- Email: [email protected]
Induction of insulin resistance by phosphatidylinositol-3-kinase inhibitor in porcine granulosa cells
Induction of insulin resistance by phosphatidylinositol-3-kinase inhibitor in porcine granulosa cells Insulin resistance on porcine granulosa cells was induced by wortmannin, the phosphatidylinositol-3-kinase inhibitor, and insulin signaling key molecules were investigated including glut4 and mitogen-activated protein kinase. Granulosa insulin resistance decreased glut4 expression but increased mitogen-activated protein kinase, indicating the cross talk between the metabolic and mitogenic pathways of insulin signaling in the ovary. (Fertil Steril 2009;92:2119–21. 2009 by American Society for Reproductive Medicine.)
Insulin is a major hormone that regulates glucogenesis in vivo, and a defect in postreceptor signaling pathways for insulin in its classic target tissues such as muscle, adipose, and liver may lead to insulin resistance (IR) and compensatory hyperinsulinemia (1). In addition, insulin has pleiotropic actions on cellular metabolism, growth, differentiation, and proliferation (2). There are two major signaling pathways for these insulin actions in target tissues: the phosphatidylinositol-3-kinase (PI-3 K) pathway, which mediates insulin’s metabolic actions on glucose uptake and utilization, and the mitogen-activated protein kinase (MAPK) pathway controlling mitogenic and other gene-regulatory actions of insulin (2). Our previous work has shown that IR also occurs in the ovary in polycystic ovary syndrome (PCOS), the main organ implicated in PCOS (3). However, little is known about the biologic significance of this metabolic phenotype to ovary dysfunction. To invesMiaoe Yan, Ph.D.a Jing Wang, M.D.a Xiaoke Wu, M.D., Ph.D.a Lihui Hou, M.D.a Haixue Kuang, Ph.D.b Yongyan Wang, M.D.c a Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, People’s Republic of China b School of Pharmacology, Heilongjiang University of Chinese Medicine, Harbin, People’s Republic of China c Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Science, Beijing, People’s Republic of China Received January 14, 2009; revised May 24, 2009; accepted June 7, 2009; published online July 31, 2009. M.Y. has nothing to disclose. J.W. has nothing to disclose. X.W. has nothing to disclose. L.H. has nothing to disclose. H.K. has nothing to disclose. Y.W. has nothing to disclose. Miaoe Yan and Jing Wang contributed equally to this work and served as coauthors. Supported by the National Clinical Research Base of Gynecology in Chinese Medicine; Heilongjiang Province Foundation for Outstanding Youths (JC200804). Reprint requests: Xiaoke Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, Heilongjiang University of Chinese Medicine, Harbin, 150040 China (FAX: 86-451-8213-0094; E-mail: xiaokewu2002@vip. sina.com).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.06.019
tigate the role of IR in the ovary, we first established the IR model in cultured porcine ovarian granulosa cells by treatment with wortmannin, a specific PI-3 K inhibitor that totally abolishes the insulin action by PI-3 K phosphorylation, and then we further studied the cross talk between two intracellular signaling pathways. Institutional Review Board approval was obtained from the Heilongjiang University of Chinese Medicine at Harbin. The granulosa cells were prepared from 32 different porcine ovaries in animals obtained at diestrus cycle from a slaughterhouse. Then the granulosa cells, 90% of which were viable, were removed from the detached follicles and incubated at 37 C under 5% CO2 for 48 hours. After the cell anchorage, the culture medium was replaced by 0.1% calf serum albumin medium (HyClone, Logan, UT). Then granulosa cells were divided into five groups. The culture medium with 0.1% dimethyl sulfoxide (DMSO; [CH3]2SO) only as vehicle was applied in the control group; the other four groups were exposed respectively to increasing doses of wortmannin (1.0, 1.5, 3.0, and 5.0 mmol/L). Subsequently, glucose uptake by granulosa cells was assessed to be insulin resistant by the method described in a previous article (4). As a result, 1.5 mmol/ L wortmannin–treated granulosa cells had significantly lower [3H]glucose uptake (49,095 13,523 counts per minute [cpm]) than the control (81,452 10,122 cpm, P<.01), indicating a defect in glucose utilization and IR. After being fixed with 4% paraformaldehyde and blocked with 3% hydrogen peroxide and 0.1% bovine serum albumin (5), the granulosa cells were treated with goat anti-glut4 (Sigma, St. Louis, MO), rat anti-MAPK (Sigma), or phosphate-buffered saline solution (negative control), respectively, and subsequently stained with fluorescein isothiocyanate–labeled diplo-antibody. Finally, these cells were visualized by phase contrast microscopy and analyzed with the Leica-Qwin system (Leica Limited, Oberkochen, Germany). Total RNA was extracted from the cells prepared with TRIzol Reagents (Invitrogen, Carlsbad, CA). Ribonucleic acid was assayed with the ThermoScript RT-PCR System (Invitrogen). The polymerase chain reaction products then were electrophoresed on agarose gel and visualized by staining with ethidium bromide. The computational method is as follows: Intensity of objective gene Area/(b-Actin intensity Area). The sequences of glut4 were 50 -GGCACCCTCACTACGCTCTGG-30 /50 -CAA TGA
Fertility and Sterility Vol. 92, No. 6, December 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
2119
FIGURE 1 Effects of wortmannin on mRNA expressions of MAPK and glut4. Cells were fixed with 4% paraformaldehyde and stained with fluorescein isothiocyanate–labeled diplo-antibody for visualization by fluorescence microscopy. Both glut4 (A) and MAPK (B) expressed at the cytosol and presented light flavovirens. Granulosa cells were cultured for 48 hours after plating on a cell culture dish and then were incubated with medium (Con), 1.5 mmol/L DMSO (DMSO), or 1.5 mmol/L DMSO and 1.5 mmol/L wortmannin (IR) for another 48 hours. Messenger RNA expression was detected by reverse transcriptase–polymerase chain reaction for glut4 (C, a), MAPK (C, b), and b-actin (C, c) in granulosa cells. The target genes were determined by the intensity of ethidium bromide Area/b-actin Area with arbitrary unit (D). *P< .01 and **P< .05 versus DMSO.
Yan. Correspondence. Fertil Steril 2009.
GGA ACC GTCCAA GAAT-30 . The sequences of MAPK were 50 AACCCAAACAAGCGCATCACAGT-30 /50 -GGAGCAGGACCA GATCCAA AAGG-30 . The sequences of b-actin were 50 -GATG AGATT GGCATGGCTTT-30 /50 -CACGAAGGCTCATTCAA-30 . We did an analysis of variance for group comparisons with Dunnett as a post hoc test with the DMSO group as the control by the SPSS 11.5 system software (SPSS, Inc., Chicago, IL). A value of P<.05 was considered statistically significant. As a result, wortmannin-treated cells at 1.5 mmol/L with well morphology and viability had nearly 40% of glucose uptake by the vehicle-treated control cells, leading to 13.76% higher glucose level left (15.55 1.62 mmol/L) in the medium than the control (13.27 1.29 mmol/L, P<.05). Although the uptake of glucose by granulosa cells treated with 3.0 mmol/L or 5.0 mmol/L wortmannin was reduced, their morphology and viability were damaged significantly. Therefore, the optimal concentration of wortmannin-induced IR in granulosa cells for the following studies was determined to be 1.5 mmol/L. In the fluorescence microscopy test, both glut4 (Fig. 1A) and MAPK (Fig. 1B) expressed at the cytoplasm and presented light flavovirens, whereas they were not detected in either nucleus or
2120
Yan et al.
Correspondence
cell membrane. However, there was no expression in the negative control group. The porcine granulosa cells were treated with medium (control), 0.1% DMSO, or 1.5 mmol/L wortmannin, of which vehicle was 0.1% DMSO (IR). The glut4 messenger RNA (mRNA) expression in wortmannin-treated cells was lower than that of 0.1% DMSO cells, with a decrease by 58% (Fig. 1C, 1D). However, as compared with 0.1% DMSO cells, IR increased MAPK expression by approximately 46% (Fig. 1C, 1D). There were not any significant differences between 0.1% DMSO and control cells for the expressions of both glut4 and MAPK. Recently some reports showed that the insulin signal also exists in the microenvironment of the ovary, and impairment of the metabolic pathway in granulosa cells in women with PCOS possibly was due to defective local insulin action (6). Moran et al. (7) isolated plasma membrane fraction of ovarian tissue. They showed defective insulin signaling in the ovaries of women with PCOS, which was similar to the findings in insulin classic target tissues. Therefore, we examined the effects of experimentally induced IR on the cultured ovarian granulosa cells. In our experiment, we confirmed the method of wortmannin induction with regard to IR (8) and clearly demonstrated for the first
Vol. 92, No. 6, December 2009
time that wortmannin can induce the granulosa cells to be IR by the inhibition of the PI-3 K activity in vitro. The model success was supported further by impaired [3-3H]glucose uptake by granulosa cells but increased the medium glucose levels. Moreover, a study shows that wortmannin not only blocks the PI-3 K activity but also consequently reduces insulin action (4). We found that IR in granulosa cells resulted in decreased glut4 expression but enhanced MAPK. This is in accordance with the reports by Gaster et al. (9) that slow-twitch fibers in subjects with type 2 diabetes have reduced glut4 content. Our results also are consistent with the conclusion of Montagnani et al. (4) that inhibition of the metabolic branch of insulin signaling leads to an enhanced mitogenic action of insulin in vascular endothelial cells. Inhibiting PI-3 K pathway can induce IR as shown in our study, but the expression of extracellular regulated protein kinases (ERK) 1/2 (a number of the MAPK family) is enhanced, which may serve as a physical feedback of insulin signaling and contribute to a compensatory mechanism to rescue resistance to insulin’s metabolic actions in the PCOS ovary (10). The reduction of glut4 of the metabolic signaling pathway and the enhancement of MAPK with respect to mitogenic potential suggest a cross talk between multiple intracellular insulin signaling pathways in ovarian cells. It is not entirely clear whether the postreceptor signaling pathway is used in insulin-mediated steroidogenesis, but signaling by both the mitogen-activated kinase and the PI-3 K pathways are thought to be involved. One study proposed that inhibition of ERK activity, by its inhibitor U0126, significantly decreased the expression of P450c17 with concomitant reduction of androstenedione production. It suggests that ERK activity plays a positive role in promotion of steroidogenesis in thecal cells (11). However, another study found that P450c17 activity increased after specific inhibition of mitogen-activated ERK kinase (MEK)/ERK phosphorylation in normal human theca cells (12). As for granulosa cells, one study found that the addition of PI-3 K inhibitor significantly decreased insulin-stimulated aromatase
mRNA levels and E2 accumulation, whereas inhibition of the MAPK pathway significantly increased aromatase mRNA abundance (13). Our study showed that inhibition of PI-3 K contributed to the reduction of glut4 and enhancement of MAPK. In addition, our recent work found that IR could directly exaggerate androgenic potential within theca cells, suggesting a possible involvement of this ovarian metabolic phenotype in PCOS hyperandrogenemia (14). Therefore, insulin-induced granulosa steroidogenesis may be mediated by both PI-3 K and MAPK pathway activation. Such an association is further evidence that insulin sensitizer, such as troglitazone and metformin, effects via the MAPK pathway. Troglitazone could divergently alter expression of various insulin-receptor substrate molecules and insulin actions and could be used as an ovarian insulin sensitizer and mitogen/steroidogenic inhibitor in PCOS (3). Furthermore, metformin has a direct effect on granulosa cells and makes phosphorylated protein kinase B (AKT) and MAPK expressions enhanced or decreased respectively in these cells (15). However, the MAPK pathway is more complicated. We cannot rule out the possibility that the granulosa steroidogenesis also is modulated by an alternative signaling mechanism of MAPK pathway such as activation of mitogen-activated protein kinase kinase (MKK)3/p38 or Jun N-terminal kinase (JNK)1/2. Therefore, our study only offered a concomitant change of MAKP and steroidogenesis, both of which resulted from the inhibition of PI-3 K, other than the casual association between MAKP and androgenesis. A weakness of this study is that protein quantification has not been done because of insufficient cell availability, but semiquantification of the mRNA of the insulin signaling molecules was detected instead. The glut4 translation by confocal microscopy as a future direction deserves to be investigated to understand its significance to altered insulin signaling. In summary, IR induced by wortmannin could directly reduce the expression of glut4 but enhance MAPK expression, indicating the cross talk between two pathways of insulin signaling in ovarian cells.
REFERENCES 1. Diamanti-Kandarakis E, Papavassiliou AG. Molecular mechanisms of insulin resistance in polycystic ovary syndrome. Trends Mol Med 2006;12:324–32. 2. Virkamaki A, Ueki K, Kahn CR. Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 1999;103:931–43. 3. Wu XK, Zhou SY, Liu JX, P€oll€anen P, Sallinen K, M€akinen M, et al. Selective ovary resistance to insulin signaling in women with polycystic ovary syndrome. Fertil Steril 2003;80:954–65. 4. Montagnani M, Golovchenko I, Kim I, Koh GY, Goalstone ML, Mundhekar AN, et al. Inhibition of phosphatidylinositol 3-kinase enhances mitogenic actions of insulin in endothelial cells. J Biol Chem 2002;277:1794–9. 5. Feteanu A. Labelled antibodies in biology and medicine. New York: McGraw-Hill, 1978. 52–190. 6. Diamanti-Kandarakis E, Argyrakopoulou G, Economou F, Kandaraki E, Koutsilieris M. Defects in insulin signaling pathways in ovarian steroidogenesis and other tissues in polycystic ovary syn-
Fertility and Sterility
7.
8.
9.
10.
11.
drome (PCOS). J Steroid Biochem Mol Biol 2008;109:242–6. Moran C, Huerta R, Conway-Myers BA, Hines GA, Azziz R. Altered autophosphorylation of the insulin receptor in the ovary of a woman with polycystic ovary syndrome. Fertil Steril 2001;75:625–8. Liu AX, Testa JR, Hamilton TC, Jove R, Nicosia SV, Cheng JQ. AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3kinase in human ovarian epithelial cancer cells. Cancer Res 1998;58:2973–7. Gaster M, Staehr P, Beck-Nielsen H, Schroder HD, Handberg A. GLUT4 is reduced in slow muscle fibers of type 2 diabetic patients: is insulin resistance in type 2 diabetes a slow, type 1 fiber disease? Diabetes 2001;50:1324–9. Corbould A, Zhao H, Mirzoeva S, Aird F, Dunaif A. Enhanced mitogenic signaling in skeletal muscle of women with polycystic ovary syndrome. Diabetes 2006;55:751–9. Tajima K, Yoshii K, Fukuda S, Orisaka M, Miyamoto K, Amsterdam A, et al. Luteinizing hor-
12.
13.
14.
15.
mone-induced extracellular-signal regulated kinase activation differently modulates progesterone and androstenedione production in bovine theca cells. Endocrinology 2005;146:2903–10. Munir I, Yen HW, Geller DH, Torbati D, Bierden RM, Weitsman SR, et al. Insulin augmentation of 17alpha-hydroxylase activity is mediated by phosphatidyl inositol 3-kinase but not extracellular signal-regulated kinase-1/2 in human ovarian theca cells. Endocrinology 2004;145:175–83. Silva JM, Hamel M, Sahmi M, Price CA. Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro. Reproduction 2006;132:909–17. Qu J, Wang Y, Wu X, Gao L, Hou L, Erkkola R. Insulin resistance directly contributes to androgenic potential within ovarian theca cells. Fertil Steril 2009;91(5 Suppl). 1990–7. Dunaif A. IR and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 1997;18:774–800.
2121
Insulin is a major hormone that regulates glucogenesis in vivo, and a defect in postreceptor signaling pathways for insulin in its classic target tissues such as muscle, adipose, and liver may lead to insulin resistance (IR) and compensatory hyperinsulinemia (1). In addition, insulin has pleiotropic actions on cellular metabolism, growth, differentiation, and proliferation (2). There are two major signaling pathways for these insulin actions in target tissues: the phosphatidylinositol-3-kinase (PI-3 K) pathway, which mediates insulin’s metabolic actions on glucose uptake and utilization, and the mitogen-activated protein kinase (MAPK) pathway controlling mitogenic and other gene-regulatory actions of insulin (2). Our previous work has shown that IR also occurs in the ovary in polycystic ovary syndrome (PCOS), the main organ implicated in PCOS (3). However, little is known about the biologic significance of this metabolic phenotype to ovary dysfunction. To invesMiaoe Yan, Ph.D.a Jing Wang, M.D.a Xiaoke Wu, M.D., Ph.D.a Lihui Hou, M.D.a Haixue Kuang, Ph.D.b Yongyan Wang, M.D.c a Department of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin, People’s Republic of China b School of Pharmacology, Heilongjiang University of Chinese Medicine, Harbin, People’s Republic of China c Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Science, Beijing, People’s Republic of China Received January 14, 2009; revised May 24, 2009; accepted June 7, 2009; published online July 31, 2009. M.Y. has nothing to disclose. J.W. has nothing to disclose. X.W. has nothing to disclose. L.H. has nothing to disclose. H.K. has nothing to disclose. Y.W. has nothing to disclose. Miaoe Yan and Jing Wang contributed equally to this work and served as coauthors. Supported by the National Clinical Research Base of Gynecology in Chinese Medicine; Heilongjiang Province Foundation for Outstanding Youths (JC200804). Reprint requests: Xiaoke Wu, M.D., Ph.D., Department of Obstetrics and Gynecology, Heilongjiang University of Chinese Medicine, Harbin, 150040 China (FAX: 86-451-8213-0094; E-mail: xiaokewu2002@vip. sina.com).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2009.06.019
tigate the role of IR in the ovary, we first established the IR model in cultured porcine ovarian granulosa cells by treatment with wortmannin, a specific PI-3 K inhibitor that totally abolishes the insulin action by PI-3 K phosphorylation, and then we further studied the cross talk between two intracellular signaling pathways. Institutional Review Board approval was obtained from the Heilongjiang University of Chinese Medicine at Harbin. The granulosa cells were prepared from 32 different porcine ovaries in animals obtained at diestrus cycle from a slaughterhouse. Then the granulosa cells, 90% of which were viable, were removed from the detached follicles and incubated at 37 C under 5% CO2 for 48 hours. After the cell anchorage, the culture medium was replaced by 0.1% calf serum albumin medium (HyClone, Logan, UT). Then granulosa cells were divided into five groups. The culture medium with 0.1% dimethyl sulfoxide (DMSO; [CH3]2SO) only as vehicle was applied in the control group; the other four groups were exposed respectively to increasing doses of wortmannin (1.0, 1.5, 3.0, and 5.0 mmol/L). Subsequently, glucose uptake by granulosa cells was assessed to be insulin resistant by the method described in a previous article (4). As a result, 1.5 mmol/ L wortmannin–treated granulosa cells had significantly lower [3H]glucose uptake (49,095 13,523 counts per minute [cpm]) than the control (81,452 10,122 cpm, P<.01), indicating a defect in glucose utilization and IR. After being fixed with 4% paraformaldehyde and blocked with 3% hydrogen peroxide and 0.1% bovine serum albumin (5), the granulosa cells were treated with goat anti-glut4 (Sigma, St. Louis, MO), rat anti-MAPK (Sigma), or phosphate-buffered saline solution (negative control), respectively, and subsequently stained with fluorescein isothiocyanate–labeled diplo-antibody. Finally, these cells were visualized by phase contrast microscopy and analyzed with the Leica-Qwin system (Leica Limited, Oberkochen, Germany). Total RNA was extracted from the cells prepared with TRIzol Reagents (Invitrogen, Carlsbad, CA). Ribonucleic acid was assayed with the ThermoScript RT-PCR System (Invitrogen). The polymerase chain reaction products then were electrophoresed on agarose gel and visualized by staining with ethidium bromide. The computational method is as follows: Intensity of objective gene Area/(b-Actin intensity Area). The sequences of glut4 were 50 -GGCACCCTCACTACGCTCTGG-30 /50 -CAA TGA
Fertility and Sterility Vol. 92, No. 6, December 2009 Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
2119
FIGURE 1 Effects of wortmannin on mRNA expressions of MAPK and glut4. Cells were fixed with 4% paraformaldehyde and stained with fluorescein isothiocyanate–labeled diplo-antibody for visualization by fluorescence microscopy. Both glut4 (A) and MAPK (B) expressed at the cytosol and presented light flavovirens. Granulosa cells were cultured for 48 hours after plating on a cell culture dish and then were incubated with medium (Con), 1.5 mmol/L DMSO (DMSO), or 1.5 mmol/L DMSO and 1.5 mmol/L wortmannin (IR) for another 48 hours. Messenger RNA expression was detected by reverse transcriptase–polymerase chain reaction for glut4 (C, a), MAPK (C, b), and b-actin (C, c) in granulosa cells. The target genes were determined by the intensity of ethidium bromide Area/b-actin Area with arbitrary unit (D). *P< .01 and **P< .05 versus DMSO.
Yan. Correspondence. Fertil Steril 2009.
GGA ACC GTCCAA GAAT-30 . The sequences of MAPK were 50 AACCCAAACAAGCGCATCACAGT-30 /50 -GGAGCAGGACCA GATCCAA AAGG-30 . The sequences of b-actin were 50 -GATG AGATT GGCATGGCTTT-30 /50 -CACGAAGGCTCATTCAA-30 . We did an analysis of variance for group comparisons with Dunnett as a post hoc test with the DMSO group as the control by the SPSS 11.5 system software (SPSS, Inc., Chicago, IL). A value of P<.05 was considered statistically significant. As a result, wortmannin-treated cells at 1.5 mmol/L with well morphology and viability had nearly 40% of glucose uptake by the vehicle-treated control cells, leading to 13.76% higher glucose level left (15.55 1.62 mmol/L) in the medium than the control (13.27 1.29 mmol/L, P<.05). Although the uptake of glucose by granulosa cells treated with 3.0 mmol/L or 5.0 mmol/L wortmannin was reduced, their morphology and viability were damaged significantly. Therefore, the optimal concentration of wortmannin-induced IR in granulosa cells for the following studies was determined to be 1.5 mmol/L. In the fluorescence microscopy test, both glut4 (Fig. 1A) and MAPK (Fig. 1B) expressed at the cytoplasm and presented light flavovirens, whereas they were not detected in either nucleus or
2120
Yan et al.
Correspondence
cell membrane. However, there was no expression in the negative control group. The porcine granulosa cells were treated with medium (control), 0.1% DMSO, or 1.5 mmol/L wortmannin, of which vehicle was 0.1% DMSO (IR). The glut4 messenger RNA (mRNA) expression in wortmannin-treated cells was lower than that of 0.1% DMSO cells, with a decrease by 58% (Fig. 1C, 1D). However, as compared with 0.1% DMSO cells, IR increased MAPK expression by approximately 46% (Fig. 1C, 1D). There were not any significant differences between 0.1% DMSO and control cells for the expressions of both glut4 and MAPK. Recently some reports showed that the insulin signal also exists in the microenvironment of the ovary, and impairment of the metabolic pathway in granulosa cells in women with PCOS possibly was due to defective local insulin action (6). Moran et al. (7) isolated plasma membrane fraction of ovarian tissue. They showed defective insulin signaling in the ovaries of women with PCOS, which was similar to the findings in insulin classic target tissues. Therefore, we examined the effects of experimentally induced IR on the cultured ovarian granulosa cells. In our experiment, we confirmed the method of wortmannin induction with regard to IR (8) and clearly demonstrated for the first
Vol. 92, No. 6, December 2009
time that wortmannin can induce the granulosa cells to be IR by the inhibition of the PI-3 K activity in vitro. The model success was supported further by impaired [3-3H]glucose uptake by granulosa cells but increased the medium glucose levels. Moreover, a study shows that wortmannin not only blocks the PI-3 K activity but also consequently reduces insulin action (4). We found that IR in granulosa cells resulted in decreased glut4 expression but enhanced MAPK. This is in accordance with the reports by Gaster et al. (9) that slow-twitch fibers in subjects with type 2 diabetes have reduced glut4 content. Our results also are consistent with the conclusion of Montagnani et al. (4) that inhibition of the metabolic branch of insulin signaling leads to an enhanced mitogenic action of insulin in vascular endothelial cells. Inhibiting PI-3 K pathway can induce IR as shown in our study, but the expression of extracellular regulated protein kinases (ERK) 1/2 (a number of the MAPK family) is enhanced, which may serve as a physical feedback of insulin signaling and contribute to a compensatory mechanism to rescue resistance to insulin’s metabolic actions in the PCOS ovary (10). The reduction of glut4 of the metabolic signaling pathway and the enhancement of MAPK with respect to mitogenic potential suggest a cross talk between multiple intracellular insulin signaling pathways in ovarian cells. It is not entirely clear whether the postreceptor signaling pathway is used in insulin-mediated steroidogenesis, but signaling by both the mitogen-activated kinase and the PI-3 K pathways are thought to be involved. One study proposed that inhibition of ERK activity, by its inhibitor U0126, significantly decreased the expression of P450c17 with concomitant reduction of androstenedione production. It suggests that ERK activity plays a positive role in promotion of steroidogenesis in thecal cells (11). However, another study found that P450c17 activity increased after specific inhibition of mitogen-activated ERK kinase (MEK)/ERK phosphorylation in normal human theca cells (12). As for granulosa cells, one study found that the addition of PI-3 K inhibitor significantly decreased insulin-stimulated aromatase
mRNA levels and E2 accumulation, whereas inhibition of the MAPK pathway significantly increased aromatase mRNA abundance (13). Our study showed that inhibition of PI-3 K contributed to the reduction of glut4 and enhancement of MAPK. In addition, our recent work found that IR could directly exaggerate androgenic potential within theca cells, suggesting a possible involvement of this ovarian metabolic phenotype in PCOS hyperandrogenemia (14). Therefore, insulin-induced granulosa steroidogenesis may be mediated by both PI-3 K and MAPK pathway activation. Such an association is further evidence that insulin sensitizer, such as troglitazone and metformin, effects via the MAPK pathway. Troglitazone could divergently alter expression of various insulin-receptor substrate molecules and insulin actions and could be used as an ovarian insulin sensitizer and mitogen/steroidogenic inhibitor in PCOS (3). Furthermore, metformin has a direct effect on granulosa cells and makes phosphorylated protein kinase B (AKT) and MAPK expressions enhanced or decreased respectively in these cells (15). However, the MAPK pathway is more complicated. We cannot rule out the possibility that the granulosa steroidogenesis also is modulated by an alternative signaling mechanism of MAPK pathway such as activation of mitogen-activated protein kinase kinase (MKK)3/p38 or Jun N-terminal kinase (JNK)1/2. Therefore, our study only offered a concomitant change of MAKP and steroidogenesis, both of which resulted from the inhibition of PI-3 K, other than the casual association between MAKP and androgenesis. A weakness of this study is that protein quantification has not been done because of insufficient cell availability, but semiquantification of the mRNA of the insulin signaling molecules was detected instead. The glut4 translation by confocal microscopy as a future direction deserves to be investigated to understand its significance to altered insulin signaling. In summary, IR induced by wortmannin could directly reduce the expression of glut4 but enhance MAPK expression, indicating the cross talk between two pathways of insulin signaling in ovarian cells.
REFERENCES 1. Diamanti-Kandarakis E, Papavassiliou AG. Molecular mechanisms of insulin resistance in polycystic ovary syndrome. Trends Mol Med 2006;12:324–32. 2. Virkamaki A, Ueki K, Kahn CR. Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 1999;103:931–43. 3. Wu XK, Zhou SY, Liu JX, P€oll€anen P, Sallinen K, M€akinen M, et al. Selective ovary resistance to insulin signaling in women with polycystic ovary syndrome. Fertil Steril 2003;80:954–65. 4. Montagnani M, Golovchenko I, Kim I, Koh GY, Goalstone ML, Mundhekar AN, et al. Inhibition of phosphatidylinositol 3-kinase enhances mitogenic actions of insulin in endothelial cells. J Biol Chem 2002;277:1794–9. 5. Feteanu A. Labelled antibodies in biology and medicine. New York: McGraw-Hill, 1978. 52–190. 6. Diamanti-Kandarakis E, Argyrakopoulou G, Economou F, Kandaraki E, Koutsilieris M. Defects in insulin signaling pathways in ovarian steroidogenesis and other tissues in polycystic ovary syn-
Fertility and Sterility
7.
8.
9.
10.
11.
drome (PCOS). J Steroid Biochem Mol Biol 2008;109:242–6. Moran C, Huerta R, Conway-Myers BA, Hines GA, Azziz R. Altered autophosphorylation of the insulin receptor in the ovary of a woman with polycystic ovary syndrome. Fertil Steril 2001;75:625–8. Liu AX, Testa JR, Hamilton TC, Jove R, Nicosia SV, Cheng JQ. AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3kinase in human ovarian epithelial cancer cells. Cancer Res 1998;58:2973–7. Gaster M, Staehr P, Beck-Nielsen H, Schroder HD, Handberg A. GLUT4 is reduced in slow muscle fibers of type 2 diabetic patients: is insulin resistance in type 2 diabetes a slow, type 1 fiber disease? Diabetes 2001;50:1324–9. Corbould A, Zhao H, Mirzoeva S, Aird F, Dunaif A. Enhanced mitogenic signaling in skeletal muscle of women with polycystic ovary syndrome. Diabetes 2006;55:751–9. Tajima K, Yoshii K, Fukuda S, Orisaka M, Miyamoto K, Amsterdam A, et al. Luteinizing hor-
12.
13.
14.
15.
mone-induced extracellular-signal regulated kinase activation differently modulates progesterone and androstenedione production in bovine theca cells. Endocrinology 2005;146:2903–10. Munir I, Yen HW, Geller DH, Torbati D, Bierden RM, Weitsman SR, et al. Insulin augmentation of 17alpha-hydroxylase activity is mediated by phosphatidyl inositol 3-kinase but not extracellular signal-regulated kinase-1/2 in human ovarian theca cells. Endocrinology 2004;145:175–83. Silva JM, Hamel M, Sahmi M, Price CA. Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro. Reproduction 2006;132:909–17. Qu J, Wang Y, Wu X, Gao L, Hou L, Erkkola R. Insulin resistance directly contributes to androgenic potential within ovarian theca cells. Fertil Steril 2009;91(5 Suppl). 1990–7. Dunaif A. IR and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 1997;18:774–800.
2121