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Endocrinology Vol. 142, No. 8 3590-3597
Copyright © 2001 by The Endocrine Society

ARTICLES

Saturated FFAs, Palmitic Acid and Stearic Acid, Induce Apoptosis in Human Granulosa Cells

Yi-Ming Mu, Toshihiko Yanase, Yoshihiro Nishi, Atsushi Tanaka, Masayuki Saito, Cheng-Hao Jin, Chizu Mukasa, Taijiro Okabe, Masatoshi Nomura, Kiminobu Goto and Hajime Nawata

Third Department of Internal Medicine (Y.-M.M., T.Y., M.S., C.M., T.O., M.N., K.G., H.N.), Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; Department of Endocrinology (Y.-M.M.), Chinese PLA General Hospital, Beijing 100853, People’s Republic of China; Division of Internal Medicine (Y.N.), National Kokura Hospital, Kokura-Minami-Ku, Kitakyushu, 802-0803, Japan; Department of Obstetrics and Gynecology (A.T.), Saint Mother Hospital, Kitakyushu City, Fukuoka, 807-0825, Japan; Department of Pediatrics (C.-H.J.), Faculty of Medicine, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan; and Core Research for Evolutional Science and Technology (T.Y., K.G., H.N.), Kawaguchi 332-0012, Japan

Address all correspondence and requests for reprints to: Toshihiko Yanase, M.D., Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: yanase{at}mail.server.med.kyushu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Obesity is associated with insulin resistance and some reproductive abnormalities. Circulating FFAs are often elevated in obese subjects and are also closely linked to insulin resistance. In this study, we demonstrated that saturated FFAs, such as palmitic acid and stearic acid, markedly suppressed the granulosa cell survival in a time- and dose-dependent manner. Polyunsaturated FFA, arachidonic acid, had no effect on the cell survival, even at supraphysiological concentrations. The suppressive effect of saturated FFAs on cell survival was caused by apoptosis, as evidenced by DNA ladder formation and annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effects of palmitic acid and stearic acid were unrelated to the increase of ceramide generation or nitric oxide production and were also completely blocked by Triacsin C, an inhibitor of acylcoenzyme A synthetase. In addition, acylcoenzyme A, pamitoylcoenzyme A, and stearylcoenzyme A markedly suppressed granulosa cell survival, whereas arachidonoylcoenzyme A had no such effect, and this finding was consistent with the effect of the respective FFA form. Surprisingly, arachidonic acid instead showed a protective effect on palmitic acid- and stearic acid-induced cell apoptosis. A Western blot analysis showed the apoptosis of the granulosa cells induced by palmitic acid to be accompanied by the down-regulation of an apoptosis inhibitor, Bcl-2, and the up-regulation of an apoptosis effector, Bax. These results indicate that saturated FFAs induce apoptosis in human granulosa cells caused by the metabolism of the respective acylcoenzyme A form, and the actual composition of circulating FFAs may thus play a critical role in the apoptotic events of human granulosa cells. These effects of FFAs on granulosa cell survival may be a possible mechanism for reproductive abnormalities, such as amenorrhea, which is frequently observed in obese women.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LONG CHAIN FFAs play an important role in the cellular biological functions. They serve as a source of metabolic energy, as the substrates for cell membrane biogenesis (glyco- and phospholipid), and as precursors of many intracellular signaling molecules such as PGs, leukotrienes, thromboxanes, platelet-activating factor, and others (1, 2, 3). Under normal conditions, the plasma total FFA concentrations range from 200–600 µM in women. This wide variation is induced by an individual’s diet and changes in the serum insulin level (4, 5, 6). In pathological states such as diabetes, obesity, and cancer, the plasma total FFA concentrations increase to levels ranging from 400-2000 µM (7, 8, 9). The elevation of the plasma FFA is one important link between obesity and insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM), and also increases the risk of cardiovascular diseases (10, 11, 12). The FFA pattern was reported to differ between the normal control and obese patients, in which obese patients had a low unsaturated-to-saturated ratio (13). More recent studies indicate that an increase in FFAs inhibits T lymphocyte signaling and induces pancreatic ß-cell apoptosis (14, 15).

Obese women are frequently associated with reproductive abnormalities, including amenorrhea (16, 17). This abnormality in obese women is generally thought to be attributable to the dysregulation of the hypothalamic-pituitary axis, regarding the ovarian function (16, 18). Hyperandrogenism that is secondary to hyperinsulinemia may also be another cause for such reproductive abnormalities in obese women. A few molecules such as FFA, leptin, and TNF{alpha} have been reported to be elevated in obese subjects and have also been intensively discussed as candidates that may contribute to insulin resistance (19, 20, 21, 22, 23). Especially, a close relationship between the elevated plasma FFA level and insulin resistance is commonly reported in obese subjects (24, 25, 26). In the present study, to test the hypothesis that these factors may also cause ovarian dysfunction, we examined the effect of these molecules on the cell proliferation of human ovarian granulosa cells obtained from in vitro fertilization. We found that saturated FFAs affect the cell survival of human granulosa cells by inducing apoptosis, whereas a polyunsaturated FFA, arachidonic acid (AA), instead antagonizes saturated FFA-induced apoptosis. Because the mechanism of FFA-induced apoptosis has not been well elucidated, we also investigated this mechanism in our system and discussed the clinical implications of this phenomenon.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
FFAs [including palmitic acid (PA) and stearic acid (SA)], oleic acid (OA), linoleic acid (LA) and AA, palmitoylcoenzyme A (palmitoyl-CoA) stearoyl-CoA and arachidonoyl-CoA, fumonisin B1, C2-ceramide, bovine insulin, and S-nitroso-N-acetylpenicillamine (SNAP) were purchased from Sigma (St. Louis, MO). PA and SA were dissolved in 0.1 N NaOH, and the final concentration of NaOH in the cell growth medium was 0.1 mN. Triacsin C was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). DME/F12, FCS, and proteinase K were purchased from Life Technologies, Inc. (Grand Island, NY). All antibodies, including antihuman Bcl-2, Bax, and ß-actin used for the Western blot analysis, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Annexin V-EGFP/PI Apoptosis Detection Kit was obtained from the MBL Co., Ltd. (Nagoya, Japan). TNF{alpha} and leptin were purchased from PeproTech LTD (London, UK).

Cells and cell survival assay
Human ovarian granulosa cells were obtained from patients who underwent in vitro fertilization. Written informed consent was obtained from each subject before starting the study. The cells were maintained in DME/F12 supplemented with 10% FCS in an atmosphere of 5% CO2 at 37 C. The purity of the human granulosa cells was estimated to be almost 90% of all cells, based on the expression of cytochrome P450arom examined by immunohistochemical staining of the cultured cells with the antibody against human cytochrome P450 (27).

For a cell survival assay, the cells were plated onto a 24-well plate, at 1 x 104 cells/well, in DME/F12 supplemented with 10% FCS, 1 d before treatment. The cells (30–40% confluence) were treated with various FFAs or other reagents, whereas control cells were incubated in medium with carrier (0.1 mN NaOH), and the medium with or without FFAs was changed every 2 d. After washing, the cells were trypsinized and then counted using a hemocytometer. Cell viability was assessed by the trypan blue dye exclusion method.

DNA fragmentation assay
DNA fragmentation was examined as previously described (28). Briefly, the cells treated with or without FFAs were trypsinized and washed with PBS and then were spinned down and resuspended in 100 µl lysing buffer (50 mM Tris-HCl, pH 7.8, 10 mM EDTA-2Na, 0.5% SDS). Heat-treated ribonuclease A (QIAGEN, GmbH, Germany) was then added to a concentration of 0.5 mg/ml and incubated at 37 C for 60 min. Next, the protein was degraded using 0.5 mg/ml proteinase K at 37 C for 60 min. DNA was extracted using two extractions, first with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and then with chloroform-isoamyl alcohol (24:1). DNA was then precipitated from the upper aqueous phase, using 0.1 vol of 3 M sodium acetate with 2.5 vol of ice-cold ethanol, and left at -20 C for 60 min before centrifugation. DNA pellets were resuspended in 50 µl sterile water. DNA fragments (10–12 µg) were separated by electrophoresis in 1.2% agarose gel and then visualized by staining with ethidium bromide.

Detection of apoptosis by fluorescence microscopy
Early and late apoptotic changes in human granulosa cells were also determined using an Annexin V-EGFP/PI Apoptosis Detection Kit. One day before treatment, the cells were divided into 35-mm glass-bottom dishes (MatTek Corporation, Ashland, MA) and then were treated with 300 µM PA or SA for 24 h and 48 h, respectively. After the treatment, the cells were washed once with PBS and then were incubated with 200 µl 1x binding buffer containing 1 µl annexin V-EGFP and 1 µl propidium iodide (PI) at room temperature, for 5 min, in the dark. The cells were then scanned using confocal laser scanning microscopy (TCS-SP system, Leica Corp. Microsystems, Heidelberg, Germany) using a dual-filter set for fluorescein isothiocyanate and TRITC. The cell membrane was imaged for green fluorescence (stained by annexin V-EGFP) by excitation with the 488-nm line from an argon laser, and the emission was viewed through a 460- to 505-nm band pass filter. The cell nucleus was imaged for red fluorescence (stained by PI) by excitation with the 560-nm line from an argon laser, and then the emission was viewed through a 520- to 580-nm band pass filter.

Western blot analysis
The expression of apoptosis-related proteins, Bax and Bcl-2, and ß-actin were examined by Western blot analysis, as described previously (28), using specific antibodies. Human granulosa cells were cultured with 100 µM or 300 µM PA for 48 h. The cells were then washed with PBS and lysed with SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.2 mM/liter phenylmethylsulfonlyfluoride supplemented with 0.2 trypsin inhibitory units/ml aprotinin, 0.7 µg/ml pepstatin, and 1 µg/ml leupeptin]. The samples were sonicated for a few seconds to shear the DNA and reduce the viscosity. Twenty micrograms of protein were mixed with an equal volume of 2x electrophoresis buffer, then boiled for 3 min. The samples were electrophoresed on a 12% SDS-polyacrylamide gel, and proteins were transferred to a Hybond-P, polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). Blots were rinsed in Tris-buffered saline-0.1% Tween-20 and blocked with 4% BSA overnight at 4 C. The blots were incubated with rabbit antihuman bax (1:2000), human bcl-2 (1:2000), and goat antihuman ß-actin (1:200) antibodies, respectively, for 1 h at room temperature. After washing with Tris-buffered saline-0.1% Tween-20 for three times, blots were probed with horseradish peroxidase-labeled antirabbit IgG (1:2000) or antigoat IgG (1:500), respectively, in blocking buffer. Proteins were detected using a chemiluminescence luminol reagent (Santa Cruz Biotechnology, Inc.), and the bands were visualized by autoradiography.

Statistical analysis
For the cell survival assay, experiments were performed in triplicate, and the results were expressed as the mean ± SD from three or four independent experiments. A statistical analysis was performed using the Statview software. All data were evaluated for unpaired variables to compare two or more groups. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Saturated FFAs induce a dose-dependent reduction in cell survival
To determine whether FFAs induce a reduction in granulosa cell survival, we investigated the cell viability, by trypan blue exclusion, using a hemocytometer. The cells were treated with or without various concentrations of FFAs for 3 d, and then the cell viability was determined. As shown in Fig. 1Go, a dose-dependent reduction in the cell survival was observed at doses of PA (C16:0) and SA (C18:0) ranging from 50–300 µM. The cell survival was less than 20% after exposure to 300 µM PA or SA for 3 d. PA and SA seemed to be similar in the potency to reduce the cell survival. The unsaturated FFAs present different effects on cell survival. Namely, OA (C18:1) and LA (C18:2) induced a 39% and 31% reduction, respectively, in survival at a 300-µM concentration, which is about 2–3 times the normal plasma level (13). AA (C20:4) had no effect on the granulosa cell survival at concentrations ranging from 1–10 µM.



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  		Figure 1. The effects of FFAs on granulosa cell viability. Human granulosa cells were treated with various concentrations of saturated FFAs (including PA and SA) or unsaturated FFAs (including OA, LA, and AA). After treatment for 3 d, the cell viability was determined as described in Materials and Methods. The data represent the mean ± SD of four independent experiments with triplicate wells and are presented as the percentage of the control cell number. *, P < 0.05 vs. control cells; **, P < 0.01 vs. control cells.

 
Neither physiological nor supraphysiological concentration of TNF{alpha} (1–20 ng/ml), leptin (10–200 ng/ml), and insulin (100–2000 ng/ml) had an effect on cell survival (data not shown). These results are consistent with previous studies (29, 30).

The effect of saturated FFAs was also time-dependent, because the cell survival declined dramatically as the time of treatment increased from 1 d to 3 d (Fig. 2Go). After 3 d of treatment, the cell viabilities were 9% and 12% in the cells exposed to 300 µM PA and SA, respectively, whereas the cell viability was more than 60% in cells treated with 300 µM of unsaturated FFAs like OA and LA. Another polyunsaturated FFA, AA, had no effect on the cell survival within the 6-d treatment period.



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  		Figure 2. The effects of various FFAs on the cell survival. Human granulosa cells were incubated in media containing various FFAs indicated, whereas the control cells were incubated in the medium with the carrier (0.1 mM NaOH). The cells were harvested at 24-h intervals and counted, and viability was assessed by trypan blue exclusion. The data represent the mean ± SD of three separate experiments with triplicate wells and are presented as the percentage of the control cell number.

 
Evidence that apoptosis mediates the saturated FFAs-induced reduction in granulosa cell survival
To determine whether apoptosis was the cause of the loss of cell survival after saturated FFAs treatment, we measured the effects of PA and SA on DNA fragmentation using DNA electrophoresis and fluorescent staining of the cells. After treating the cells with 300 µM PA or SA, respectively, for 72 h, the genomic DNA extracted from cells was subjected to 1.2% agarose gel electrophoresis. In each case, DNA ladders, which are typical of apoptosis, were visible on agarose gel after staining with ethidium bromide in granulosa cells (Fig. 3Go). In contrast, in the control cells treated with carrier (0.1 mN NaOH), no DNA fragmentation was observed. Saturated FFA-induced apoptosis was further confirmed using Annexin V-EGFP/PI staining. In healthy cells, the phospholipids of the plasma membrane are distributed asymmetrically over the two leaflets of the bilayer. Phosphatidylserine (PS) is located exclusively in the inner membrane leaflet because of an inhibition of aminophospholipid translocase and a subsequent activation of scramblase (31, 32). Because annexin V binds with a high affinity to negatively charged PS, it has been used in combination with PI to detect early and late apoptotic or necrosis cells (33). In unexposed control cells, most cells stained for neither annexin V-EGFP (green) nor PI (red). After being treated with 300 µM PA or SA, early apoptotic cells with a cell membrane stained by green color (annexin V positive/PI negative) were detected at 24 h; and late apoptotic cells, with a cell membrane demonstrating green staining and red staining for nuclear staining (annexin V positive/PI positive) and chromatin fragmentation were seen (Fig. 4Go).



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  		Figure 3. The induction of DNA fragmentation by saturated FFAs in granulosa cells. The cells were cultured for 3 d with saturated FFAs (300 µM each) or carrier (0.1 mM NaOH) alone as control. DNA was extracted as described in Materials and Methods and separated by electrophoresis in 1.2% agarous gel. Band formation from a DNA molecular weight marker is shown in lane M, and the sizes of the migration fragments are indicated. Both PA (lane 2) and SA (lane 3) induced DNA fragmentation in granulosa cells, whereas the carrier did not induce DNA fragmentation (lane 1).

 


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  		Figure 4. Morphological evidence for apoptosis induced by saturated FFAs. After incubating the granulosa cells with 300 µM PA or SA for 24 h and 48 h, as indicated, the cells were stained by annexin V-EGFP and PI, as described in Materials and Methods. The cells were then scanned using a confocal laser scanning microscopy. The early apoptotic cells, with the cell membrane stained by annexin V-EGFP (green), and the late apoptotic cells, with both the cell membrane stained by annexin V-EGFP and nucleus stained by PI (red), were detected (0 h, 24 h, and 48 h indicate before treatment, 24 h after treatment, and 48 h after treatment, respectively.

 
FFA-induced apoptosis is not mediated by an increase of ceramide generation or nitric oxide (NO) production
Because FFA has been demonstrated to induce pancreatic ß-cell apoptosis by increasing both ceramide generation and NO production (15), we investigated whether DNA fragmentation in granulosa cells was caused by exogenous ceramide and NO donor SNAP. As shown in Fig. 5Go, 100 µM C2-ceramide induced DNA fragmentation in CEM leukemic cells (lane 3) but did not induce apoptosis in the granulosa cells (lane 4); and 50 µM fumonisin B1, the ceramide synthase inhibitor, did not prevent the PA (300 µM)-induced apoptosis (lane 5). These results indicate that PA-induced apoptosis in granulosa cells is not by an increase of ceramide generation. We further determined that the increase of NO production was not the cause of PA- or SA-induced apoptosis in granulosa cells, because: 1) a high concentration (1 mM) of NO donor SNAP did not induce DNA fragmentation (lane 6); 2) aminoguanidine (AG), an inducible NO synthase (iNOS) inhibitor (34), which has been reported to profoundly reduce NO production in vitro and effectively prevent ß-cell loss (15), did not block the apoptotic effect of PA in granulosa cells (lane 7); and 3) even PA and SA increased NO production and iNOS protein level determined by Western blot analysis in granulosa cells (data not shown), but the NO levels in culture medium were much lower than those in the cells treated with 1 mM SNAP (about 1:200) (data not shown). The doses of C2-ceramide, fumonisin B1, and AG used in this experiment were optimal, above which levels these reagents themselves were toxic to the cells. From the above results, we concluded that saturated FFA-induced apoptosis in granulosa cells is not mediated by either the increase of ceramide generation or NO production.



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  		Figure 5. The effects of ceramide and NO donor on DNA fragmentation induced by PA. The cells were incubated with the reagents described below, for 3 d, and the DNA from the cells was extracted and electrophoresed in 1.2% agarose gel. Lane M, DNA molecular weight marker; lane 1, control; lane 2, 300 µM PA; lane 3, treatment of CEM leukemic cells with 100 µM C2-ceramide; lane 4, treatment of granulosa cells with 100 µM C2-ceramide; lane 5, 300 µM PA plus 100 µM fumonisin B1; lane 6, 1 mM SNAP; lane 7, 300 µM PA plus 1 mM AG.

 
Effects of fatty acyl-CoA blockade on DNA fragmentation
Next, to examine whether FFA-induced apoptosis is caused by FFA itself or by its metabolites, we investigated the effects of fatty acyl-CoA blockade on granulosa cell survival. Exogenous FFAs traverse the cellular membrane by means of nonmediated passive diffusion (35, 36). Upon entry into the cell, FFAs are esterified into their active form, acyl-CoA, and then are further metabolized. Triacsin C has been shown to be an effective inhibitor of acyl-CoA synthetase (37, 38, 39). As shown in Fig. 6Go, though Triacsin C itself had no effect on cell survival, 5 µM of this compound completely prevented the apoptotic effects of various concentrations (ranging from 100–500 µM) of PA and SA. To further determine whether acyl-CoA can directly induce granulosa cell death, we treated the cells with various concentrations of palmitoyl-CoA, stearoyl-CoA, and arachidonoyl-CoA for 3 d As shown in Fig. 6Go, both palmitoyl-CoA and stearoyl-CoA induced a reduction in cell survival in a dose-dependent manner; 200 µM of either palmitoyl-CoA or stearoyl-CoA nearly killed all of the cells. In line with the observed effect of AA, arachidonoyl-CoA itself had no effect on granulosa cell survival (Fig. 7Go). On the other hand, Triacsin C had no effect on palmitoyl-CoA- and stearoyl-CoA-induced apoptosis (data not shown).



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  		Figure 6. The inhibitory effect of Triacsin C on saturated FFA-induced apoptosis in granulosa cells. The cells were treated with various concentrations of PA or SA alone or with PA or SA plus 5 µM Triacsin C for 3 d. The cell viability was determined as described in Materials and Methods. The data represent the mean ± SD of three independent experiments with triplicate wells and are presented as the percentage of control cell number. *, P < 0.05 vs. control cells; **, P < 0.01 vs. control cells.

 


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  		Figure 7. The effects of palmitoyl-CoA, stearoyl-CoA, and arachidonoyl-CoA on granulosa cell viability. Cells were incubated in media with various concentrations of acyl-CoAs, as indicated, for 3 d. The cell viability was determined as described in Materials and Methods. The data represent the mean ± SD of four independent experiments with triplicate wells and are presented as the percentage of the control cell number. **, P < 0.01 vs. control cells.

 
AA suppresses PA- and SA-induced cell apoptosis
AA has been determined to promote tumor growth by enhancing DNA synthesis and cell proliferation (40). To test the hypothesis that AA may also function as a survival factor for granulosa cells, we cultured the cells with various concentrations of PA or SA alone, and PA or SA plus 15 µM AA for 3 d. As shown in Fig. 8AGo, AA provided a significantly protective effect on PA- and SA-induced cell death. The AA effect was dose-dependent and was first observed at 3 µM, and later plateaued at approx. 10–15 µM and could be observed up to 20 µM, above which level AA itself was toxic to granulosa cells (Fig. 8BGo). These results indicate that AA may be important in the prevention of apoptosis induced by saturated FFAs in granulosa cells.



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  		Figure 8. The protective effect of AA on saturated FFA-induced apoptosis in granulosa cells. A, The cells were incubated in media containing 100–300 µM PA or SA, with or without 15 µM AA, for 3 d. B, The cells were incubated in media containing 300 µM PA alone or 300 µM PA plus various concentrations of AA, as indicated, for 3 d. The cell viability was determined as described in Materials and Methods. The data represent the mean ± SD of three independent experiments with triplicate wells and are presented as the percentage of the control cell number. a, P < 0.05 vs. control cells; b, P < 0.01 vs. control cells; c, P < 0.05 vs. cells treated with saturated FFA alone; *, P < 0.05 vs. cells treated with 300 µM PA; **, P < 0.01 vs. cells treated with 300 µM PA.

 
PA increases Bax and decreases Bcl-2 expression in granulosa cells
An apoptosis suppressor, Bcl-2, has been reported to be suppressed in FFA-induced pancreatic ß-cell apoptosis (41). To determine whether saturated FFA-induced apoptosis in granulosa cells is related to the suppression of an apoptosis suppressor, Bcl-2, or the induction of an apoptosis effector, Bax, we measured the expression levels of Bcl-2 and Bax in granulosa cells, either treated or not treated with PA, by a Western blot analysis. As shown in Fig. 9Go, PA treatment resulted in a dramatic down-regulation of Bcl-2 and an up-regulation of Bax.



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  		Figure 9. Expression of Bcl-2 and Bax during apoptosis induced by PA in the granulosa cells. A, The cells were treated with 100 or 300 µM PA for 2 d. The cell lysates were then prepared, and a Western blot analysis was performed, with the use of specific antibodies to Bcl-2, Bax, and ß-actin. B, The relative expression levels of Bcl-2 and Bax were determined by measuring the intensity using ImageQuant. The data represent the mean ± SD of three independent experiments and are presented as the fold of ß-actin. *, P < 0.05 vs. cells treated with 300 µM PA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Obesity is the most common cause of insulin resistance and is frequently associated with reproductive abnormalities, especially in women. Obese women show ovarian dysfunction, leading to various degrees of menstrual abnormalities, including amenorrhea. At least three candidate molecules, which are increased in the circulation of obese subjects, have been implicated in the development of insulin resistance (19, 20, 21, 22, 23, 24). One is FFA, which inhibits the glucose uptake and is used in muscle (19, 24). Second, TNF{alpha} has been reported to be at least partially responsible for the insulin resistance caused by obesity (22, 23). Third, leptin may cause insulin resistance, especially in the liver and adipocytes (20, 21); however, this issue remains highly controversial. We tested the possibility that these candidate molecules for insulin resistance may also be a possible cause for the ovarian dysfunction by affecting ovarian granulosa cell survival. Leptin and TNF{alpha} caused no effect on the cell survival of human granulosa cells, which also correlated with the results observed by others (41, 42). In the present study, we demonstrated that mainly saturated FFAs, such as PA (C16:0) and SA (C18:0), induce cell death via apoptosis in human ovarian granulosa cells, whereas unsaturated FFAs do not essentially have such an effect. The same phenomenon was also observed in a human granulosa cell line, KGN, which was most recently established by our group (27) (data not shown). These results indicate that the human granulosa cell is truly a target for apoptosis by FFAs.

The nonfasting serum FFA levels in patients with hyperlipidemia are reported to be 140.9 ± 77.7 µM, 109.79 ± 70.0 µM, 89.5 ± 74.5 µM, 45.9 ± 44.0 µM, and 0.8 ± 1.3 µM for PA, SA, OA, LA, and AA, respectively (13). As a result, the dose-dependent reduction in cellular viability observed at doses of PA and SA ranging from 100–300 µM is considered to mimic the hyperlipidemic condition. The unsaturated FFAs had only a slight effect on cell survival. Namely, OA (C18:1) and LA (C18:2) caused a 39% and 31% reduction, respectively, in survival at a 300-µM concentration, which is a level 2–3 times the normal plasma level (13). On the other hand, AA had no effect on granulosa cell survival at concentrations ranging from 1–20 µM. The predominant effect of saturated FFAs over unsaturated FFAs, regarding the suppression of human granulosa cell proliferation, is very similar to the situation observed in insulin resistance; namely, a higher proportion of saturated FFAs, relative to unsaturated FFAs, is reported to be associated with insulin resistance (42). As a result, we may safely say that obese women with insulin resistance may be susceptible to ovarian dysfunction via elevated saturated FFA concentration in the circulation. Interestingly, although AA itself has no effect on the cell survival of human granulosa cells, it had a highly protective effect against PA- or SA-induced apoptosis of human granulosa cells. This protective effect is dose-dependent at concentrations from 1–15 µM. The suppressive effect of AA on apoptosis induced by lipoxygenase inhibitor, nordihydroguaiaretic acid, is also reported in the W256 carcinosarcoma cell (40). These results may clinically suggest that not only the circulating levels of FFAs, but also the composition of FFAs, may play an important role in the apoptotic event. Interestingly, it is also reported that FFA destabilizes LH receptor and inhibits LH responsiveness in porcine ovarian granulosa cells (43). Such an effect of FFA may also be one additional mechanism for the ovarian dysfunction in insulin resistance. However, to elucidate the exact role of FFAs on human granulosa cells, the examinations of actual concentrations of FFAs in human follicular fluid may be needed.

FFA-induced apoptosis has been reported in the pancreatic ß-cells (15), hepatocytes (44), and brain tumors (45). However, the mechanism of FFA-induced apoptosis is still not well understood. It is not clear even whether FFA-induced apoptosis is a direct effect of FFA or an indirect effect through its metabolite. The complete block of FFA-induced apoptosis in human granulosa cells by fatty acyl-CoA synthetase inhibitor, Triacsin C, and the direct apoptotic effects of acyl-CoA form in the cells clearly indicate that FFA itself does not have an apoptotic effect; instead, it shows that the metabolite acyl-CoA form plays an important role in the induction of apoptosis. This is the first demonstration of the important fact that FFA induces apoptosis through its metabolite to acyl-CoA forms.

Ceramide, which is one of the known sphingolipids, has recently attracted much attention attributable, in part, to its role as a second messenger of cell death. Ceramide can be generated by the hydrolysis of the membrane phospholipid and sphingomelin, which is enriched in the outer leaflet of the plasma membrane (46, 47), or derived from de novo synthesis in response to inducers of apoptosis (48). Fatty acid-induced apoptosis has been reported in the pancreatic ß-cells (15) of Zucker diabetic fatty rats. Under these conditions, the increase of ceramide generation or NO production was implicated as a mechanism of FFA-induced ß-cell apoptosis (15). However, in human granulosa cells, neither mechanism seems to be involved, because neither exogenous ceramide nor NO donor SNAP-induced apoptosis and because the ceramide synthase inhibitor, fumonisin B1, which has been reported to effectively block the apoptotic effect of ceramide (15, 49), or AG, an iNOS inhibitor, did not prevent FFA-induced apoptosis. The difference in the mechanism between human granulosa cells and ß-cells may be attributable to a difference in the cell types or to a difference in the FFA concentration used in the experiment, because a relatively higher concentration of FFAs, namely 1mM long-chain FFAs, was used in a previous study of ß-cell apoptosis, than that (1–300 µM) in our study. Several recent papers have suggested rather that NO production by iNOS suppresses apoptosis in granulosa cells (50) and may act as a cytostatic factor in ovarian follicles (51).

FFAs are known to be endogenous ligands for peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) (52). However, another PPAR{alpha} ligand, fenofibrate (10 µM), did not cause apoptosis of human granulosa cells, suggesting that PPAR{alpha} is not involved in the mechanism.

Bcl-2 is known to extend cell survival by suppressing apoptosis, thus suggesting that a reduction in Bcl-2 might play a role in promoting the apoptotic process. Conversely, the overexpression of one of the apoptosis effector genes, Bax, can antagonize the function of Bcl-2 and increase the susceptibility of the cells to apoptosis. Both Bcl-2 and Bax are expressed in ovarian granulosa cells. The gonadotropin- induced follicular growth and survival were associated with a relatively constitutive level of Bcl-2 and Bcl-XL expression but markedly reduced levels of Bax mRNA (53, 54). In our study, the induction of apoptosis by the treatment with PA in human granulosa cells was accompanied by a dramatic reduction in the expression of Bcl-2 and by the induction of the heterodimer partner Bax proteins. As a result, the PA-induced apoptotic mechanism in human granulosa cells seems to be clearly explained by the observed changes in Bcl-2 and Bax. Recently, both tumor suppressor genes, such as p53 (55, 56), and death genes, such as Fas antigen/Fas ligand (57, 58), were found to play an important role in controlling granulosa cell death during follicular atresia. In the present study, FFA-induced apoptosis was not accompanied by any change in the expression levels of p53 and Fas antigen, based on the findings of either a Western blot or a flow cytometry analysis of human granulosa cells (data not shown).

In summary, we demonstrated, for the first time, that FFAs, especially saturated FFAs, induce apoptosis in human granulosa cells, not directly but through the metabolite of acyl-CoA forms. These effects of FFAs on granulosa cell proliferation may therefore be a possible mechanism for the reproductive abnormalities, including amenorrhea, which are frequently observed in obese women.


   Acknowledgments
 
We thank Dr. L. Zhang and Dr. J.-X. Liao for valuable comments and discussion. We are grateful to Prof. Brian Quinn for critically reading the manuscript.


   Footnotes
 
Abbreviations: AA, Arachidonic acid; AG, aminoguanidine; CoA, coenzyme A; EGFP, enhanced green fluorescent protein; iNOS, aminoguanidine; LA, linoleic acid; NIDDM, non-insulin-dependent diabetes mellitus; NO, nitric oxide; OA, oleic acid; PA, palmitic acid; PI, propidium iodide; PPAR, peroxisome proliferator-activated receptor {alpha}; SA, stearic acid; SNAP, S-nitroso-N-acetylpenicillamine.

Received November 6, 2000.

Accepted for publication April 4, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bergstrom S 1967 Prostaglandins: members of a new hormonal system. These physiologically very potent compounds of ubiquitous occurrence are formed from essential fatty acids. Science157:382–391
  2. Grimminger F, Mayer K, Kiss L, Wahn H, Walmrath D, Seeger W 1997 Synthesis of 4- and 5-series leukotrienes in the lung microvasculature challenged with Escherichia coli hemolysin: critical dependence on exogenous free fatty acid supply. Am J Respir Cell Mol Biol 16:317–324[Abstract]
  3. McArthur MJ, Atshaves BP, Frolov A, Foxworth WD, Kier AB, Schroeder F 1999 Cellular uptake and intracellular trafficking of long chain fatty acids. J Lipid Res 40:1371–1383[Abstract/Free Full Text]
  4. Wolever TM, Bentum-Williams A, Jenkins DJ 1995 Physiological modulation of plasma free fatty acid concentrations by diet. Metabolic implications in nondiabetic subjects. Diabetes Care 18:962–970[Abstract]
  5. Murphy MC, Isherwood SG, Sethi S, et al. 1995 Postprandial lipid and hormone responses to meals of varying fat contents: modulatory role of lipoprotein lipase? Eur J Clin Nutr 49:578–588[Medline]
  6. Singer P, Godicke W, Voigt S, Hajdu I, Weiss M 1985 Postprandial hyperinsulinemia in patients with mild essential hypertension. Hypertension 7:182–186[Abstract/Free Full Text]
  7. Hall SE, Saunders J, Sonksen PH 1979 Glucose and free fatty acid turnover in normal subjects and in diabetic patients before and after insulin treatment. Diabetologia 16:297–306[CrossRef][Medline]
  8. Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD 1988 Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020–1024[Abstract]
  9. Stolba P, Kvapil M, Wichterle D, Dvorak P 1993 Kinetics of free fatty acids in hypertriglyceridemia. Evidence for different types of insulin resistance. Ann NY Acad Sci 683:373–374[CrossRef][Medline]
  10. Smith U, Axelsen M, Carvalho E, Eliasson B, Jansson PA, Wesslau C 1999 Insulin signaling and action in fat cells: associations with insulin resistance and type 2 diabetes. Ann NY Acad Sci 892:119–126[Abstract/Free Full Text]
  11. Belfiore F, Iannello S 1998 Insulin resistance in obesity: metabolic mechanisms and measurement methods. Mol Genet Metab 65:121–128[CrossRef][Medline]
  12. Lewis GF, Steiner G 1996 Hypertriglyceridemia and its metabolic consequences as a risk factor for atherosclerotic cardiovascular disease in non-insulin-dependent diabetes mellitus. Diabetes Metab Rev 12:37–56[CrossRef][Medline]
  13. Mitropoulos KA, Armitage JM, Collins R, et al. 1997 Randomized placebo-controlled study of the effects of simvastatin on haemostatic variables, lipoproteins and free fatty acids. The Oxford Cholesterol Study Group. Eur Heart J 18:235–241[Abstract/Free Full Text]
  14. Stulnig TM, Berger M, Roden M, Stingl H, Raederstorff D, Waldhausl W 2000 Elevated serum free fatty acid concentrations inhibit T lymphocyte signaling. FASEB J 14:939–947[Abstract/Free Full Text]
  15. Shimabukuro M, Zhou YT, Levi M, Unger RH 1998 Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci USA 95:2498–2502[Abstract/Free Full Text]
  16. Zhang YW, Stern B, Rebar RW 1984 Endocrine comparison of obese menstruating and amenorrheic women. J Clin Endocrinol Metab 58:1077–1083[Abstract]
  17. Glass AR, Dahms WT, Abraham G, Atkinson RL, Bray GA, Swerdloff RS 1978 Secondary amenorrhea in obesity: etiologic role of weight-related androgen excess. Fertil Steril 30:243–244[Medline]
  18. Loughlin T, Cunningham SK, Culliton M, Smyth PP, Meagher DJ, McKenna TJ 1985 Altered androstenedione and estrone dynamics associated with abnormal hormonal profiles in amenorrheic subjects with weight loss or obesity. Fertil Steril 43:720–725[Medline]
  19. Boden G 1997 Role of fatty acids in the pathogenesis of insulin resistance and NIDDM. Diabetes 46:3–10[Abstract]
  20. Taylor SI, Barr V, Reitman M 1996 Does leptin contribute to diabetes caused by obesity? Science 274:1151–1152[Free Full Text]
  21. Cohen B, Novic D, Rubinstein M 1996 Modulation of insulin activities by leptin. Science 274:1185–1188[Abstract/Free Full Text]
  22. Hotamisligil GS, Shargill NS, Spiegelman BM 1993 Adipose expression of tumor necrosis factor-{alpha}; direct role in obesity-linked insulin resistance. Science 259:87–91[Abstract/Free Full Text]
  23. Hotamisligil GS, Murray DL, Choy LN, Spiegelman BM 1994 Tumor necrosis factor-1 inhibits signaling from the insulin receptor. Proc Natl Acad Sci USA 91:4854–4858[Abstract/Free Full Text]
  24. Ferrannini E, Barrett EJ, Bevilacqua S, DeFronzo RA 1983 Effect of fatty acids on glucose production and utilization in man. J Clin Invest 72:1737–1747
  25. Groop LC, Bonadonna RC, DelPrato S, et al. 1989 Glucose and free fatty acid metabolism in non-insulin-dependent diabetes mellitus. Evidence for multiple sites of insulin resistance. J Clin Invest 84:205–213
  26. Manco M, Mingrone G, Greco AV, et al. 2000 Insulin resistance directly correlates with increased saturated fatty acids in skeletal muscle triglycerides. Metabolism 49:220–224[CrossRef][Medline]
  27. Nishi Y, Yanase T, Mu Y-M, et al. 2001 Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology 142:437–445[Abstract/Free Full Text]
  28. Hirase N, Yanase T, Mu Y, et al. 1999 Thiazolidinedione induces apoptosis and monocytic differentiation in the promyelocytic leukemia cell line HL60. Oncology 57(Suppl 2):17–26
  29. Spicer LJ, Francisco CC 1997 The adipose obese gene product, leptin: evidence of a direct inhibitory role in ovarian function. Endocrinology 138:3374–3379[Abstract/Free Full Text]
  30. Best CL, Pudney J, Anderson DJ, Hill JA 1994 Modulation of human granulosa cell steroid production in vitro by tumor necrosis factor alpha: implications of white blood cells in culture. Obstet Gynecol 84:121–127[Abstract/Free Full Text]
  31. Zwaal RF, Schroit AJ 1997 Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89:1121–1132[Free Full Text]
  32. Verhoven B, Schlegel RA, Williamson P 1995 Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182:1597–1601[Abstract/Free Full Text]
  33. Koopman G, Reutelingsperger CPM, Kuijten GAM, Keehnen RMJ, Pals ST, van Oers MHG 1994 Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415–1420[Abstract/Free Full Text]
  34. Corbett JA, McDaniel ML 1996 Selective inhibition of inducible nitric oxide synthase by aminoguanidine. Methods Enzymol 268:398–408[Medline]
  35. Hamilton JA, Civelek VN, Kamp F, Tornheim K, Corkey BE 1994 Changes in internal pH caused by movement of fatty acids into and out of clonal pancreatic beta-cells (HIT). J Biol Chem 269:20852–20856[Abstract/Free Full Text]
  36. Kamp F, Hamilton JA 1992 pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids. Proc Natl Acad Sci USA 89:11367–11370[Abstract/Free Full Text]
  37. Knoll L J, Schall OF, Suzuki I, Gokel GW, Gordon JI 1995 Comparison of the reactivity of tetradecenoic acids, a triacsin, and unsaturated oximes with four purified Saccharomyces cerevisiae fatty acid activation proteins. J Biol Chem 270:20090–20097[Abstract/Free Full Text]
  38. Igal RA, Coleman RA 1996 Acylglycerol recycling from triacylglycerol to phospholipid, not lipase activity, is defective in neutral lipid storage disease fibroblasts. J Biol Chem 271:16644–16651[Abstract/Free Full Text]
  39. Noel RJ, Antinozzi PA, McGarry JD, Newgard CB 1997 Engineering of glycerol-stimulated insulin secretion in islet beta cells. Differential metabolic fates of glucose and glycerol provide insight into mechanisms of stimulus-secretion coupling. J Biol Chem 272:18621–18627[Abstract/Free Full Text]
  40. Tang DG, Guan KL, Li L, et al. 1997 Suppression of W256 carcinosarcoma cell apoptosis by arachidonic acid and other polyunsaturated fatty acids. Int J Cancer 72:1078–1087[CrossRef][Medline]
  41. Shimabukuro M, Wang MY, Zhou YT, Newgard CB, Unger RH 1998 Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression. Proc Natl Acad Sci USA 95:9558–9561[Abstract/Free Full Text]
  42. Pan DA, Lilloja S, Milner MR 1995 Skeletal muscle membrane lipid composition is related to adiposity and insulin action. J Clin Invest 96:2802–2808
  43. Scsukova S, Jezova M, Vranova J, Kolena J 2000 Involvement of membrane surface charge in thermal tability of the rat ovarian LH/hCG receptor. Biochim Biophys Acta 1468:15–19[Medline]
  44. Wu D, Cederbaum AI 2000 Ethanol and arachidonic acid produce toxicity in hepatocytes from pyrazole-treated rats with high levels of CYP2E1. Mol Cell Biochem 204:157–67[CrossRef][Medline]
  45. Willoams JR, Leaver HA, Ironside JW, Miller EP, Whittle IR, Gregor A 1998 Apoptosis in human primary brain tumours: actions of arachidonic acid. Prostaglandins Leukot Essent Fatty Acids 58:193–200[CrossRef][Medline]
  46. Shaul PW, Anderson RG 1998 Role of plasmalemmal caveolae in signal transduction. Am J Physiol 275:L843–L851
  47. Morita Y, Tilly JL 2000 Sphingolipid regulation of female gonadal cell apoptosis. Ann NY Acad Sci 905:209–220[Abstract/Free Full Text]
  48. Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Kolesnick R 1995 Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 82:405–414[CrossRef][Medline]
  49. Merrill Jr AH, van Echten G, Wang E, Sandhoff K 1993 Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J Biol Chem 268:27299–27306[Abstract/Free Full Text]
  50. Matsumi H, Koji T, Yano T, et al. 1998 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 atrsia. Endocr J 45:745–751[Medline]
  51. Matsumi H, Yano T, Osuga Y, et al. 2000 Regulation of nitric oxide synthase to promote cytostasis in ovarian follicular development. Biol Reprod 63:141–146[Abstract/Free Full Text]
  52. Desvergne B, Wahli W 1999 Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr Rev 20:649–688[Abstract/Free Full Text]
  53. Tilly JL, Tilly KI, Kenton ML, Johnson AL 1995 Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels. Endocrinology 136:232–241[Abstract]
  54. Kugu K, Ratts VS, Piquette GN, et al. 1998 Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary. Cell Death Differ 5:67–76[CrossRef][Medline]
  55. Keren-Tal I, Suh BS, Dantes A, Linder S, Oren M, Amsterdam A 1995 Involvement of p53 expression in cAMP mediated apoptosis in immortalized granulosa cells. Exp Cell Res 218:283–296[CrossRef][Medline]
  56. Amsterdam A, Gold RS, Hosokawa K, et al. 1999 Cross-talk among multiple signaling pathways controlling ovarian cell death. Trends Endocrinol Metab 10:255–262[CrossRef][Medline]
  57. Quirk SM, Cowan RG, Joshi SG, Henrikson KP 1995 Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biol Reprod 52:279–287[Abstract]
  58. Kondo H, Maruo T, Peng X, Mochizuki M 1996 Immunological evidence for the expression of the Fas antigen in the infant and adult human ovary during follicular regression and atresia. J Clin Endocrinol Metab 81:2702–2710[Abstract]



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