translocation, and glucose uptake in granulosa cells. These findings suggest that NO increases cellular glucose uptake via GLUT upregulation and translocation, which are mediated through the activation of the cGMP/PKG pathway. Meanwhile, the activated CREB is also involved in the regulation. These findings indicate that NO has an important influence on the glucose uptake of granulosa cells. Folliculogenesis and follicular development are complex and highly selective processes; >99% of the follicles undergo atresia during the ovarian cycle in life. Follicular development is regulated by endocrine, autocrine, and paracrine factors. The transition from preantral to early antral is the penultimate stage in terms of gonadotropin dependence (1). It is also a critical stage of follicular development, as it is highly susceptible to atretogenic signals that determine follicle destiny (survival/growth vs atresia). Glucose as a major energy substrate is essential to ovarian growth. The utilization of glucose is facilitated by a family of glucose transporter proteins (GLUTs), including 14 identified members in the GLUT family (2, 3). These transporters exhibit differential tissue localization and functional characteristics, with considerable interspecies differences. However, GLUT1–4 are major contributors to glucose uptake owing to their high affinity for glucose in various tissues (4–8). It has been reported that GLUT1–4 are detected in rat ovaries (9–11). Our previous results showed that 3,5,3′-triiodothyronine (T3) combined with follicle-stimulating hormone (FSH) to potentiate cellular glucose uptake via regulating the expression and translocation of GLUT1/4 upregulation in granulosa cells. Nitric oxide synthase (NOS)/nitric oxide (NO) are also involved in this regulatory network (12). NO is a multifunctional gaseous molecule. Accumulating evidence suggests that NO is involved in multiple cellular and biological processes (13). Moreover, NO is also important to female reproduction including follicle growth, ovulation, oocyte maturation, corpus luteum function and steroidogenesis (14–17). It has been reported that NO has multiple effects, such as inhibiting granulosa cell apoptosis, increasing estradiol levels and promoting follicular development (18–20). NO is synthesized from l-arginine, reduced form of NAD phosphate, and oxygen by a family of enzymes termed NOS. There are three isoforms of NOS: neuronal NOS (nNOS/NOS1), inducible NOS (iNOS/NOS2), and endothelial NOS (eNOS/NOS3). Although NOSs are detected in the ovary, their expression and activity are highly dependent on species, cell type, and stage of ovarian development (21, 22). Only NOS3 is regulated by FSH and T3 in granulosa cells. Our previous study also showed that NO is also involved in T3- and FSH-induced follicular development via GLUT upregulation and glucose uptake (12). However, the precise underlying mechanism by which NO regulates glucose uptake in granulosa cells is not understood. It is well known that NO regulates many biological functions through cyclic guanosine monophosphate (cGMP)–dependent signaling in different cells. NO binds to soluble guanylyl cyclase (sGC) and sharply increases sGC activity. sGC converts guanosine triphosphate into cGMP, increasing the intracellular concentrations of cGMP. The latter initiates a variety of downstream signaling events, one of which is to combine and activate its major cellular receptor, cGMP-dependent protein kinase (PKG) (23). However, whether and how NO/cGMP/PKG signaling modulates follicular development is not known. The transcription factor cyclic adenosine monophosphate response element binding factor (CREB) plays a critical role in many physiological activities. Many reports have noted that CREB is phosphorylated by PKG in different cells (24–27). Furthermore, the expression of GLUT1 is elevated by CREB/CBP (CREB protein) in mouse embryonic stem cells (28). However, the role of activated CREB in GLUT expression remains uncertain. In the present study, we investigated the cellular mechanisms of NO-induced granulosa cell GLUT expression and the possible involvement of cGMP/PKG in these processes. We demonstrated that NO increases the expression of GLUT1 and GLUT4, which are associated with increased glucose uptake by granulosa cells. Additionally, NO increased GLUT translocation to the plasma membrane. These responses are mediated by the activation of cGMP/PKG pathways, and CREB also plays a regulatory role in this system. Materials and Methods Reagents and antibodies All chemicals and culture media components used in the present study were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Culture media were purchased from Gibco Bethesda Research Laboratories (Grand Island, NY). The enhanced chemiluminescence detection kit was obtained from Amersham Life Science (Oakville, ON, Canada). Acrylamide (electrophoresis grade), N,N′-methylene-bis-acrylamide, ammonium persulfate, glycine, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis prestained molecular mass standards were products of Bio-Rad Laboratories (Richmond, CA). Rabbit polyclonal anti-GLUT1 (ab652) and rabbit polyclonal anti-GLUT4 (ab654) antibodies were purchased from Abcam (Cambridge, MA). Rabbit monoclonal anti–PKG-1 (3248), rabbit polyclonal anti–Na,K-adenosine triphosphatase (3010), rabbit monoclonal anti-CREB (9197), and phospho-CREB (Ser133) antibody were from Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti–β-actin (sc-81178) and horseradish peroxidase (HRP)–conjugated anti-rabbit and anti-mouse IgG were from Santa Cruz Biotechnology (Beijing, China). A RevertAid first-strand complementary DNA (cDNA) synthesis kit and TRIzol reagent were obtained from Thermo Fisher Scientific (Waltham, MA), and a SYBR Green polymerase chain reaction (PCR) kit was purchased from Bio-Rad Laboratories. PCR primers for GLUT1, GLUT4 and 18S ribosomal RNA (rRNA) were from Beijing Sunbiotech (Beijing, China). PKG inhibitor KT5823 was purchased from Beyotime Biotechnology (Beijing China). A cGMP assay kit purchased from Cell Signaling Technology was used to determine cGMP levels in cells. The 2-deoxyglucose (2-DG) uptake measurement kit purchased from Cosmo Bio (Tokyo Japan) was used to detect glucose uptake level. The cell counting kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Lipofectamine 3000 was purchased from Invitrogen (Carlsbad, CA). Animal treatments Twenty-one-day-old Sprague-Dawley rats were (Beijing Vital Laboratory Animal Technology, Beijing, China) maintained on 12-hour light/12-hour dark cycle and received pathogen-free water and food. Rats were injected subcutaneously with diethylstilbestrol (1 mg/d; 3 days), which induces more preantral and early antral follicles (12, 29, 30), and ovaries were collected at 72 hours after euthanization by cervical dislocation. All procedures were carried out in accordance with the Principles of the Care and Use of Laboratory Animals and China Council on Animal Care and were approved by the Institutional Animal Care and Use Committee of Capital Normal University (approval no. 2016017). Preantral follicle isolation and culture Ovaries of 14-day-old rats were removed and placed in 2.5 ml of Leibowitz L15 medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Large preantral follicles (diameter, 150 to 180 μm) were isolated using a 26-gauge needle. To minimize the experimental variation caused by damage incurred during the isolation procedures, only round follicles with an intact basement membrane and thecal layer were selected for the present studies. Follicles were cultured individually in a 96-well plate in 100 μL of α–minimum essential medium supplemented with HEPES (10 mM), bovine serum albumin (0.1%, weight-to-volume), bovine insulin (5 μg/mL), transferrin (2 μg/mL), ascorbic acid (25 μg/mL), sodium selenite anhydrous (2 ng/mL), l-glutamine (3 mM), sodium pyruvate (100 μg/mL), streptomycin (100 μg/mL), and penicillin (100 U/mL), with or without S-nitroso-N-acetyl-dl-penicillamine (SNAP; 100 μM). The diameter of the follicle was measured every day as the average of distance between the outer edges of the basement in two perpendicular planes. The results were calculated as follows: the percentage change of follicular volume = [(Vn − V0)/V0] × 100%, where Vn is the follicular volume on day n and V0 is the initial volume of the follicles. The culture medium was changed every other day. Rat granulosa cell isolation and culture Rat granulosa cell isolation and culture processes were performed as described previously (12). Simply, granulosa cells were released by follicular puncture from large preantral and early antral follicles. The cell clumps and oocytes were removed by filtering the cell suspensions through a 40-μm nylon cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ; no. 352340). The viability of cells was estimated by a Trypan blue dye exclusion test. Granulosa cells (9 × 105 viable granulosa cells per plate in a six-well plate) were cultured in M199 with fetal bovine serum (10%, weight-to-volume) for 6 hours. The media were then replaced with serum-free M199 supplemented as above for 12 hours thereafter and cells were treated with SNAP, a nitrosothiol derivative. In some experiments, cells were pretreated with a cell-permeable cGMP analog, 8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cGMP, 1 mM), sGC inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 μM), or PKG inhibitor KT5823 (1 μM) 1 hour before SNAP treatment, respectively. Cells were maintained at 37°C under humidified atmosphere (5% CO2). For RNA interference, granulosa cells were transfected (48 hours) with PKG small interfering RNA (siRNA) or CREB siRNA and scrambled sequence control (GenePharma, Shanghai, China), respectively, using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Western blot analysis Western blot analysis was performed as described previously (12). Whole-cell lysates were prepared by incubating cell pellets for 30 minutes at 0°C in lysis buffer (Beyotime Biotechnology, Shanghai, China), and protease and phosphatase inhibitor cocktails (Sigma-Aldrich) were added to buffers before use. The supernatant was collected by centrifugation (15,000 × g, 4°C, 30 minutes) and protein concentration was determined with the BCA protein assay kit (Beyotime Biotechnology). The protein samples (15 to 80 µg, depending on individual experiments) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto the nitrocellulose membrane. The membranes were then blocked in tris-buffered saline with Tween 20 buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20; pH 7.5) containing 5% dehydrated nonfat milk at room temperature for 1 hour and subsequently incubated (4°C, overnight) with diluted primary antibody [polyclonal anti-GLUT1 (1:500), polyclonal anti-GLUT4 (1:2000), rabbit monoclonal anti–PKG-I (1:1000), rabbit polyclonal anti–Na,K-ATPase (1:1000), rabbit monoclonal anti-CREB (1:500), or β-actin (1:10,000)], followed by HRP-conjugated secondary antibody (1:1000 to 1:10,000; 1.5 hours, room temperature). Peroxidase activity was visualized with the enhanced chemiluminescence kit according to the manufacturer’s instructions. Protein content was determined by densitometrically scanning the exposed x-ray film. Immunoreaction signals were analyzed using Gel-Pro Analyzer 4.0. RNA extraction, cDNA synthesis, and real-time PCR analysis Total RNAs were extracted from cultured granulosa cells with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, and then washed in 75% ethanol and dissolved in RNase-free distilled water. cDNAs were produced from 0.2 μg of total RNAs by reverse transcription. Gene expression was measured by real-time PCR and normalized to 18S rRNA. Specific primer pairs used in the experiments are listed in Table 1. Data were analyzed by the 2−ΔΔCT method (31). Table 1. Primer Sequence Used for Real-Time Quantitative PCR Target Gene  GenBank Accession No.  Primer Sequence  Product Size, bp  Annealing Temperature, °C  GLUT-1  NM_138827.1  F: 5′-TGGCCAAGGACACACGAATACTGA-3′  144  60  R: 5′-TGGAAGAGACAGGAATGGGCGAAT-3′  GLUT-4  NM_012751.1  F: 5′-TGTTGCGGATGCTATGGGTC-3′  123  60  R: 5′-GCCGAGATCTGGTCAAATGT-3′  18S rRNA  NR_046237.1  F: 5′-CGCGGTTCTATTTTGTTGGT-3′  219  60  R: 5′-AGTCGGCATCGTTTATGGTC-3′  Target Gene  GenBank Accession No.  Primer Sequence  Product Size, bp  Annealing Temperature, °C  GLUT-1  NM_138827.1  F: 5′-TGGCCAAGGACACACGAATACTGA-3′  144  60  R: 5′-TGGAAGAGACAGGAATGGGCGAAT-3′  GLUT-4  NM_012751.1  F: 5′-TGTTGCGGATGCTATGGGTC-3′  123  60  R: 5′-GCCGAGATCTGGTCAAATGT-3′  18S rRNA  NR_046237.1  F: 5′-CGCGGTTCTATTTTGTTGGT-3′  219  60  R: 5′-AGTCGGCATCGTTTATGGTC-3′  View Large cGMP level assay Granulosa cells were cultured with or without SNAP (100 μM). The cells were harvested at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 18 hours, respectively. The cGMP level was detected by a cGMP assay kit according to the manufacturer’s protocol. Briefly, cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and kept in lysis buffer on ice for 5 to 10 minutes. The 50-μL sample and 50-μL HRP-linked cGMP solution were transferred to the cGMP assay plate. Next, the plate contents were discarded and washed four times with wash buffer. After that, the sample was incubated with 100 μL of tetramethylbenzidine substrate for 30 minutes at room temperature. After adding 100 μL of STOP solutions, the absorbance was measured at 450 nm. The cGMP concentration was calculated according to the standard curve. Plasma membrane protein isolation To investigate whether NO regulated GLUT1 and GLUT4 translocation to the plasma membrane, the plasma membrane of a granulosa cell was isolated by a Minute plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions. Briefly, granulosa cells were first sensitized followed by a series of centrifugation steps to remove the nucleus, cytosol, and organelle membrane, respectively. Thus, the cell membrane was obtained. Immunofluorescence cell staining The rat granulosa cells were cultured in poly-d-lysine–coated (0.05% weight-to-volume; Sigma-Aldrich) eight-well glass culture slides (Becton Dickinson and Co.) used for immunofluorescence analysis. After treatment with or without SNAP for 24 hours, the cells were fixed in 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and then blocked in PBS buffer containing 5% bovine serum albumin at room temperature for 30 min. The cells were subsequently incubated with polyclonal anti-GLUT1 (1:200 dilution in blocking solution) or polyclonal anti-GLUT4 (1:200 dilution in blocking solution), respectively, at 4°C overnight. The samples were then incubated with Alexa Fluor 488–conjugated secondary antibody (1:400 dilution in blocking solution; Jackson ImmunoResearch Laboratories, West Grove, PA) at 37°C for 1.5 hours. After washing with PBS three times, samples were incubated with DiI (red, membrane stain) (32, 33) and 4′,6-diamidino-2-phenylindole (blue, nuclear stain) for 10 minutes at room temperature and then washed in PBS three times. After that, the coverslips were mounted on object slides using fluorescent mounting medium. Immunofluorescence was visualized using an immunofluorescence microscope (Olympus BX51), and images were recorded by using a DP70 digital camera (Olympus Optical Co., Tokyo, Japan). Glucose uptake assay Glucose uptake levels were determined using a 2-DG measurement kit (Cosmo Bio Co., Tokyo, Japan) in accordance with the manufacturer’s protocol. Briefly, granulosa cells were cultured up to 24 hours in the presence of SNAP with or without inhibitors as mentioned above, and then the cells were incubated in serum-free medium for 6 hours. After washing three times in PBS, the cells were incubated in 2-DG solution at 37°C for 20 minutes. Cell lysates were collected after washing the cells three times again and then heated at 80°C for 15 min. The samples were centrifuged (4°C, 15,000 × g) for 20 minutes and transferred to a new tube. The optical density (OD) value was detected using a microplate detector at 420 nm. The concentration of each sample was measured in triplicates and calculated using the standard curve supplied in the kit. Analysis of cell viability Cell activity was detected by the CCK-8 (Dojindo), which is based on the dehydrogenase activity detection in viable cells. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. In the present study, granulosa cells were cultured with SNAP in 96-well plates and then incubated with 10 µL of CCK-8 solution at 37°C for 2 hours. The OD values in each well were recorded using a microplate reader at 450 nm. The mean OD values for each treatment were used as the index of cell viability. Statistical analysis The experiments were repeated at least three times, as detailed in the figure legends. Experimental data are presented as means ± standard error of the mean. The statistical differences between treatments were calculated with t test, one-way analysis of variance, or two-way (repeated-measures) analysis of variance (Prism 5.0 statistical software; GraphPad Software, San Diego, CA). When significant differences were found, means were compared by the Bonferroni posttest. A P value < 0.05 was considered to be significant. Results Effect of NO on follicular growth and GLUT expression in vitro To evaluate the effect of NO on preantral follicle development, follicles were cotreated with SNAP for 4 days. The follicular volume change curve during the 4-day culture period is presented in Fig. 1A. Preantral follicles cultured in the presence of SNAP were significantly increased (day 4, 119.54 ± 1.37 vs 59.93 ± 2.59, P < 0.001). Meanwhile, SNAP also gradually increased glucose uptake (P < 0.01, Fig. 1B). Moreover, SNAP statistically increased GLUT1 (1.74 ± 0.11 vs 1.00 ± 0.09, P < 0.05, Fig. 1C) and GLUT4 (1.41 ± 0.17 vs 1.02 ± 0.07, P < 0.05, Fig. 1D) messenger RNA (mRNA) levels. However, the upregulation of GLUT1 and GLUT4 by SNAP was significantly attenuated by ODQ and KT5823 (Fig. 1C and 1D). Figure 1. View largeDownload slide Effect of NO on preantral follicular growth in vitro. Preantral follicles were cultured for 4 days with or without SNAP (100 μM). Follicles cultured in the absence of SNAP were named as the control group. (A) Follicular diameter was measured daily and results are expressed as change in follicular volume. (B) Follicular glucose uptake was assessed. (C and D) Follicles were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The mRNA abundance of GLUT1 and GLUT4 were analyzed by real-time PCR. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. CTL, control. Figure 1. View largeDownload slide Effect of NO on preantral follicular growth in vitro. Preantral follicles were cultured for 4 days with or without SNAP (100 μM). Follicles cultured in the absence of SNAP were named as the control group. (A) Follicular diameter was measured daily and results are expressed as change in follicular volume. (B) Follicular glucose uptake was assessed. (C and D) Follicles were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The mRNA abundance of GLUT1 and GLUT4 were analyzed by real-time PCR. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. CTL, control. NO increased granulosa cell development To determine whether NO affected the growth of granuloasa cells, we investigated cellular development after treatment with various concentrations (0, 50, 100, 200, and 500 μM) of SNAP. As shown in Fig. 2, SNAP induced a peak of cellular viability at 100 μM compared with the control group [2.01 ± 0.11 (SNAP) vs 0.88 ± 0.01 (control), P < 0.001]. Figure 2. View largeDownload slide NO increased granulosa cell development in vitro. Immature rats were treated with diethylstilbestrol (1 mg/d, 3 consecutive days) prior to granulosa cell isolation from preantral and early antral follicles. The cells were collected and cocultured with SNAP (0, 50, 100, 200, and 500 µM) for 24 hours, and cell viability was analyzed by CCK-8 assay. ***P < 0.001, compared with control (without SNAP). Figure 2. View largeDownload slide NO increased granulosa cell development in vitro. Immature rats were treated with diethylstilbestrol (1 mg/d, 3 consecutive days) prior to granulosa cell isolation from preantral and early antral follicles. The cells were collected and cocultured with SNAP (0, 50, 100, 200, and 500 µM) for 24 hours, and cell viability was analyzed by CCK-8 assay. ***P < 0.001, compared with control (without SNAP). Effects of NO on granulosa cell GLUT expression In our previous study we showed that T3- and FSH-induced GLUT1 and GLUT4 expressions are attenuated by NOS3 knockdown in rat granulosa cells. To examine whether NO regulates GLUT1 and GLUT4, we cultured granulosa cells with SNAP for 24 hours. As shown in Fig. 3A and 3B, SNAP treatment significantly increased GLUT1 [18 hours, 0.94 ± 0.03 (SNAP) vs 0.55 ± 0.03 (control), P < 0.05; 24 hours, 1.66 ± 0.11 (SNAP) vs 0.99 ± 0.06 (control), P < 0.01] and GLUT4 [18 hours, 1.06 ± 0.08 (SNAP) vs 0.67 ± 0.02 (control), P < 0.05; 24 hours, 1.55 ± 0.05 (SNAP) vs 0.98 ± 0.13 (control), P < 0.05] protein levels. Figure 3. View largeDownload slide Effects of NO on granulosa cell GLUT expression in vitro. (A and B) Granulosa cells were collected after cotreatment with SNAP (100 μM) for 18 hours and 24 hours, and GLUT1 and GLUT4 proteins were detected by western blot analysis. (C and D) Cells were treated with SNAP (100 μM) up to 24 hours (6, 12, 18, and 24 hours) for GLUT1 (B) and GLUT4 (C) mRNA analysis by real-time PCR. mRNA abundance was normalized using 18S rRNA. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. CTL, control. Figure 3. View largeDownload slide Effects of NO on granulosa cell GLUT expression in vitro. (A and B) Granulosa cells were collected after cotreatment with SNAP (100 μM) for 18 hours and 24 hours, and GLUT1 and GLUT4 proteins were detected by western blot analysis. (C and D) Cells were treated with SNAP (100 μM) up to 24 hours (6, 12, 18, and 24 hours) for GLUT1 (B) and GLUT4 (C) mRNA analysis by real-time PCR. mRNA abundance was normalized using 18S rRNA. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. CTL, control. To understand more fully whether the content of GLUT1 and GLUT4 is regulated at the mRNA level by NO, we determined GLUT mRNA abundance using real-time PCR analysis. Granulosa cells were cultured with SNAP for 24 hours. The results showed that GLUT1 and GLUT4 mRNA levels were significantly increased at 12 hours [GLUT1, 3.73 ± 0.04 (SNAP) vs 1.01 ± 0.11 (control), P < 0.001, Fig. 3C; GLUT4, 3.89 ± 0.06 (SNAP) vs 0.98 ± 0.54 (control), P < 0.001, Fig. 3D] and 18 hours [GLUT1, 5.26 ± 0.05 (SNAP) vs 1.10 ± 0.14 (control), P < 0.001, Fig. 3C; GLUT4, 5.69 ± 0.25 (SNAP) vs 1.05 ± 0.06 (control), P < 0.001, Fig. 3D] after SNAP treatment. These results suggest that increased gene transcription may at least partly account for the increased protein content induced by SNAP. Involvement of the cGMP/PKG pathway in NO-induced GLUT expression and glucose uptake To determine whether the cGMP/PKG signaling pathway is involved in the regulation of GLUT expression by NO, we next examined the intracellular cGMP levels after SNAP treatment. We noticed that intracellular cGMP levels were increased with the duration of treatment, reaching a peak at 1 hour after SNAP treatment [425.13 ± 9.97 (SNAP) vs 10.63 ± 0.66 (CTL), P < 0.001, Fig. 4A]. We also detected phospho–vasodilator-stimulated phosphoprotein content after SNAP treatment, which is originally characterized as a substrate of PKG (34) (Supplemental Fig. 2). We then pretreated cells with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. Western blot analysis carried out after 24 hours revealed that the expression of GLUT1 and GLUT4 were significantly higher in the 8-Br-cGMP group, which was similar to the effect of SNAP treatment. However, these effects were abolished after coculture with ODQ or KT5823 [GLUT1, 1.19 ± 0.11 (8-Br-cGMP) vs 0.62 ± 0.03 (control), P < 0.01; GLUT4, 1.24 ± 0.04 (8-Br-cGMP) vs 0.86 ± 0.02 (control), P < 0.05, Fig. 4B]. Glucose levels followed a pattern similar to GLUT1/4 [3.02 ± 0.13 (8-Br-cGMP) vs 1.76 ± 0.14 (control), P < 0.001; 2.80 ± 0.09 (SNAP) vs 1.76 ±0.14 (control), P < 0.01, Fig. 4C]. SNAP-induced GLUT expression and glucose uptake were also significantly attenuated by PKG knockdown [GLUT1, 0.85 ± 0.03 (PKG siRNA) vs 1.59 ± 0.09 (SNAP), P < 0.05; GLUT4, 1.13 ± 0.08 (PKG siRNA) vs 1.68 ± 0.04 (SNAP), P < 0.05, Fig. 5A; glucose, 1.45 ± 0.18 (PKG siRNA) vs 2.86 ± 0.12 (SNAP), P < 0.01, Fig. 5B]. Figure 4. View largeDownload slide Involvement of the cGMP/PKG pathway in NO-induced GLUT expression and glucose uptake. (A) After cotreatment with SNAP (100 μM), granulosa cells were harvested at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 18 hours, respectively. Next, the cGMP level was detected by a cGMP assay kit. (B and C) Granulosa cells were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The proteins levels of GLUT1 and GLUT4 contents (B) and cellular glucose uptake (C) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. Figure 4. View largeDownload slide Involvement of the cGMP/PKG pathway in NO-induced GLUT expression and glucose uptake. (A) After cotreatment with SNAP (100 μM), granulosa cells were harvested at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 18 hours, respectively. Next, the cGMP level was detected by a cGMP assay kit. (B and C) Granulosa cells were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The proteins levels of GLUT1 and GLUT4 contents (B) and cellular glucose uptake (C) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. Figure 5. View largeDownload slide Role of PKG in NO-induced GLUT expression and glucose uptake in vitro. Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents (A) and cellular glucose uptake (B) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. Figure 5. View largeDownload slide Role of PKG in NO-induced GLUT expression and glucose uptake in vitro. Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents (A) and cellular glucose uptake (B) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. cGMP/PKG mediated NO-induced GLUT translocation The previous results showed that NO increased glucose uptake through cGMP/PKG signaling. Because glucose is actively transported into cells via GLUTs, we next investigated whether NO regulated the plasma membrane translocation of GLUT1 and GLUT4. Western blot analysis experiments indicated that the GLUT1 and GLUT4 expression on the cell membrane were increased after SNAP treatment [GLUT1, 1.17 ± 0.12 (SNAP) vs 0.66 ± 0.04 (control), P < 0.05; GLUT4, 1.20 ± 0.06 (SNAP) vs 0.72 ± 0.06 (control), P < 0.05, Fig. 6A], but that GLUT translocation was blocked by PKG siRNA [GLUT1, 0.70 ± 0.07 (PKG siRNA), P < 0.01; GLUT4, 0.74 ± 0.03 (PKG siRNA), P < 0.01, Fig. 6A]. We confirmed these results by immunofluorescence cell staining. As shown in Fig. 6, we found that both GLUT1 and GLUT4 were weakly present on the cell membrane and in the cytoplasm in the control group. After SNAP treatment of 24 hours, expression of GLUT1 (Fig. 6B) and GLUT4 (Fig. 6C) on the cell membrane was increased, and GLUT translocation was also attenuated by PKG knockdown. Figure 6. View largeDownload slide cGMP/PKG mediated NO-induced GLUT translocation. (A) Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. The plasma membrane was obtained by a Minute plasma membrane protein isolation kit. Membrane proteins of GLUT1 and GLUT4 were detected by western blot analysis. (B and C) Granulosa cells were cultured on poly-d-lysine-coated (0.05% weight-to-volume; Sigma-Aldrich) eight-well glass culture slides and transfected with PKG siRNA for 48 hours, and then treated with SNAP for another 24 hours. As described in detail in Materials and Methods, the specimens were fixed and subjected to immunofluorescent staining and analyzed by confocal microscopy for GLUT1 (B, green) and GLUT4 (C, green). The nuclei were stained with DAPI (blue), and the membrane was stained with DiI (red). Scale bars, 10 μm. *P < 0.05 compared with control (without SNAP); ++P < 0.01, compared with SNAP treatment group. CTL, control. Figure 6. View largeDownload slide cGMP/PKG mediated NO-induced GLUT translocation. (A) Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. The plasma membrane was obtained by a Minute plasma membrane protein isolation kit. Membrane proteins of GLUT1 and GLUT4 were detected by western blot analysis. (B and C) Granulosa cells were cultured on poly-d-lysine-coated (0.05% weight-to-volume; Sigma-Aldrich) eight-well glass culture slides and transfected with PKG siRNA for 48 hours, and then treated with SNAP for another 24 hours. As described in detail in Materials and Methods, the specimens were fixed and subjected to immunofluorescent staining and analyzed by confocal microscopy for GLUT1 (B, green) and GLUT4 (C, green). The nuclei were stained with DAPI (blue), and the membrane was stained with DiI (red). Scale bars, 10 μm. *P < 0.05 compared with control (without SNAP); ++P < 0.01, compared with SNAP treatment group. CTL, control. Role of CREB in NO-induced GLUT expression To determine whether NO activates CREB in granulosa cells in vitro, granulosa cells were incubated with SNAP for 12 hours and phospho-CREB content was determined. We found that phospho-CREB content was significantly higher in granulosa cells incubated with SNAP [1.05 ± 0.06 (SNAP) vs 0.47 ± 0.04 (control), P < 0.05, Fig. 7A]. However, gene knockdown of PKG decreased the levels of phospho-CREB [0.61 ± 0.02 (PKG siRNA), P < 0.05, Fig. 7A]. Figure 7. View largeDownload slide Role of CREB in NO induced GLUT expression. (A) PKG was knockdown, and phospho-CREB content was analyzed by western blot analysis after SNAP treatment. (B) Granulosa cells were transfected with CREB siRNA, and then cells were treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents were detected by western blot analysis. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, compared with SNAP treatment group. Figure 7. View largeDownload slide Role of CREB in NO induced GLUT expression. (A) PKG was knockdown, and phospho-CREB content was analyzed by western blot analysis after SNAP treatment. (B) Granulosa cells were transfected with CREB siRNA, and then cells were treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents were detected by western blot analysis. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, compared with SNAP treatment group. To determine the role of the activated CREB in GLUT expression, granulosa cells were transfected with CREB siRNA and then treated with SNAP for 24 hours (Fig. 7B). Although GLUT1 and GLUT4 protein content were upregulated by the presence of SNAP, this effect was significantly attenuated by CREB knockdown [GLUT1, 0.64 ± 0.09 (PKG siRNA) vs 1.38 ± 0.07 (SNAP), P < 0.01; GLUT4, 1.29 ± 0.08 (PKG siRNA) vs 1.63 ± 0.06 (SNAP), P < 0.05, Fig. 7B]. Discussion In the present study, we investigated the role of NO in rat granulosa cells. We demonstrated that SNAP, as an NO donor, increased GLUT1 and GLUT4 expression and translocation through the cGMP/PKG pathway in granulosa cells in vitro, and that activated CREB also played a regulatory role. To the best of our knowledge, this is the first study to show that NO, PKG, and CREB have important roles in the expression of GLUTs in rat granulosa cells in vitro. Glucose is the main energy substrate in the rat ovary, and a sufficient supply of glucose is essential for the maintenance of ovarian function. It has been reported that glucose cannot freely pass through the cell membrane and that glucose uptake is mediated by transporter proteins. Therefore, GLUT expression and activity play vital roles in ovary physiological function (5, 35). In our previous study, we detected GLUT1–4 in the granulosa cells. At the same time, we also found that GLUT1 and GLUT4 are regulated by T3 and FSH, and these processes are mediated by NO (12). In the present study, we found that NO increased GLUT1 and GLUT4 expression at both the transcriptional and translational levels, the results of which are consistent with our previous results. It is well known that GLUT1 is ubiquitously expressed in all organs, including ovaries, and that its expression is closely related to the basal level of glucose uptake in most cell types (5, 35, 36). GLUT4 is abundantly expressed in many tissues, and it plays important roles in whole-body glucose homeostasis (7). GLUT4 is a primary factor that determines the maximal rate of glucose transport (37). Moreover, most GLUT4 is stored in the membranous vesicles in the cytoplasm under the normal resting state. Once stimulated, GLUT4 is transported to the cell membrane, where it increases the cellular glucose uptake. Our results showed that SNAP increased the expression of GLUT1 and GLUT4 on the cell membrane, which indicates that NO promoted GLUT translocation. The increased GLUT content and translocation stimulate cellular glucose utilization. However, the underlying mechanisms for the expression and translocation of GLUT1 and GLUT4 induced by NO have not yet been elucidated. NO exerts a variety of physiological effects that are important for the regulation of mammalian reproductive functions, including follicular growth and development (38). In the present study, SNAP stimulated preantral follicular growth, which may have been partly caused by the upregulation of GLUT1 and GLUT4, as well as increased glucose uptake. It has been reported that NO exerts antiapoptotic effects in rat granulosa cells, which prevent ovarian follicle atresia (39–41). The results of the present study showed that NO increased granulosa cell development at a lower dose, which indicates that the effect of NO occurs in a dose-dependent manner. This pattern is consistent with the previous reports that lower concentrations of NO stimulated the survival, growth, and antrum formation of preantral follicles in buffalo. In contrast, the higher NO concentrations inhibited preantral follicle development (42, 43). These results indicate that NO plays different roles (inhibiting or stimulating) in granulosa cell growth depending on its concentration (15). It has been reported that NO activates sGC, then catalyzes guanosine triphosphate to generate cGMP. PKG is a serine/threonine-specific protein kinase that is triggered by cGMP, followed by phosphorylation of a number of biologically important targets. Many reports have demonstrated that the NO/cGMP/PKG signaling pathway plays an important role in promoting cell survival or preventing spontaneous apoptosis in human renal carcinoma cells (44), bone marrow stromal cells (45), neural stem/progenitor cells derived from embryonic hippocampus (46), retinal neuroglial progenitor cells (47), neural cells (48), and human ovarian cancer cells (49). However, it is not known whether the cGMP/PKG pathway is involved in NO-induced GLUT expression and glucose uptake in ovarian cells. In the present study, we found that NO elevated levels of intracellular cGMP. Inhibition of the cGMP/PKG pathway decreased NO-induced GLUT expression and glucose uptake in follicles and granulosa cells. Our results suggest that the cGMP/PKG pathway plays a regulatory role in the stimulation of glucose transport and glucose uptake by NO. Moreover, our results showed that SNAP upregulated the phosphorylation level of CREB, which was blocked by PKG siRNA. CREB siRNA knockdown also decreased the expression of GLUTs and glucose levels in granulosa cells. These results suggest that NO may activate the cGMP/PKG pathway and, subsequently, the phosphorylation of CREB, then promoting the expression and translocation of GLUTs. In conclusion, our findings demonstrate that NO is a novel positive regulator of granulosa cell development in the early stages of follicular development. As indicated by our model (Fig. 8), NO increases GLUT1/4 expression and translocation, which in turn contributes to glucose uptake by granulosa cells. Moreover, the cGMP/PKG pathway mediated these regulations induced by NO, and phospho-CREB is also involved in this system. Our results enhance our understanding of the role of NO in ovarian cells. Figure 8. View largeDownload slide Schematic diagram of the role of NO on GLUT expression and translocation. Figure 8. View largeDownload slide Schematic diagram of the role of NO on GLUT expression and translocation. Table. Antibody Table Peptide/Protein Target  Antigen Sequence  Name of Antibody  Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID   GLUT1  Synthetic peptide conjugated to KLH, corresponding to amino acids 478 to 492 of human glucose transporter GLUT1  Anti-glucose transporter GLUT1 antibody  ab652  Rabbit; polyclonal  1:500  AB_305540  GLUT4  Synthetic peptide corresponding to the C-terminal 15 residues of rat Glut4 cross-linked to KLH  Anti-glucose transporter GLUT4 antibody  ab654  Rabbit; polyclonal  1:2000  AB_305554  PKG-1    PKG-1 (C8A4) rabbit monoclonal antibody  3248  Rabbit; monoclonal  1:1000  AB_2067450  Na,K-ATPase α    Na,K-ATPase antibody  3010  Rabbit; polyclonal  1:1000  AB_2060983  Phospho-CREB (Ser133)    Phospho-CREB (Ser133) (87G3) rabbit monoclonal antibody  9198  Rabbit; monoclonal  1:1000  AB_2561044  CREB    CREB (48H2) rabbit monoclonal antibody  9197  Rabbit; monoclonal  1:1000  AB_331277  β-Actin    β-Actin (ACTBD11B7)  sc-81178  Mouse; monoclonal  1:1000  AB_2223230  Peptide/Protein Target  Antigen Sequence  Name of Antibody  Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID   GLUT1  Synthetic peptide conjugated to KLH, corresponding to amino acids 478 to 492 of human glucose transporter GLUT1  Anti-glucose transporter GLUT1 antibody  ab652  Rabbit; polyclonal  1:500  AB_305540  GLUT4  Synthetic peptide corresponding to the C-terminal 15 residues of rat Glut4 cross-linked to KLH  Anti-glucose transporter GLUT4 antibody  ab654  Rabbit; polyclonal  1:2000  AB_305554  PKG-1    PKG-1 (C8A4) rabbit monoclonal antibody  3248  Rabbit; monoclonal  1:1000  AB_2067450  Na,K-ATPase α    Na,K-ATPase antibody  3010  Rabbit; polyclonal  1:1000  AB_2060983  Phospho-CREB (Ser133)    Phospho-CREB (Ser133) (87G3) rabbit monoclonal antibody  9198  Rabbit; monoclonal  1:1000  AB_2561044  CREB    CREB (48H2) rabbit monoclonal antibody  9197  Rabbit; monoclonal  1:1000  AB_331277  β-Actin    β-Actin (ACTBD11B7)  sc-81178  Mouse; monoclonal  1:1000  AB_2223230  Abbreviation: RRID, research resource identifier. View Large Abbreviations: 2-DG 2-deoxyglucose 8-Br-cGMP 8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt CCK-8 cell counting kit-8 cDNA complementary DNA cGMP cyclic guanosine monophosphate CREB cyclic adenosine monophosphate response element binding factor FSH follicle-stimulating hormone GLUT glucose transporter protein HRP horseradish peroxidase mRNA messenger RNA NO nitric oxide NOS nitric oxide synthase OD optical density ODQ 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one PBS phosphate-buffered saline PCR polymerase chain reaction PKG cyclic guanosine monophosphate–dependent protein kinase rRNA ribosomal RNA sGC soluble guanylyl cyclase siRNA small interfering RNA SNAP S-nitroso-N-acetyl-dl-penicillamine T3 3,5,3′-triiodothyronine. Acknowledgments Financial Support: This work was supported by the National Natural Science Foundation of China Grant 31671555 (to C.Z.) and Scientific Research Program of Beijing Municipal Commission of Education Grant KM201610028011 (to C.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Hsu SY, Hsueh AJ. Hormonal regulation of apoptosis an ovarian perspective. Trends Endocrinol Metab . 1997; 8( 5): 207– 213. Google Scholar CrossRef Search ADS PubMed  2. Wood IS, Trayhurn P. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br J Nutr . 2003; 89( 1): 3– 9. Google Scholar CrossRef Search ADS PubMed  3. Hresko RC, Kraft TE, Quigley A, Carpenter EP, Hruz PW. Mammalian glucose transporter activity is dependent upon anionic and conical phospholipids. J Biol Chem . 2016; 291( 33): 17271– 17282. Google Scholar CrossRef Search ADS PubMed  4. Carnagarin R, Dharmarajan AM, Dass CR. PEDF attenuates insulin-dependent molecular pathways of glucose homeostasis in skeletal myocytes. Mol Cell Endocrinol . 2016; 422: 115– 124. Google Scholar CrossRef Search ADS PubMed  5. Nishimoto H, Matsutani R, Yamamoto S, Takahashi T, Hayashi KG, Miyamoto A, Hamano S, Tetsuka M. Gene expression of glucose transporter (GLUT) 1, 3 and 4 in bovine follicle and corpus luteum. J Endocrinol . 2006; 188( 1): 111– 119. Google Scholar CrossRef Search ADS PubMed  6. Berry GT, Baynes JW, Wells-Knecht KJ, Szwergold BS, Santer R. Elements of diabetic nephropathy in a patient with GLUT 2 deficiency. Mol Genet Metab . 2005; 86( 4): 473– 477. Google Scholar CrossRef Search ADS PubMed  7. MacLean PS, Zheng D, Jones JP, Olson AL, Dohm GL. Exercise-induced transcription of the muscle glucose transporter (GLUT 4) gene. Biochem Biophys Res Commun . 2002; 292( 2): 409– 414. Google Scholar CrossRef Search ADS PubMed  8. Shimada Y, Sawada S, Hojo S, Okumura T, Nagata T, Nomoto K, Tsukada K. Glucose transporter 3 and 1 may facilitate high uptake of 18F-FDG in gastric schwannoma. Clin Nucl Med . 2013; 38( 11): e417– e420. Google Scholar CrossRef Search ADS PubMed  9. Kol S, Ben-Shlomo I, Ruutiainen K, Ando M, Davies-Hill TM, Rohan RM, Simpson IA, Adashi EY. The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process. J Clin Invest . 1997; 99( 9): 2274– 2283. Google Scholar CrossRef Search ADS PubMed  10. Kodaman PH, Behrman HR. Hormone-regulated and glucose-sensitive transport of dehydroascorbic acid in immature rat granulosa cells. Endocrinology . 1999; 140( 8): 3659– 3665. Google Scholar CrossRef Search ADS PubMed  11. Zhang C, Niu W, Wang Z, Wang X, Xia G. The effect of gonadotropin on glucose transport and apoptosis in rat ovary. PLoS One . 2012; 7( 8): e42406. Google Scholar CrossRef Search ADS PubMed  12. Tian Y, Ding Y, Liu J, Heng D, Xu K, Liu W, Zhang C. Nitric oxide–mediated regulation of GLUT by T3 and follicle-stimulating hormone in rat granulosa cells. Endocrinology . 2017; 158( 6): 1898– 1915. Google Scholar CrossRef Search ADS PubMed  13. Koesling D, Mergia E, Russwurm M. Physiological functions of NO-sensitive guanylyl cyclase isoforms. Curr Med Chem . 2016; 23( 24): 2653– 2665. Google Scholar CrossRef Search ADS PubMed  14. Dixit VD, Parvizi N. Nitric oxide and the control of reproduction. Anim Reprod Sci . 2001; 65( 1-2): 1– 16. Google Scholar CrossRef Search ADS PubMed  15. Basini G, Grasselli F. Nitric oxide in follicle development and oocyte competence. Reproduction . 2015; 150( 1): R1– R9. Google Scholar CrossRef Search ADS PubMed  16. Zamberlam G, Sahmi F, Price CA. Nitric oxide synthase activity is critical for the preovulatory epidermal growth factor-like cascade induced by luteinizing hormone in bovine granulosa cells. Free Radic Biol Med . 2014; 74: 237– 244. Google Scholar CrossRef Search ADS PubMed  17. Masuda M, Kubota T, Aso T. Effects of nitric oxide on steroidogenesis in porcine granulosa cells during different stages of follicular development. Eur J Endocrinol . 2001; 144( 3): 303– 308. Google Scholar CrossRef Search ADS PubMed  18. Dineva JD, Vangelov IM, Nikolov GG, Konakchieva RTs, Ivanova MD. Nitric oxide stimulates the production of atrial natriuretic peptide and progesterone by human granulosa luteinized cells with an antiapoptotic effect. Endocr Regul . 2008; 42( 2-3): 45– 51. Google Scholar PubMed  19. Jee BC, Kim SH, Moon SY. The role of nitric oxide on apoptosis in human luteinized granulosa cells. Immunocytochemical evidence. Gynecol Obstet Invest . 2003; 56( 3): 143– 147. Google Scholar CrossRef Search ADS PubMed  20. Matsumi H, Yano T, Osuga Y, Kugu K, Tang X, Xu JP, Yano N, Kurashima Y, Ogura T, Tsutsumi O, Koji T, Esumi H, Taketani Y. Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development. Biol Reprod . 2000; 63( 1): 141– 146. Google Scholar CrossRef Search ADS PubMed  21. Jablonka-Shariff A, Olson LM. Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology . 1997; 138( 1): 460– 468. Google Scholar CrossRef Search ADS PubMed  22. Zackrisson U, Mikuni M, Wallin A, Delbro D, Hedin L, Brännström M. Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation. Hum Reprod . 1996; 11( 12): 2667– 2673. Google Scholar CrossRef Search ADS PubMed  23. El-Sehemy A, Postovit LM, Fu Y. Nitric oxide signaling in human ovarian cancer: a potential therapeutic target. Nitric Oxide . 2016; 54: 30– 37. Google Scholar CrossRef Search ADS PubMed  24. Colbran JL, Roach PJ, Fiol CJ, Dixon JE, Andrisani OM, Corbin JD. cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates delta-CREB, and a synthetic delta-CREB peptide. Biochem Cell Biol . 1992; 70( 10-11): 1277– 1282. Google Scholar CrossRef Search ADS PubMed  25. Wong JC, Bathina M, Fiscus RR. Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells. J Cell Biochem . 2012; 113( 11): 3587– 3598. Google Scholar CrossRef Search ADS PubMed  26. Gudi T, Casteel DE, Vinson C, Boss GR, Pilz RB. NO activation of fos promoter elements requires nuclear translocation of G-kinase I and CREB phosphorylation but is independent of MAP kinase activation. Oncogene . 2000; 19( 54): 6324– 6333. Google Scholar CrossRef Search ADS PubMed  27. Chen Y, Zhuang S, Cassenaer S, Casteel DE, Gudi T, Boss GR, Pilz RB. Synergism between calcium and cyclic GMP in cyclic AMP response element-dependent transcriptional regulation requires cooperation between CREB and C/EBP-beta. Mol Cell Biol . 2003; 23( 12): 4066– 4082. Google Scholar CrossRef Search ADS PubMed  28. Kim MO, Lee YJ, Park JH, Ryu JM, Yun SP, Han HJ. PKA and cAMP stimulate proliferation of mouse embryonic stem cells by elevating GLUT1 expression mediated by the NF-κB and CREB/CBP signaling pathways. Biochim Biophys Acta . 2012; 1820( 10): 1636– 1646. Google Scholar CrossRef Search ADS PubMed  29. Rao IM, Mills TM, Anderson E, Mahesh VB. Heterogeneity in granulosa cells of developing rat follicles. Anat Rec . 1991; 229( 2): 177– 185. Google Scholar CrossRef Search ADS PubMed  30. Wang Q, Leader A, Tsang BK. Follicular stage-dependent regulation of apoptosis and steroidogenesis by prohibitin in rat granulosa cells. J Ovarian Res . 2013; 6( 1): 23. Google Scholar CrossRef Search ADS PubMed  31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method. Methods . 2001; 25( 4): 402– 408. Google Scholar CrossRef Search ADS PubMed  32. Liu M, Li ZH, Xu FJ, Lai LH, Wang QQ, Tang GP, Yang WT. An oligopeptide ligand-mediated therapeutic gene nanocomplex for liver cancer-targeted therapy. Biomaterials . 2012; 33( 7): 2240– 2250. Google Scholar CrossRef Search ADS PubMed  33. Li RJ, Ying X, Zhang Y, Ju RJ, Wang XX, Yao HJ, Men Y, Tian W, Yu Y, Zhang L, Huang RJ, Lu WL. All-trans retinoic acid stealth liposomes prevent the relapse of breast cancer arising from the cancer stem cells. J Control Release . 2011; 149( 3): 281– 291. Google Scholar CrossRef Search ADS PubMed  34. Oelze M, Mollnau H, Hoffmann N, Warnholtz A, Bodenschatz M, Smolenski A, Walter U, Skatchkov M, Meinertz T, Münzel T. Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction. Circ Res . 2000; 87( 11): 999– 1005. Google Scholar CrossRef Search ADS PubMed  35. Shi T, Papay RS, Perez DM. α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake. J Recept Signal Transduct Res . 2016; 36( 3): 261– 270. Google Scholar CrossRef Search ADS PubMed  36. Deng D, Xu C, Sun P, Wu J, Yan C, Hu M, Yan N. Crystal structure of the human glucose transporter GLUT1. Nature . 2014; 510( 7503): 121– 125. Google Scholar CrossRef Search ADS PubMed  37. Morato PN, Lollo PC, Moura CS, Batista TM, Camargo RL, Carneiro EM, Amaya-Farfan J. Whey protein hydrolysate increases translocation of GLUT-4 to the plasma membrane independent of insulin in wistar rats. PLoS One . 2013; 8( 8): e71134. Google Scholar CrossRef Search ADS PubMed  38. Fedail JS, Zheng K, Wei Q, Kong L, Shi F. Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats. Endocrine . 2014; 46( 3): 594– 604. Google Scholar CrossRef Search ADS PubMed  39. Chen Q, Yano T, Matsumi H, Osuga Y, Yano N, Xu J, Wada O, Koga K, Fujiwara T, Kugu K, Taketani Y. Cross-talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia. Endocrinology . 2005; 146( 2): 808– 815. Google Scholar CrossRef Search ADS PubMed  40. Yoon SJ, Choi KH, Lee KA. Nitric oxide-mediated inhibition of follicular apoptosis is associated with HSP70 induction and Bax suppression. Mol Reprod Dev . 2002; 61( 4): 504– 510. Google Scholar CrossRef Search ADS PubMed  41. Matsumi H, Koji T, Yano T, Yano N, Tsutsumi O, Momoeda M, Osuga Y, Taketani Y. Evidence for an inverse relationship between apoptosis and inducible nitric oxide synthase expression in rat granulosa cells: a possible role of nitric oxide in ovarian follicle atresia. Endocr J . 1998; 45( 6): 745– 751. Google Scholar CrossRef Search ADS PubMed  42. Dubey PK, Tripathi V, Singh RP, Saikumar G, Nath A, Pratheesh, Gade N, Sharma GT. Expression of nitric oxide synthase isoforms in different stages of buffalo (Bubalus bubalis) ovarian follicles: effect of nitric oxide on in vitro development of preantral follicle. Theriogenology . 2012; 77( 2): 280– 291. Google Scholar CrossRef Search ADS PubMed  43. Dubey PK, Tripathi V, Singh RP, Sharma GT. Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles. J Vet Sci . 2011; 12( 3): 257– 265. Google Scholar CrossRef Search ADS PubMed  44. Ren Y, Zheng J, Yao X, Weng G, Wu L. Essential role of the cGMP/PKG signaling pathway in regulating the proliferation and survival of human renal carcinoma cells. Int J Mol Med . 2014; 34( 5): 1430– 1438. Google Scholar CrossRef Search ADS PubMed  45. Wong JC, Fiscus RR. Essential roles of the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells. J Cell Biochem . 2011; 112( 3): 829– 839. Google Scholar CrossRef Search ADS PubMed  46. Yoneyama M, Kawada K, Shiba T, Ogita K. Endogenous nitric oxide generation linked to ryanodine receptors activates cyclic GMP/protein kinase G pathway for cell proliferation of neural stem/progenitor cells derived from embryonic hippocampus. J Pharmacol Sci . 2011; 115( 2): 182– 195. Google Scholar CrossRef Search ADS PubMed  47. Nagai-Kusuhara A, Nakamura M, Mukuno H, Kanamori A, Negi A, Seigel GM. cAMP-responsive element binding protein mediates a cGMP/protein kinase G-dependent anti-apoptotic signal induced by nitric oxide in retinal neuro-glial progenitor cells. Exp Eye Res . 2007; 84( 1): 152– 162. Google Scholar CrossRef Search ADS PubMed  48. Fiscus RR. Involvement of cyclic GMP and protein kinase G in the regulation of apoptosis and survival in neural cells. Neurosignals . 2002; 11( 4): 175– 190. Google Scholar CrossRef Search ADS PubMed  49. Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR. Protein kinase G type Iα activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Mol Cancer Res . 2010; 8( 4): 578– 591. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Endocrinology Oxford University Press

cGMP/PKG-I Pathway–Mediated GLUT1/4 Regulation by NO in Female Rat Granulosa Cells

Loading next page...
 
/lp/ou_press/cgmp-pkg-i-pathway-mediated-glut1-4-regulation-by-no-in-female-rat-Ixghp2aJ4y
Publisher
Endocrine Society
Copyright
Copyright © 2018 Endocrine Society
ISSN
0013-7227
eISSN
1945-7170
D.O.I.
10.1210/en.2017-00863
Publisher site
See Article on Publisher Site

Abstract

Abstract Nitric oxide (NO) is a multifunctional gaseous molecule that plays important roles in mammalian reproductive functions, including follicular growth and development. Although our previous study showed that NO mediated 3,5,3′-triiodothyronine and follicle-stimulating hormone–induced granulosa cell development via upregulation of glucose transporter protein (GLUT)1 and GLUT4 in granulosa cells, little is known about the precise mechanisms regulating ovarian development via glucose. The objective of the present study was to determine the cellular and molecular mechanism by which NO regulates GLUT expression and glucose uptake in granulosa cells. Our results indicated that NO increased GLUT1/GLUT4 expression and translocation in cells, as well as glucose uptake. These changes were accompanied by upregulation of cyclic guanosine monophosphate (cGMP) level and cGMP-dependent protein kinase (PKG)-I protein content. The results of small interfering RNA (siRNA) analysis showed that knockdown of PKG-I significantly attenuated gene expression, translocation, and glucose uptake. Moreover, the PKG-I inhibitor also blocked the above processes. Furthermore, NO induced cyclic adenosine monophosphate response element binding factor (CREB) phosphorylation, and CREB siRNA attenuated NO-induced GLUT expression, translocation, and glucose uptake in granulosa cells. These findings suggest that NO increases cellular glucose uptake via GLUT upregulation and translocation, which are mediated through the activation of the cGMP/PKG pathway. Meanwhile, the activated CREB is also involved in the regulation. These findings indicate that NO has an important influence on the glucose uptake of granulosa cells. Folliculogenesis and follicular development are complex and highly selective processes; >99% of the follicles undergo atresia during the ovarian cycle in life. Follicular development is regulated by endocrine, autocrine, and paracrine factors. The transition from preantral to early antral is the penultimate stage in terms of gonadotropin dependence (1). It is also a critical stage of follicular development, as it is highly susceptible to atretogenic signals that determine follicle destiny (survival/growth vs atresia). Glucose as a major energy substrate is essential to ovarian growth. The utilization of glucose is facilitated by a family of glucose transporter proteins (GLUTs), including 14 identified members in the GLUT family (2, 3). These transporters exhibit differential tissue localization and functional characteristics, with considerable interspecies differences. However, GLUT1–4 are major contributors to glucose uptake owing to their high affinity for glucose in various tissues (4–8). It has been reported that GLUT1–4 are detected in rat ovaries (9–11). Our previous results showed that 3,5,3′-triiodothyronine (T3) combined with follicle-stimulating hormone (FSH) to potentiate cellular glucose uptake via regulating the expression and translocation of GLUT1/4 upregulation in granulosa cells. Nitric oxide synthase (NOS)/nitric oxide (NO) are also involved in this regulatory network (12). NO is a multifunctional gaseous molecule. Accumulating evidence suggests that NO is involved in multiple cellular and biological processes (13). Moreover, NO is also important to female reproduction including follicle growth, ovulation, oocyte maturation, corpus luteum function and steroidogenesis (14–17). It has been reported that NO has multiple effects, such as inhibiting granulosa cell apoptosis, increasing estradiol levels and promoting follicular development (18–20). NO is synthesized from l-arginine, reduced form of NAD phosphate, and oxygen by a family of enzymes termed NOS. There are three isoforms of NOS: neuronal NOS (nNOS/NOS1), inducible NOS (iNOS/NOS2), and endothelial NOS (eNOS/NOS3). Although NOSs are detected in the ovary, their expression and activity are highly dependent on species, cell type, and stage of ovarian development (21, 22). Only NOS3 is regulated by FSH and T3 in granulosa cells. Our previous study also showed that NO is also involved in T3- and FSH-induced follicular development via GLUT upregulation and glucose uptake (12). However, the precise underlying mechanism by which NO regulates glucose uptake in granulosa cells is not understood. It is well known that NO regulates many biological functions through cyclic guanosine monophosphate (cGMP)–dependent signaling in different cells. NO binds to soluble guanylyl cyclase (sGC) and sharply increases sGC activity. sGC converts guanosine triphosphate into cGMP, increasing the intracellular concentrations of cGMP. The latter initiates a variety of downstream signaling events, one of which is to combine and activate its major cellular receptor, cGMP-dependent protein kinase (PKG) (23). However, whether and how NO/cGMP/PKG signaling modulates follicular development is not known. The transcription factor cyclic adenosine monophosphate response element binding factor (CREB) plays a critical role in many physiological activities. Many reports have noted that CREB is phosphorylated by PKG in different cells (24–27). Furthermore, the expression of GLUT1 is elevated by CREB/CBP (CREB protein) in mouse embryonic stem cells (28). However, the role of activated CREB in GLUT expression remains uncertain. In the present study, we investigated the cellular mechanisms of NO-induced granulosa cell GLUT expression and the possible involvement of cGMP/PKG in these processes. We demonstrated that NO increases the expression of GLUT1 and GLUT4, which are associated with increased glucose uptake by granulosa cells. Additionally, NO increased GLUT translocation to the plasma membrane. These responses are mediated by the activation of cGMP/PKG pathways, and CREB also plays a regulatory role in this system. Materials and Methods Reagents and antibodies All chemicals and culture media components used in the present study were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Culture media were purchased from Gibco Bethesda Research Laboratories (Grand Island, NY). The enhanced chemiluminescence detection kit was obtained from Amersham Life Science (Oakville, ON, Canada). Acrylamide (electrophoresis grade), N,N′-methylene-bis-acrylamide, ammonium persulfate, glycine, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis prestained molecular mass standards were products of Bio-Rad Laboratories (Richmond, CA). Rabbit polyclonal anti-GLUT1 (ab652) and rabbit polyclonal anti-GLUT4 (ab654) antibodies were purchased from Abcam (Cambridge, MA). Rabbit monoclonal anti–PKG-1 (3248), rabbit polyclonal anti–Na,K-adenosine triphosphatase (3010), rabbit monoclonal anti-CREB (9197), and phospho-CREB (Ser133) antibody were from Cell Signaling Technology (Danvers, MA). Mouse monoclonal anti–β-actin (sc-81178) and horseradish peroxidase (HRP)–conjugated anti-rabbit and anti-mouse IgG were from Santa Cruz Biotechnology (Beijing, China). A RevertAid first-strand complementary DNA (cDNA) synthesis kit and TRIzol reagent were obtained from Thermo Fisher Scientific (Waltham, MA), and a SYBR Green polymerase chain reaction (PCR) kit was purchased from Bio-Rad Laboratories. PCR primers for GLUT1, GLUT4 and 18S ribosomal RNA (rRNA) were from Beijing Sunbiotech (Beijing, China). PKG inhibitor KT5823 was purchased from Beyotime Biotechnology (Beijing China). A cGMP assay kit purchased from Cell Signaling Technology was used to determine cGMP levels in cells. The 2-deoxyglucose (2-DG) uptake measurement kit purchased from Cosmo Bio (Tokyo Japan) was used to detect glucose uptake level. The cell counting kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Lipofectamine 3000 was purchased from Invitrogen (Carlsbad, CA). Animal treatments Twenty-one-day-old Sprague-Dawley rats were (Beijing Vital Laboratory Animal Technology, Beijing, China) maintained on 12-hour light/12-hour dark cycle and received pathogen-free water and food. Rats were injected subcutaneously with diethylstilbestrol (1 mg/d; 3 days), which induces more preantral and early antral follicles (12, 29, 30), and ovaries were collected at 72 hours after euthanization by cervical dislocation. All procedures were carried out in accordance with the Principles of the Care and Use of Laboratory Animals and China Council on Animal Care and were approved by the Institutional Animal Care and Use Committee of Capital Normal University (approval no. 2016017). Preantral follicle isolation and culture Ovaries of 14-day-old rats were removed and placed in 2.5 ml of Leibowitz L15 medium supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Large preantral follicles (diameter, 150 to 180 μm) were isolated using a 26-gauge needle. To minimize the experimental variation caused by damage incurred during the isolation procedures, only round follicles with an intact basement membrane and thecal layer were selected for the present studies. Follicles were cultured individually in a 96-well plate in 100 μL of α–minimum essential medium supplemented with HEPES (10 mM), bovine serum albumin (0.1%, weight-to-volume), bovine insulin (5 μg/mL), transferrin (2 μg/mL), ascorbic acid (25 μg/mL), sodium selenite anhydrous (2 ng/mL), l-glutamine (3 mM), sodium pyruvate (100 μg/mL), streptomycin (100 μg/mL), and penicillin (100 U/mL), with or without S-nitroso-N-acetyl-dl-penicillamine (SNAP; 100 μM). The diameter of the follicle was measured every day as the average of distance between the outer edges of the basement in two perpendicular planes. The results were calculated as follows: the percentage change of follicular volume = [(Vn − V0)/V0] × 100%, where Vn is the follicular volume on day n and V0 is the initial volume of the follicles. The culture medium was changed every other day. Rat granulosa cell isolation and culture Rat granulosa cell isolation and culture processes were performed as described previously (12). Simply, granulosa cells were released by follicular puncture from large preantral and early antral follicles. The cell clumps and oocytes were removed by filtering the cell suspensions through a 40-μm nylon cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ; no. 352340). The viability of cells was estimated by a Trypan blue dye exclusion test. Granulosa cells (9 × 105 viable granulosa cells per plate in a six-well plate) were cultured in M199 with fetal bovine serum (10%, weight-to-volume) for 6 hours. The media were then replaced with serum-free M199 supplemented as above for 12 hours thereafter and cells were treated with SNAP, a nitrosothiol derivative. In some experiments, cells were pretreated with a cell-permeable cGMP analog, 8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt (8-Br-cGMP, 1 mM), sGC inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 μM), or PKG inhibitor KT5823 (1 μM) 1 hour before SNAP treatment, respectively. Cells were maintained at 37°C under humidified atmosphere (5% CO2). For RNA interference, granulosa cells were transfected (48 hours) with PKG small interfering RNA (siRNA) or CREB siRNA and scrambled sequence control (GenePharma, Shanghai, China), respectively, using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Western blot analysis Western blot analysis was performed as described previously (12). Whole-cell lysates were prepared by incubating cell pellets for 30 minutes at 0°C in lysis buffer (Beyotime Biotechnology, Shanghai, China), and protease and phosphatase inhibitor cocktails (Sigma-Aldrich) were added to buffers before use. The supernatant was collected by centrifugation (15,000 × g, 4°C, 30 minutes) and protein concentration was determined with the BCA protein assay kit (Beyotime Biotechnology). The protein samples (15 to 80 µg, depending on individual experiments) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto the nitrocellulose membrane. The membranes were then blocked in tris-buffered saline with Tween 20 buffer (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20; pH 7.5) containing 5% dehydrated nonfat milk at room temperature for 1 hour and subsequently incubated (4°C, overnight) with diluted primary antibody [polyclonal anti-GLUT1 (1:500), polyclonal anti-GLUT4 (1:2000), rabbit monoclonal anti–PKG-I (1:1000), rabbit polyclonal anti–Na,K-ATPase (1:1000), rabbit monoclonal anti-CREB (1:500), or β-actin (1:10,000)], followed by HRP-conjugated secondary antibody (1:1000 to 1:10,000; 1.5 hours, room temperature). Peroxidase activity was visualized with the enhanced chemiluminescence kit according to the manufacturer’s instructions. Protein content was determined by densitometrically scanning the exposed x-ray film. Immunoreaction signals were analyzed using Gel-Pro Analyzer 4.0. RNA extraction, cDNA synthesis, and real-time PCR analysis Total RNAs were extracted from cultured granulosa cells with TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, and then washed in 75% ethanol and dissolved in RNase-free distilled water. cDNAs were produced from 0.2 μg of total RNAs by reverse transcription. Gene expression was measured by real-time PCR and normalized to 18S rRNA. Specific primer pairs used in the experiments are listed in Table 1. Data were analyzed by the 2−ΔΔCT method (31). Table 1. Primer Sequence Used for Real-Time Quantitative PCR Target Gene  GenBank Accession No.  Primer Sequence  Product Size, bp  Annealing Temperature, °C  GLUT-1  NM_138827.1  F: 5′-TGGCCAAGGACACACGAATACTGA-3′  144  60  R: 5′-TGGAAGAGACAGGAATGGGCGAAT-3′  GLUT-4  NM_012751.1  F: 5′-TGTTGCGGATGCTATGGGTC-3′  123  60  R: 5′-GCCGAGATCTGGTCAAATGT-3′  18S rRNA  NR_046237.1  F: 5′-CGCGGTTCTATTTTGTTGGT-3′  219  60  R: 5′-AGTCGGCATCGTTTATGGTC-3′  Target Gene  GenBank Accession No.  Primer Sequence  Product Size, bp  Annealing Temperature, °C  GLUT-1  NM_138827.1  F: 5′-TGGCCAAGGACACACGAATACTGA-3′  144  60  R: 5′-TGGAAGAGACAGGAATGGGCGAAT-3′  GLUT-4  NM_012751.1  F: 5′-TGTTGCGGATGCTATGGGTC-3′  123  60  R: 5′-GCCGAGATCTGGTCAAATGT-3′  18S rRNA  NR_046237.1  F: 5′-CGCGGTTCTATTTTGTTGGT-3′  219  60  R: 5′-AGTCGGCATCGTTTATGGTC-3′  View Large cGMP level assay Granulosa cells were cultured with or without SNAP (100 μM). The cells were harvested at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 18 hours, respectively. The cGMP level was detected by a cGMP assay kit according to the manufacturer’s protocol. Briefly, cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and kept in lysis buffer on ice for 5 to 10 minutes. The 50-μL sample and 50-μL HRP-linked cGMP solution were transferred to the cGMP assay plate. Next, the plate contents were discarded and washed four times with wash buffer. After that, the sample was incubated with 100 μL of tetramethylbenzidine substrate for 30 minutes at room temperature. After adding 100 μL of STOP solutions, the absorbance was measured at 450 nm. The cGMP concentration was calculated according to the standard curve. Plasma membrane protein isolation To investigate whether NO regulated GLUT1 and GLUT4 translocation to the plasma membrane, the plasma membrane of a granulosa cell was isolated by a Minute plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN) according to the manufacturer’s instructions. Briefly, granulosa cells were first sensitized followed by a series of centrifugation steps to remove the nucleus, cytosol, and organelle membrane, respectively. Thus, the cell membrane was obtained. Immunofluorescence cell staining The rat granulosa cells were cultured in poly-d-lysine–coated (0.05% weight-to-volume; Sigma-Aldrich) eight-well glass culture slides (Becton Dickinson and Co.) used for immunofluorescence analysis. After treatment with or without SNAP for 24 hours, the cells were fixed in 4% paraformaldehyde for 30 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and then blocked in PBS buffer containing 5% bovine serum albumin at room temperature for 30 min. The cells were subsequently incubated with polyclonal anti-GLUT1 (1:200 dilution in blocking solution) or polyclonal anti-GLUT4 (1:200 dilution in blocking solution), respectively, at 4°C overnight. The samples were then incubated with Alexa Fluor 488–conjugated secondary antibody (1:400 dilution in blocking solution; Jackson ImmunoResearch Laboratories, West Grove, PA) at 37°C for 1.5 hours. After washing with PBS three times, samples were incubated with DiI (red, membrane stain) (32, 33) and 4′,6-diamidino-2-phenylindole (blue, nuclear stain) for 10 minutes at room temperature and then washed in PBS three times. After that, the coverslips were mounted on object slides using fluorescent mounting medium. Immunofluorescence was visualized using an immunofluorescence microscope (Olympus BX51), and images were recorded by using a DP70 digital camera (Olympus Optical Co., Tokyo, Japan). Glucose uptake assay Glucose uptake levels were determined using a 2-DG measurement kit (Cosmo Bio Co., Tokyo, Japan) in accordance with the manufacturer’s protocol. Briefly, granulosa cells were cultured up to 24 hours in the presence of SNAP with or without inhibitors as mentioned above, and then the cells were incubated in serum-free medium for 6 hours. After washing three times in PBS, the cells were incubated in 2-DG solution at 37°C for 20 minutes. Cell lysates were collected after washing the cells three times again and then heated at 80°C for 15 min. The samples were centrifuged (4°C, 15,000 × g) for 20 minutes and transferred to a new tube. The optical density (OD) value was detected using a microplate detector at 420 nm. The concentration of each sample was measured in triplicates and calculated using the standard curve supplied in the kit. Analysis of cell viability Cell activity was detected by the CCK-8 (Dojindo), which is based on the dehydrogenase activity detection in viable cells. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. In the present study, granulosa cells were cultured with SNAP in 96-well plates and then incubated with 10 µL of CCK-8 solution at 37°C for 2 hours. The OD values in each well were recorded using a microplate reader at 450 nm. The mean OD values for each treatment were used as the index of cell viability. Statistical analysis The experiments were repeated at least three times, as detailed in the figure legends. Experimental data are presented as means ± standard error of the mean. The statistical differences between treatments were calculated with t test, one-way analysis of variance, or two-way (repeated-measures) analysis of variance (Prism 5.0 statistical software; GraphPad Software, San Diego, CA). When significant differences were found, means were compared by the Bonferroni posttest. A P value < 0.05 was considered to be significant. Results Effect of NO on follicular growth and GLUT expression in vitro To evaluate the effect of NO on preantral follicle development, follicles were cotreated with SNAP for 4 days. The follicular volume change curve during the 4-day culture period is presented in Fig. 1A. Preantral follicles cultured in the presence of SNAP were significantly increased (day 4, 119.54 ± 1.37 vs 59.93 ± 2.59, P < 0.001). Meanwhile, SNAP also gradually increased glucose uptake (P < 0.01, Fig. 1B). Moreover, SNAP statistically increased GLUT1 (1.74 ± 0.11 vs 1.00 ± 0.09, P < 0.05, Fig. 1C) and GLUT4 (1.41 ± 0.17 vs 1.02 ± 0.07, P < 0.05, Fig. 1D) messenger RNA (mRNA) levels. However, the upregulation of GLUT1 and GLUT4 by SNAP was significantly attenuated by ODQ and KT5823 (Fig. 1C and 1D). Figure 1. View largeDownload slide Effect of NO on preantral follicular growth in vitro. Preantral follicles were cultured for 4 days with or without SNAP (100 μM). Follicles cultured in the absence of SNAP were named as the control group. (A) Follicular diameter was measured daily and results are expressed as change in follicular volume. (B) Follicular glucose uptake was assessed. (C and D) Follicles were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The mRNA abundance of GLUT1 and GLUT4 were analyzed by real-time PCR. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. CTL, control. Figure 1. View largeDownload slide Effect of NO on preantral follicular growth in vitro. Preantral follicles were cultured for 4 days with or without SNAP (100 μM). Follicles cultured in the absence of SNAP were named as the control group. (A) Follicular diameter was measured daily and results are expressed as change in follicular volume. (B) Follicular glucose uptake was assessed. (C and D) Follicles were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The mRNA abundance of GLUT1 and GLUT4 were analyzed by real-time PCR. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. CTL, control. NO increased granulosa cell development To determine whether NO affected the growth of granuloasa cells, we investigated cellular development after treatment with various concentrations (0, 50, 100, 200, and 500 μM) of SNAP. As shown in Fig. 2, SNAP induced a peak of cellular viability at 100 μM compared with the control group [2.01 ± 0.11 (SNAP) vs 0.88 ± 0.01 (control), P < 0.001]. Figure 2. View largeDownload slide NO increased granulosa cell development in vitro. Immature rats were treated with diethylstilbestrol (1 mg/d, 3 consecutive days) prior to granulosa cell isolation from preantral and early antral follicles. The cells were collected and cocultured with SNAP (0, 50, 100, 200, and 500 µM) for 24 hours, and cell viability was analyzed by CCK-8 assay. ***P < 0.001, compared with control (without SNAP). Figure 2. View largeDownload slide NO increased granulosa cell development in vitro. Immature rats were treated with diethylstilbestrol (1 mg/d, 3 consecutive days) prior to granulosa cell isolation from preantral and early antral follicles. The cells were collected and cocultured with SNAP (0, 50, 100, 200, and 500 µM) for 24 hours, and cell viability was analyzed by CCK-8 assay. ***P < 0.001, compared with control (without SNAP). Effects of NO on granulosa cell GLUT expression In our previous study we showed that T3- and FSH-induced GLUT1 and GLUT4 expressions are attenuated by NOS3 knockdown in rat granulosa cells. To examine whether NO regulates GLUT1 and GLUT4, we cultured granulosa cells with SNAP for 24 hours. As shown in Fig. 3A and 3B, SNAP treatment significantly increased GLUT1 [18 hours, 0.94 ± 0.03 (SNAP) vs 0.55 ± 0.03 (control), P < 0.05; 24 hours, 1.66 ± 0.11 (SNAP) vs 0.99 ± 0.06 (control), P < 0.01] and GLUT4 [18 hours, 1.06 ± 0.08 (SNAP) vs 0.67 ± 0.02 (control), P < 0.05; 24 hours, 1.55 ± 0.05 (SNAP) vs 0.98 ± 0.13 (control), P < 0.05] protein levels. Figure 3. View largeDownload slide Effects of NO on granulosa cell GLUT expression in vitro. (A and B) Granulosa cells were collected after cotreatment with SNAP (100 μM) for 18 hours and 24 hours, and GLUT1 and GLUT4 proteins were detected by western blot analysis. (C and D) Cells were treated with SNAP (100 μM) up to 24 hours (6, 12, 18, and 24 hours) for GLUT1 (B) and GLUT4 (C) mRNA analysis by real-time PCR. mRNA abundance was normalized using 18S rRNA. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. CTL, control. Figure 3. View largeDownload slide Effects of NO on granulosa cell GLUT expression in vitro. (A and B) Granulosa cells were collected after cotreatment with SNAP (100 μM) for 18 hours and 24 hours, and GLUT1 and GLUT4 proteins were detected by western blot analysis. (C and D) Cells were treated with SNAP (100 μM) up to 24 hours (6, 12, 18, and 24 hours) for GLUT1 (B) and GLUT4 (C) mRNA analysis by real-time PCR. mRNA abundance was normalized using 18S rRNA. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. CTL, control. To understand more fully whether the content of GLUT1 and GLUT4 is regulated at the mRNA level by NO, we determined GLUT mRNA abundance using real-time PCR analysis. Granulosa cells were cultured with SNAP for 24 hours. The results showed that GLUT1 and GLUT4 mRNA levels were significantly increased at 12 hours [GLUT1, 3.73 ± 0.04 (SNAP) vs 1.01 ± 0.11 (control), P < 0.001, Fig. 3C; GLUT4, 3.89 ± 0.06 (SNAP) vs 0.98 ± 0.54 (control), P < 0.001, Fig. 3D] and 18 hours [GLUT1, 5.26 ± 0.05 (SNAP) vs 1.10 ± 0.14 (control), P < 0.001, Fig. 3C; GLUT4, 5.69 ± 0.25 (SNAP) vs 1.05 ± 0.06 (control), P < 0.001, Fig. 3D] after SNAP treatment. These results suggest that increased gene transcription may at least partly account for the increased protein content induced by SNAP. Involvement of the cGMP/PKG pathway in NO-induced GLUT expression and glucose uptake To determine whether the cGMP/PKG signaling pathway is involved in the regulation of GLUT expression by NO, we next examined the intracellular cGMP levels after SNAP treatment. We noticed that intracellular cGMP levels were increased with the duration of treatment, reaching a peak at 1 hour after SNAP treatment [425.13 ± 9.97 (SNAP) vs 10.63 ± 0.66 (CTL), P < 0.001, Fig. 4A]. We also detected phospho–vasodilator-stimulated phosphoprotein content after SNAP treatment, which is originally characterized as a substrate of PKG (34) (Supplemental Fig. 2). We then pretreated cells with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. Western blot analysis carried out after 24 hours revealed that the expression of GLUT1 and GLUT4 were significantly higher in the 8-Br-cGMP group, which was similar to the effect of SNAP treatment. However, these effects were abolished after coculture with ODQ or KT5823 [GLUT1, 1.19 ± 0.11 (8-Br-cGMP) vs 0.62 ± 0.03 (control), P < 0.01; GLUT4, 1.24 ± 0.04 (8-Br-cGMP) vs 0.86 ± 0.02 (control), P < 0.05, Fig. 4B]. Glucose levels followed a pattern similar to GLUT1/4 [3.02 ± 0.13 (8-Br-cGMP) vs 1.76 ± 0.14 (control), P < 0.001; 2.80 ± 0.09 (SNAP) vs 1.76 ±0.14 (control), P < 0.01, Fig. 4C]. SNAP-induced GLUT expression and glucose uptake were also significantly attenuated by PKG knockdown [GLUT1, 0.85 ± 0.03 (PKG siRNA) vs 1.59 ± 0.09 (SNAP), P < 0.05; GLUT4, 1.13 ± 0.08 (PKG siRNA) vs 1.68 ± 0.04 (SNAP), P < 0.05, Fig. 5A; glucose, 1.45 ± 0.18 (PKG siRNA) vs 2.86 ± 0.12 (SNAP), P < 0.01, Fig. 5B]. Figure 4. View largeDownload slide Involvement of the cGMP/PKG pathway in NO-induced GLUT expression and glucose uptake. (A) After cotreatment with SNAP (100 μM), granulosa cells were harvested at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 18 hours, respectively. Next, the cGMP level was detected by a cGMP assay kit. (B and C) Granulosa cells were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The proteins levels of GLUT1 and GLUT4 contents (B) and cellular glucose uptake (C) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. Figure 4. View largeDownload slide Involvement of the cGMP/PKG pathway in NO-induced GLUT expression and glucose uptake. (A) After cotreatment with SNAP (100 μM), granulosa cells were harvested at 15 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 18 hours, respectively. Next, the cGMP level was detected by a cGMP assay kit. (B and C) Granulosa cells were pretreated with or without 8-Br-cGMP (cGMP analog, 1 mM), ODQ (sGC inhibitor, 10 μM), or KT5823 (PKG inhibitor, 1 μM) before SNAP treatment. The proteins levels of GLUT1 and GLUT4 contents (B) and cellular glucose uptake (C) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control. Figure 5. View largeDownload slide Role of PKG in NO-induced GLUT expression and glucose uptake in vitro. Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents (A) and cellular glucose uptake (B) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. Figure 5. View largeDownload slide Role of PKG in NO-induced GLUT expression and glucose uptake in vitro. Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents (A) and cellular glucose uptake (B) were assessed by western blot analysis and 2-DG measurement, respectively. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, ++P < 0.01, compared with SNAP treatment group. cGMP/PKG mediated NO-induced GLUT translocation The previous results showed that NO increased glucose uptake through cGMP/PKG signaling. Because glucose is actively transported into cells via GLUTs, we next investigated whether NO regulated the plasma membrane translocation of GLUT1 and GLUT4. Western blot analysis experiments indicated that the GLUT1 and GLUT4 expression on the cell membrane were increased after SNAP treatment [GLUT1, 1.17 ± 0.12 (SNAP) vs 0.66 ± 0.04 (control), P < 0.05; GLUT4, 1.20 ± 0.06 (SNAP) vs 0.72 ± 0.06 (control), P < 0.05, Fig. 6A], but that GLUT translocation was blocked by PKG siRNA [GLUT1, 0.70 ± 0.07 (PKG siRNA), P < 0.01; GLUT4, 0.74 ± 0.03 (PKG siRNA), P < 0.01, Fig. 6A]. We confirmed these results by immunofluorescence cell staining. As shown in Fig. 6, we found that both GLUT1 and GLUT4 were weakly present on the cell membrane and in the cytoplasm in the control group. After SNAP treatment of 24 hours, expression of GLUT1 (Fig. 6B) and GLUT4 (Fig. 6C) on the cell membrane was increased, and GLUT translocation was also attenuated by PKG knockdown. Figure 6. View largeDownload slide cGMP/PKG mediated NO-induced GLUT translocation. (A) Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. The plasma membrane was obtained by a Minute plasma membrane protein isolation kit. Membrane proteins of GLUT1 and GLUT4 were detected by western blot analysis. (B and C) Granulosa cells were cultured on poly-d-lysine-coated (0.05% weight-to-volume; Sigma-Aldrich) eight-well glass culture slides and transfected with PKG siRNA for 48 hours, and then treated with SNAP for another 24 hours. As described in detail in Materials and Methods, the specimens were fixed and subjected to immunofluorescent staining and analyzed by confocal microscopy for GLUT1 (B, green) and GLUT4 (C, green). The nuclei were stained with DAPI (blue), and the membrane was stained with DiI (red). Scale bars, 10 μm. *P < 0.05 compared with control (without SNAP); ++P < 0.01, compared with SNAP treatment group. CTL, control. Figure 6. View largeDownload slide cGMP/PKG mediated NO-induced GLUT translocation. (A) Granulosa cells were transfected with PKG siRNA (scrambled sequence as control) for 48 hours using Lipofectamine 3000 and then treated with SNAP for another 24 hours. The plasma membrane was obtained by a Minute plasma membrane protein isolation kit. Membrane proteins of GLUT1 and GLUT4 were detected by western blot analysis. (B and C) Granulosa cells were cultured on poly-d-lysine-coated (0.05% weight-to-volume; Sigma-Aldrich) eight-well glass culture slides and transfected with PKG siRNA for 48 hours, and then treated with SNAP for another 24 hours. As described in detail in Materials and Methods, the specimens were fixed and subjected to immunofluorescent staining and analyzed by confocal microscopy for GLUT1 (B, green) and GLUT4 (C, green). The nuclei were stained with DAPI (blue), and the membrane was stained with DiI (red). Scale bars, 10 μm. *P < 0.05 compared with control (without SNAP); ++P < 0.01, compared with SNAP treatment group. CTL, control. Role of CREB in NO-induced GLUT expression To determine whether NO activates CREB in granulosa cells in vitro, granulosa cells were incubated with SNAP for 12 hours and phospho-CREB content was determined. We found that phospho-CREB content was significantly higher in granulosa cells incubated with SNAP [1.05 ± 0.06 (SNAP) vs 0.47 ± 0.04 (control), P < 0.05, Fig. 7A]. However, gene knockdown of PKG decreased the levels of phospho-CREB [0.61 ± 0.02 (PKG siRNA), P < 0.05, Fig. 7A]. Figure 7. View largeDownload slide Role of CREB in NO induced GLUT expression. (A) PKG was knockdown, and phospho-CREB content was analyzed by western blot analysis after SNAP treatment. (B) Granulosa cells were transfected with CREB siRNA, and then cells were treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents were detected by western blot analysis. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, compared with SNAP treatment group. Figure 7. View largeDownload slide Role of CREB in NO induced GLUT expression. (A) PKG was knockdown, and phospho-CREB content was analyzed by western blot analysis after SNAP treatment. (B) Granulosa cells were transfected with CREB siRNA, and then cells were treated with SNAP for another 24 hours. GLUT1 and GLUT4 contents were detected by western blot analysis. *P < 0.05, **P < 0.01, compared with control (without SNAP); +P < 0.05, compared with SNAP treatment group. To determine the role of the activated CREB in GLUT expression, granulosa cells were transfected with CREB siRNA and then treated with SNAP for 24 hours (Fig. 7B). Although GLUT1 and GLUT4 protein content were upregulated by the presence of SNAP, this effect was significantly attenuated by CREB knockdown [GLUT1, 0.64 ± 0.09 (PKG siRNA) vs 1.38 ± 0.07 (SNAP), P < 0.01; GLUT4, 1.29 ± 0.08 (PKG siRNA) vs 1.63 ± 0.06 (SNAP), P < 0.05, Fig. 7B]. Discussion In the present study, we investigated the role of NO in rat granulosa cells. We demonstrated that SNAP, as an NO donor, increased GLUT1 and GLUT4 expression and translocation through the cGMP/PKG pathway in granulosa cells in vitro, and that activated CREB also played a regulatory role. To the best of our knowledge, this is the first study to show that NO, PKG, and CREB have important roles in the expression of GLUTs in rat granulosa cells in vitro. Glucose is the main energy substrate in the rat ovary, and a sufficient supply of glucose is essential for the maintenance of ovarian function. It has been reported that glucose cannot freely pass through the cell membrane and that glucose uptake is mediated by transporter proteins. Therefore, GLUT expression and activity play vital roles in ovary physiological function (5, 35). In our previous study, we detected GLUT1–4 in the granulosa cells. At the same time, we also found that GLUT1 and GLUT4 are regulated by T3 and FSH, and these processes are mediated by NO (12). In the present study, we found that NO increased GLUT1 and GLUT4 expression at both the transcriptional and translational levels, the results of which are consistent with our previous results. It is well known that GLUT1 is ubiquitously expressed in all organs, including ovaries, and that its expression is closely related to the basal level of glucose uptake in most cell types (5, 35, 36). GLUT4 is abundantly expressed in many tissues, and it plays important roles in whole-body glucose homeostasis (7). GLUT4 is a primary factor that determines the maximal rate of glucose transport (37). Moreover, most GLUT4 is stored in the membranous vesicles in the cytoplasm under the normal resting state. Once stimulated, GLUT4 is transported to the cell membrane, where it increases the cellular glucose uptake. Our results showed that SNAP increased the expression of GLUT1 and GLUT4 on the cell membrane, which indicates that NO promoted GLUT translocation. The increased GLUT content and translocation stimulate cellular glucose utilization. However, the underlying mechanisms for the expression and translocation of GLUT1 and GLUT4 induced by NO have not yet been elucidated. NO exerts a variety of physiological effects that are important for the regulation of mammalian reproductive functions, including follicular growth and development (38). In the present study, SNAP stimulated preantral follicular growth, which may have been partly caused by the upregulation of GLUT1 and GLUT4, as well as increased glucose uptake. It has been reported that NO exerts antiapoptotic effects in rat granulosa cells, which prevent ovarian follicle atresia (39–41). The results of the present study showed that NO increased granulosa cell development at a lower dose, which indicates that the effect of NO occurs in a dose-dependent manner. This pattern is consistent with the previous reports that lower concentrations of NO stimulated the survival, growth, and antrum formation of preantral follicles in buffalo. In contrast, the higher NO concentrations inhibited preantral follicle development (42, 43). These results indicate that NO plays different roles (inhibiting or stimulating) in granulosa cell growth depending on its concentration (15). It has been reported that NO activates sGC, then catalyzes guanosine triphosphate to generate cGMP. PKG is a serine/threonine-specific protein kinase that is triggered by cGMP, followed by phosphorylation of a number of biologically important targets. Many reports have demonstrated that the NO/cGMP/PKG signaling pathway plays an important role in promoting cell survival or preventing spontaneous apoptosis in human renal carcinoma cells (44), bone marrow stromal cells (45), neural stem/progenitor cells derived from embryonic hippocampus (46), retinal neuroglial progenitor cells (47), neural cells (48), and human ovarian cancer cells (49). However, it is not known whether the cGMP/PKG pathway is involved in NO-induced GLUT expression and glucose uptake in ovarian cells. In the present study, we found that NO elevated levels of intracellular cGMP. Inhibition of the cGMP/PKG pathway decreased NO-induced GLUT expression and glucose uptake in follicles and granulosa cells. Our results suggest that the cGMP/PKG pathway plays a regulatory role in the stimulation of glucose transport and glucose uptake by NO. Moreover, our results showed that SNAP upregulated the phosphorylation level of CREB, which was blocked by PKG siRNA. CREB siRNA knockdown also decreased the expression of GLUTs and glucose levels in granulosa cells. These results suggest that NO may activate the cGMP/PKG pathway and, subsequently, the phosphorylation of CREB, then promoting the expression and translocation of GLUTs. In conclusion, our findings demonstrate that NO is a novel positive regulator of granulosa cell development in the early stages of follicular development. As indicated by our model (Fig. 8), NO increases GLUT1/4 expression and translocation, which in turn contributes to glucose uptake by granulosa cells. Moreover, the cGMP/PKG pathway mediated these regulations induced by NO, and phospho-CREB is also involved in this system. Our results enhance our understanding of the role of NO in ovarian cells. Figure 8. View largeDownload slide Schematic diagram of the role of NO on GLUT expression and translocation. Figure 8. View largeDownload slide Schematic diagram of the role of NO on GLUT expression and translocation. Table. Antibody Table Peptide/Protein Target  Antigen Sequence  Name of Antibody  Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID   GLUT1  Synthetic peptide conjugated to KLH, corresponding to amino acids 478 to 492 of human glucose transporter GLUT1  Anti-glucose transporter GLUT1 antibody  ab652  Rabbit; polyclonal  1:500  AB_305540  GLUT4  Synthetic peptide corresponding to the C-terminal 15 residues of rat Glut4 cross-linked to KLH  Anti-glucose transporter GLUT4 antibody  ab654  Rabbit; polyclonal  1:2000  AB_305554  PKG-1    PKG-1 (C8A4) rabbit monoclonal antibody  3248  Rabbit; monoclonal  1:1000  AB_2067450  Na,K-ATPase α    Na,K-ATPase antibody  3010  Rabbit; polyclonal  1:1000  AB_2060983  Phospho-CREB (Ser133)    Phospho-CREB (Ser133) (87G3) rabbit monoclonal antibody  9198  Rabbit; monoclonal  1:1000  AB_2561044  CREB    CREB (48H2) rabbit monoclonal antibody  9197  Rabbit; monoclonal  1:1000  AB_331277  β-Actin    β-Actin (ACTBD11B7)  sc-81178  Mouse; monoclonal  1:1000  AB_2223230  Peptide/Protein Target  Antigen Sequence  Name of Antibody  Catalog No.  Species Raised in; Monoclonal or Polyclonal  Dilution Used  RRID   GLUT1  Synthetic peptide conjugated to KLH, corresponding to amino acids 478 to 492 of human glucose transporter GLUT1  Anti-glucose transporter GLUT1 antibody  ab652  Rabbit; polyclonal  1:500  AB_305540  GLUT4  Synthetic peptide corresponding to the C-terminal 15 residues of rat Glut4 cross-linked to KLH  Anti-glucose transporter GLUT4 antibody  ab654  Rabbit; polyclonal  1:2000  AB_305554  PKG-1    PKG-1 (C8A4) rabbit monoclonal antibody  3248  Rabbit; monoclonal  1:1000  AB_2067450  Na,K-ATPase α    Na,K-ATPase antibody  3010  Rabbit; polyclonal  1:1000  AB_2060983  Phospho-CREB (Ser133)    Phospho-CREB (Ser133) (87G3) rabbit monoclonal antibody  9198  Rabbit; monoclonal  1:1000  AB_2561044  CREB    CREB (48H2) rabbit monoclonal antibody  9197  Rabbit; monoclonal  1:1000  AB_331277  β-Actin    β-Actin (ACTBD11B7)  sc-81178  Mouse; monoclonal  1:1000  AB_2223230  Abbreviation: RRID, research resource identifier. View Large Abbreviations: 2-DG 2-deoxyglucose 8-Br-cGMP 8-bromoguanosine 3′,5′-cyclic monophosphate sodium salt CCK-8 cell counting kit-8 cDNA complementary DNA cGMP cyclic guanosine monophosphate CREB cyclic adenosine monophosphate response element binding factor FSH follicle-stimulating hormone GLUT glucose transporter protein HRP horseradish peroxidase mRNA messenger RNA NO nitric oxide NOS nitric oxide synthase OD optical density ODQ 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one PBS phosphate-buffered saline PCR polymerase chain reaction PKG cyclic guanosine monophosphate–dependent protein kinase rRNA ribosomal RNA sGC soluble guanylyl cyclase siRNA small interfering RNA SNAP S-nitroso-N-acetyl-dl-penicillamine T3 3,5,3′-triiodothyronine. Acknowledgments Financial Support: This work was supported by the National Natural Science Foundation of China Grant 31671555 (to C.Z.) and Scientific Research Program of Beijing Municipal Commission of Education Grant KM201610028011 (to C.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Disclosure Summary: The authors have nothing to disclose. References 1. Hsu SY, Hsueh AJ. Hormonal regulation of apoptosis an ovarian perspective. Trends Endocrinol Metab . 1997; 8( 5): 207– 213. Google Scholar CrossRef Search ADS PubMed  2. Wood IS, Trayhurn P. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br J Nutr . 2003; 89( 1): 3– 9. Google Scholar CrossRef Search ADS PubMed  3. Hresko RC, Kraft TE, Quigley A, Carpenter EP, Hruz PW. Mammalian glucose transporter activity is dependent upon anionic and conical phospholipids. J Biol Chem . 2016; 291( 33): 17271– 17282. Google Scholar CrossRef Search ADS PubMed  4. Carnagarin R, Dharmarajan AM, Dass CR. PEDF attenuates insulin-dependent molecular pathways of glucose homeostasis in skeletal myocytes. Mol Cell Endocrinol . 2016; 422: 115– 124. Google Scholar CrossRef Search ADS PubMed  5. Nishimoto H, Matsutani R, Yamamoto S, Takahashi T, Hayashi KG, Miyamoto A, Hamano S, Tetsuka M. Gene expression of glucose transporter (GLUT) 1, 3 and 4 in bovine follicle and corpus luteum. J Endocrinol . 2006; 188( 1): 111– 119. Google Scholar CrossRef Search ADS PubMed  6. Berry GT, Baynes JW, Wells-Knecht KJ, Szwergold BS, Santer R. Elements of diabetic nephropathy in a patient with GLUT 2 deficiency. Mol Genet Metab . 2005; 86( 4): 473– 477. Google Scholar CrossRef Search ADS PubMed  7. MacLean PS, Zheng D, Jones JP, Olson AL, Dohm GL. Exercise-induced transcription of the muscle glucose transporter (GLUT 4) gene. Biochem Biophys Res Commun . 2002; 292( 2): 409– 414. Google Scholar CrossRef Search ADS PubMed  8. Shimada Y, Sawada S, Hojo S, Okumura T, Nagata T, Nomoto K, Tsukada K. Glucose transporter 3 and 1 may facilitate high uptake of 18F-FDG in gastric schwannoma. Clin Nucl Med . 2013; 38( 11): e417– e420. Google Scholar CrossRef Search ADS PubMed  9. Kol S, Ben-Shlomo I, Ruutiainen K, Ando M, Davies-Hill TM, Rohan RM, Simpson IA, Adashi EY. The midcycle increase in ovarian glucose uptake is associated with enhanced expression of glucose transporter 3. Possible role for interleukin-1, a putative intermediary in the ovulatory process. J Clin Invest . 1997; 99( 9): 2274– 2283. Google Scholar CrossRef Search ADS PubMed  10. Kodaman PH, Behrman HR. Hormone-regulated and glucose-sensitive transport of dehydroascorbic acid in immature rat granulosa cells. Endocrinology . 1999; 140( 8): 3659– 3665. Google Scholar CrossRef Search ADS PubMed  11. Zhang C, Niu W, Wang Z, Wang X, Xia G. The effect of gonadotropin on glucose transport and apoptosis in rat ovary. PLoS One . 2012; 7( 8): e42406. Google Scholar CrossRef Search ADS PubMed  12. Tian Y, Ding Y, Liu J, Heng D, Xu K, Liu W, Zhang C. Nitric oxide–mediated regulation of GLUT by T3 and follicle-stimulating hormone in rat granulosa cells. Endocrinology . 2017; 158( 6): 1898– 1915. Google Scholar CrossRef Search ADS PubMed  13. Koesling D, Mergia E, Russwurm M. Physiological functions of NO-sensitive guanylyl cyclase isoforms. Curr Med Chem . 2016; 23( 24): 2653– 2665. Google Scholar CrossRef Search ADS PubMed  14. Dixit VD, Parvizi N. Nitric oxide and the control of reproduction. Anim Reprod Sci . 2001; 65( 1-2): 1– 16. Google Scholar CrossRef Search ADS PubMed  15. Basini G, Grasselli F. Nitric oxide in follicle development and oocyte competence. Reproduction . 2015; 150( 1): R1– R9. Google Scholar CrossRef Search ADS PubMed  16. Zamberlam G, Sahmi F, Price CA. Nitric oxide synthase activity is critical for the preovulatory epidermal growth factor-like cascade induced by luteinizing hormone in bovine granulosa cells. Free Radic Biol Med . 2014; 74: 237– 244. Google Scholar CrossRef Search ADS PubMed  17. Masuda M, Kubota T, Aso T. Effects of nitric oxide on steroidogenesis in porcine granulosa cells during different stages of follicular development. Eur J Endocrinol . 2001; 144( 3): 303– 308. Google Scholar CrossRef Search ADS PubMed  18. Dineva JD, Vangelov IM, Nikolov GG, Konakchieva RTs, Ivanova MD. Nitric oxide stimulates the production of atrial natriuretic peptide and progesterone by human granulosa luteinized cells with an antiapoptotic effect. Endocr Regul . 2008; 42( 2-3): 45– 51. Google Scholar PubMed  19. Jee BC, Kim SH, Moon SY. The role of nitric oxide on apoptosis in human luteinized granulosa cells. Immunocytochemical evidence. Gynecol Obstet Invest . 2003; 56( 3): 143– 147. Google Scholar CrossRef Search ADS PubMed  20. Matsumi H, Yano T, Osuga Y, Kugu K, Tang X, Xu JP, Yano N, Kurashima Y, Ogura T, Tsutsumi O, Koji T, Esumi H, Taketani Y. Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development. Biol Reprod . 2000; 63( 1): 141– 146. Google Scholar CrossRef Search ADS PubMed  21. Jablonka-Shariff A, Olson LM. Hormonal regulation of nitric oxide synthases and their cell-specific expression during follicular development in the rat ovary. Endocrinology . 1997; 138( 1): 460– 468. Google Scholar CrossRef Search ADS PubMed  22. Zackrisson U, Mikuni M, Wallin A, Delbro D, Hedin L, Brännström M. Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation. Hum Reprod . 1996; 11( 12): 2667– 2673. Google Scholar CrossRef Search ADS PubMed  23. El-Sehemy A, Postovit LM, Fu Y. Nitric oxide signaling in human ovarian cancer: a potential therapeutic target. Nitric Oxide . 2016; 54: 30– 37. Google Scholar CrossRef Search ADS PubMed  24. Colbran JL, Roach PJ, Fiol CJ, Dixon JE, Andrisani OM, Corbin JD. cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates delta-CREB, and a synthetic delta-CREB peptide. Biochem Cell Biol . 1992; 70( 10-11): 1277– 1282. Google Scholar CrossRef Search ADS PubMed  25. Wong JC, Bathina M, Fiscus RR. Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells. J Cell Biochem . 2012; 113( 11): 3587– 3598. Google Scholar CrossRef Search ADS PubMed  26. Gudi T, Casteel DE, Vinson C, Boss GR, Pilz RB. NO activation of fos promoter elements requires nuclear translocation of G-kinase I and CREB phosphorylation but is independent of MAP kinase activation. Oncogene . 2000; 19( 54): 6324– 6333. Google Scholar CrossRef Search ADS PubMed  27. Chen Y, Zhuang S, Cassenaer S, Casteel DE, Gudi T, Boss GR, Pilz RB. Synergism between calcium and cyclic GMP in cyclic AMP response element-dependent transcriptional regulation requires cooperation between CREB and C/EBP-beta. Mol Cell Biol . 2003; 23( 12): 4066– 4082. Google Scholar CrossRef Search ADS PubMed  28. Kim MO, Lee YJ, Park JH, Ryu JM, Yun SP, Han HJ. PKA and cAMP stimulate proliferation of mouse embryonic stem cells by elevating GLUT1 expression mediated by the NF-κB and CREB/CBP signaling pathways. Biochim Biophys Acta . 2012; 1820( 10): 1636– 1646. Google Scholar CrossRef Search ADS PubMed  29. Rao IM, Mills TM, Anderson E, Mahesh VB. Heterogeneity in granulosa cells of developing rat follicles. Anat Rec . 1991; 229( 2): 177– 185. Google Scholar CrossRef Search ADS PubMed  30. Wang Q, Leader A, Tsang BK. Follicular stage-dependent regulation of apoptosis and steroidogenesis by prohibitin in rat granulosa cells. J Ovarian Res . 2013; 6( 1): 23. Google Scholar CrossRef Search ADS PubMed  31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method. Methods . 2001; 25( 4): 402– 408. Google Scholar CrossRef Search ADS PubMed  32. Liu M, Li ZH, Xu FJ, Lai LH, Wang QQ, Tang GP, Yang WT. An oligopeptide ligand-mediated therapeutic gene nanocomplex for liver cancer-targeted therapy. Biomaterials . 2012; 33( 7): 2240– 2250. Google Scholar CrossRef Search ADS PubMed  33. Li RJ, Ying X, Zhang Y, Ju RJ, Wang XX, Yao HJ, Men Y, Tian W, Yu Y, Zhang L, Huang RJ, Lu WL. All-trans retinoic acid stealth liposomes prevent the relapse of breast cancer arising from the cancer stem cells. J Control Release . 2011; 149( 3): 281– 291. Google Scholar CrossRef Search ADS PubMed  34. Oelze M, Mollnau H, Hoffmann N, Warnholtz A, Bodenschatz M, Smolenski A, Walter U, Skatchkov M, Meinertz T, Münzel T. Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction. Circ Res . 2000; 87( 11): 999– 1005. Google Scholar CrossRef Search ADS PubMed  35. Shi T, Papay RS, Perez DM. α1A-Adrenergic receptor prevents cardiac ischemic damage through PKCδ/GLUT1/4-mediated glucose uptake. J Recept Signal Transduct Res . 2016; 36( 3): 261– 270. Google Scholar CrossRef Search ADS PubMed  36. Deng D, Xu C, Sun P, Wu J, Yan C, Hu M, Yan N. Crystal structure of the human glucose transporter GLUT1. Nature . 2014; 510( 7503): 121– 125. Google Scholar CrossRef Search ADS PubMed  37. Morato PN, Lollo PC, Moura CS, Batista TM, Camargo RL, Carneiro EM, Amaya-Farfan J. Whey protein hydrolysate increases translocation of GLUT-4 to the plasma membrane independent of insulin in wistar rats. PLoS One . 2013; 8( 8): e71134. Google Scholar CrossRef Search ADS PubMed  38. Fedail JS, Zheng K, Wei Q, Kong L, Shi F. Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats. Endocrine . 2014; 46( 3): 594– 604. Google Scholar CrossRef Search ADS PubMed  39. Chen Q, Yano T, Matsumi H, Osuga Y, Yano N, Xu J, Wada O, Koga K, Fujiwara T, Kugu K, Taketani Y. Cross-talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia. Endocrinology . 2005; 146( 2): 808– 815. Google Scholar CrossRef Search ADS PubMed  40. Yoon SJ, Choi KH, Lee KA. Nitric oxide-mediated inhibition of follicular apoptosis is associated with HSP70 induction and Bax suppression. Mol Reprod Dev . 2002; 61( 4): 504– 510. Google Scholar CrossRef Search ADS PubMed  41. Matsumi H, Koji T, Yano T, Yano N, Tsutsumi O, Momoeda M, Osuga Y, Taketani Y. Evidence for an inverse relationship between apoptosis and inducible nitric oxide synthase expression in rat granulosa cells: a possible role of nitric oxide in ovarian follicle atresia. Endocr J . 1998; 45( 6): 745– 751. Google Scholar CrossRef Search ADS PubMed  42. Dubey PK, Tripathi V, Singh RP, Saikumar G, Nath A, Pratheesh, Gade N, Sharma GT. Expression of nitric oxide synthase isoforms in different stages of buffalo (Bubalus bubalis) ovarian follicles: effect of nitric oxide on in vitro development of preantral follicle. Theriogenology . 2012; 77( 2): 280– 291. Google Scholar CrossRef Search ADS PubMed  43. Dubey PK, Tripathi V, Singh RP, Sharma GT. Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles. J Vet Sci . 2011; 12( 3): 257– 265. Google Scholar CrossRef Search ADS PubMed  44. Ren Y, Zheng J, Yao X, Weng G, Wu L. Essential role of the cGMP/PKG signaling pathway in regulating the proliferation and survival of human renal carcinoma cells. Int J Mol Med . 2014; 34( 5): 1430– 1438. Google Scholar CrossRef Search ADS PubMed  45. Wong JC, Fiscus RR. Essential roles of the nitric oxide (NO)/cGMP/protein kinase G type-Iα (PKG-Iα) signaling pathway and the atrial natriuretic peptide (ANP)/cGMP/PKG-Iα autocrine loop in promoting proliferation and cell survival of OP9 bone marrow stromal cells. J Cell Biochem . 2011; 112( 3): 829– 839. Google Scholar CrossRef Search ADS PubMed  46. Yoneyama M, Kawada K, Shiba T, Ogita K. Endogenous nitric oxide generation linked to ryanodine receptors activates cyclic GMP/protein kinase G pathway for cell proliferation of neural stem/progenitor cells derived from embryonic hippocampus. J Pharmacol Sci . 2011; 115( 2): 182– 195. Google Scholar CrossRef Search ADS PubMed  47. Nagai-Kusuhara A, Nakamura M, Mukuno H, Kanamori A, Negi A, Seigel GM. cAMP-responsive element binding protein mediates a cGMP/protein kinase G-dependent anti-apoptotic signal induced by nitric oxide in retinal neuro-glial progenitor cells. Exp Eye Res . 2007; 84( 1): 152– 162. Google Scholar CrossRef Search ADS PubMed  48. Fiscus RR. Involvement of cyclic GMP and protein kinase G in the regulation of apoptosis and survival in neural cells. Neurosignals . 2002; 11( 4): 175– 190. Google Scholar CrossRef Search ADS PubMed  49. Leung EL, Wong JC, Johlfs MG, Tsang BK, Fiscus RR. Protein kinase G type Iα activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation. Mol Cancer Res . 2010; 8( 4): 578– 591. Google Scholar CrossRef Search ADS PubMed  Copyright © 2018 Endocrine Society

Journal

EndocrinologyOxford University Press

Published: Feb 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 12 million articles from more than
10,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Unlimited reading

Read as many articles as you need. Full articles with original layout, charts and figures. Read online, from anywhere.

Stay up to date

Keep up with your field with Personalized Recommendations and Follow Journals to get automatic updates.

Organize your research

It’s easy to organize your research with our built-in tools.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

Monthly Plan

  • Read unlimited articles
  • Personalized recommendations
  • No expiration
  • Print 20 pages per month
  • 20% off on PDF purchases
  • Organize your research
  • Get updates on your journals and topic searches

$49/month

Start Free Trial

14-day Free Trial

Best Deal — 39% off

Annual Plan

  • All the features of the Professional Plan, but for 39% off!
  • Billed annually
  • No expiration
  • For the normal price of 10 articles elsewhere, you get one full year of unlimited access to articles.

$588

$360/year

billed annually
Start Free Trial

14-day Free Trial