Link between metformin and the peroxisome proliferator-activated receptor γ pathway in the uterine tissue of hyperandrogenized prepubertal mice
2011; Elsevier BV; Volume: 95; Issue: 8 Linguagem: Inglês
10.1016/j.fertnstert.2011.02.004
ISSN1556-5653
AutoresEvelin Elia, María Carolina Pustovrh, Sabrina Amalfi, Luigi Devoto, Alicia Beatriz Motta,
Tópico(s)Lipid metabolism and disorders
ResumoChronic hyperandrogenism alters the peroxisome proliferator-activated receptor γ (PPARγ) pathway in the uterine tissue of prepubertal mice. The gene and protein expression of PPARγ is not modified, but the gene and protein expression of 12-lipoxygenase (12-LOX), an enzyme that synthesizes PPARγ ligands, is decreased. The antihyperglycemic drug metformin can prevent this adverse effect. Chronic hyperandrogenism alters the peroxisome proliferator-activated receptor γ (PPARγ) pathway in the uterine tissue of prepubertal mice. The gene and protein expression of PPARγ is not modified, but the gene and protein expression of 12-lipoxygenase (12-LOX), an enzyme that synthesizes PPARγ ligands, is decreased. The antihyperglycemic drug metformin can prevent this adverse effect. Polycystic ovary syndrome (PCOS) is a disease characterized by hyperandrogenism, hirsutism, oligomenorrhea or amenorrhea, and anovulation (1Franks S. Polycystic ovary syndrome.N Engl J Med. 1995; 333: 853-861Crossref PubMed Scopus (1768) Google Scholar). The excess of androgens has a detrimental effect on endometrial function, which can lead to infertility (2Okon M.A. Laird S.M. Tuckerman E.M. Li T.C. Serum androgen levels in women who have recurrent miscarriages and their correlation with markers of endometrial function.Fertil Steril. 1998; 69: 682-690Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 3Tuckerman E.M. Okon M.A. Li T. Laird S.M. Do androgens have a direct effect on endometrial function? An in vitro study.Fertil Steril. 2000; 74: 771-779Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 4Sir-Petermann T. Maliqueo M. Angel B. Lara H.E. Perez-Bravo F. Recabarren S.E. Maternal serum androgens in pregnant women with polycystic ovary syndrome: possible implications in prenatal androgenization.Hum Reprod. 2002; 17: 2573-2579Crossref PubMed Scopus (301) Google Scholar) and even endometrial cancer (5Balen A.H. Tan S.L. McDougall J. Jacobs H.S. Miscarriage rates following in-vitro fertilization are increased in women with polycystic ovary syndrome and reduced by pituitary desensitization with buserelin.Hum Reprod. 1993; 8: 959-964Crossref PubMed Scopus (57) Google Scholar, 6Pillay O.C. Te Fong L.F. Crow J.C. Benjamin E. Mould T. Atiomo W. et al.The association between polycystic ovaries and endometrial cancer.Hum Reprod. 2006; 21: 924-929Crossref PubMed Scopus (101) Google Scholar, 7Elia E. Vighi S. Lombardi E. Motta A.B. Detrimental effects of hyperandrogenism on uterine functions.Int Immunopharmacol. 2008; 8: 1827-1834Crossref PubMed Scopus (12) Google Scholar, 8Elia E.M. Belgorosky D. Faut M. Vighi S. Pustovrh C. Devoto L. et al.The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.Mol Hum Reprod. 2009; 15: 421-432Crossref PubMed Scopus (20) Google Scholar). By the use of a hyperandrogenized murine model, we previously found that the excess of androgens induced embryo resorption of early pregnant mice (9Sander V. Solano M.E. Elia E. Luchetti C.G. Di Girolamo G. Gonzalez C. et al.The influence of dehydroepiandrosterone on early pregnancy in mice.Neuroimmunomodulation. 2005; 12: 285-292Crossref PubMed Scopus (24) Google Scholar, 10Solano M.E. Sander V. Elia E. Luchetti C.G. Di Girolamo G. Gonzalez C. et al.Metformin prevents embryonic resorption induced by hyperandrogenization with dehydroepiandrosterone in mice.Reprod Fertil Dev. 2006; 18: 533-544Crossref PubMed Scopus (30) Google Scholar, 11Luchetti C.G. Mikó E. Szekeres-Bartho J. Paz D.A. Motta A.B. Dehydroepiandrosterone and metformin modulate progesterone induced blocking factor (PIBF), cyclooxygenase 2 (COX2) and cytokines in early pregnant mice.J Steroid Biochem Mol Biol. 2008; 111: 200-207Crossref PubMed Scopus (23) Google Scholar) and promoted the development of uterine structures that were closely related to precancerous structures (7Elia E. Vighi S. Lombardi E. Motta A.B. Detrimental effects of hyperandrogenism on uterine functions.Int Immunopharmacol. 2008; 8: 1827-1834Crossref PubMed Scopus (12) Google Scholar, 8Elia E.M. Belgorosky D. Faut M. Vighi S. Pustovrh C. Devoto L. et al.The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.Mol Hum Reprod. 2009; 15: 421-432Crossref PubMed Scopus (20) Google Scholar).N,N′-dimethylbiguanide metformin is one of the most common drugs used for the treatment of type 2 diabetes. Metformin activates the adenosine 3′:5′ monophosphate (AMP)-dependent kinase α (AMPK-α) pathway to decrease glucose production, increase fatty acid oxidation, and promote the uptake of glucose by cells (12Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. et al.Role of AMP-activated protein kinase in mechanism of metformin action.J Clin Invest. 2001; 108: 1167-1174Crossref PubMed Scopus (4332) Google Scholar, 13Fryer L.G. Parbu-Patel A. Carling D. The anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways.J Biol Chem. 2002; 12: 25226-25232Crossref Scopus (904) Google Scholar, 14Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles W.G. Schlattner U. et al.Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo.J Biol Chem. 2004; 279: 43940-43951Crossref PubMed Scopus (415) Google Scholar). In PCOS patients, metformin decreases androgen levels, improves the frequency of ovulation and menstrual cycles (15Diamanti-Kandarakis E. Kouli C. Tsianateli T. Bergiele A. Therapeutic effects of metformin on insulin resistance and hyperandrogenism in polycystic ovary syndrome.Eur J Endocrinol. 1998; 138: 269-274Crossref PubMed Scopus (293) Google Scholar, 16Glueck C.J. Fontaine R.N. Wang P. Subbiah M.T. Weber K. Illig E. et al.Metformin reduces weight, centripetal obesity, insulin, leptin, and low-density lipoprotein cholesterol in nondiabetic, morbidly obese subjects with body mass index greater than 30.Metabolism. 2001; 50: 856-861Abstract Full Text PDF PubMed Scopus (155) Google Scholar, 17Jakubowicz D.J. Seppälä M. Jakubowicz S. Rodriguez-Armas O. Rivas-Santiago A. Koistinen H. et al.Insulin reduction with metformin increases luteal phase serum glycodelin and insulin-like growth factor-binding protein 1 concentrations and enhances uterine vascularity and blood flow in the polycystic ovary syndrome.J Clin Endocrinol Metab. 2001; 86: 1126-1133Crossref PubMed Scopus (160) Google Scholar, 18La Marca A. Morgante G. Palumbo M. Cianci A. Petraglia F. De Leo V. Insulin-lowering treatment reduces aromatase activity in response to follicle-stimulating hormone in women with polycystic ovary syndrome.Fertil Steril. 2002; 78: 1234-1239Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), and prevents abortions (17Jakubowicz D.J. Seppälä M. Jakubowicz S. Rodriguez-Armas O. Rivas-Santiago A. Koistinen H. et al.Insulin reduction with metformin increases luteal phase serum glycodelin and insulin-like growth factor-binding protein 1 concentrations and enhances uterine vascularity and blood flow in the polycystic ovary syndrome.J Clin Endocrinol Metab. 2001; 86: 1126-1133Crossref PubMed Scopus (160) Google Scholar, 19Velázquez E. Acosta A. Mendoza S.G. Menstrual cyclicity after metformin therapy in polycystic ovary syndrome.Obstet Gynecol. 1997; 90: 392-395Crossref PubMed Scopus (276) Google Scholar, 20Glueck C.J. Goldenberg N. Wang P. Loftspring M. Sherman A. Metformin during pregnancy reduces insulin, insulin resistance, insulin secretion, weight, testosterone and development of gestational diabetes: prospective longitudinal assessment of women with polycystic ovary syndrome from preconception throughout pregnancy.Hum Reprod. 2004; 19: 510-521Crossref PubMed Scopus (143) Google Scholar). However, little is known about the mechanisms by which metformin restores uterine functions in women with PCOS. In this context, we previously reported elsewhere that metformin prevents the adverse effects induced by hyperandrogenism in uterine tissue, including the appearance of abnormal endometrial structure (8Elia E.M. Belgorosky D. Faut M. Vighi S. Pustovrh C. Devoto L. et al.The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.Mol Hum Reprod. 2009; 15: 421-432Crossref PubMed Scopus (20) Google Scholar).Peroxisome proliferator-activated receptors (PPARs) are a family of transcriptional nuclear factors with three isoforms, α, β, and γ, which regulate the expression of multiple genes (21Issemann I. Green S. Activation of a member of the steroid hormonal receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Crossref PubMed Scopus (3020) Google Scholar). Lipoxygenase (LOX) metabolizes arachidonic and linoleic acids, producing eicosanoids (22Needleman P. Turk J. Jakschik B.A. Morrison A.R. Lefkowith J.B. Arachidonic acid and metabolism.Annu Rev Biochem. 1986; 55: 69-102Crossref PubMed Google Scholar, 23Kuhn H. Borngraber S. Mammalian 15-lipoxygenases: enzyme properties and biological implications.in: Nigam S. Pace-Asciak C.R. Lipoxygenases and their metabolites. 447. Plenum, New York1999: 5-28Google Scholar). The primary metabolites of arachidonic acid generated by 1-LOX are the leukotrienes and lipoxines, whereas those produced by 12-LOX and 15-LOX are hydroxyeicosatetraenoic acids (HETEs) (24Brash A.R. Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate.J Biol Chem. 1999; 274: 23679-23682Crossref PubMed Scopus (1128) Google Scholar, 25Conrad D.J. The arachidonate 12/15 lipoxygenases: a review of tissue expression and biologic function.Clin Rev Allergy Immunol. 1999; 17: 71-89Crossref PubMed Scopus (84) Google Scholar). It is known that the uterine PPARγ pathway regulates implantation in mice by modulating the 12/15 LOX system (26Li Q. Cheon Y.P. Kannan A. Shanker S. Bagchi I.C. Bagchi M.K. A novel pathway involving progesterone receptor, 12/15-lipoxygenase-derived eicosanoids, and peroxisome proliferator-activated receptor gamma regulates implantation in mice.J Biol Chem. 2004; 279: 11570-11581Crossref PubMed Scopus (77) Google Scholar). These findings, together with the fact that metformin and the PPAR system have been related in a synergistic action (27Pan Q.R. Li W.H. Wang H. Sun Q. Xiao X.H. Brock B. et al.Glucose, metformin, and AICAR regulate the expression of G protein-coupled receptor members in INS-1 beta cell.Horm Metab Res. 2009; 41: 799-804Crossref PubMed Scopus (29) Google Scholar, 28Prabhakar P.K. Doble M. Synergistic effect of phytochemicals in combination with hypoglycemic drugs on glucose uptake in myotubes.Phytomedicine. 2009; 16: 1119-1122Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), led us to study the expression of PPARγ, 12-LOX, and 15-LOX in uterine tissue from hyperandrogenized mice and their relationship with the metformin treatment.The animal model (7Elia E. Vighi S. Lombardi E. Motta A.B. Detrimental effects of hyperandrogenism on uterine functions.Int Immunopharmacol. 2008; 8: 1827-1834Crossref PubMed Scopus (12) Google Scholar, 8Elia E.M. Belgorosky D. Faut M. Vighi S. Pustovrh C. Devoto L. et al.The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.Mol Hum Reprod. 2009; 15: 421-432Crossref PubMed Scopus (20) Google Scholar, 9Sander V. Solano M.E. Elia E. Luchetti C.G. Di Girolamo G. Gonzalez C. et al.The influence of dehydroepiandrosterone on early pregnancy in mice.Neuroimmunomodulation. 2005; 12: 285-292Crossref PubMed Scopus (24) Google Scholar, 10Solano M.E. Sander V. Elia E. Luchetti C.G. Di Girolamo G. Gonzalez C. et al.Metformin prevents embryonic resorption induced by hyperandrogenization with dehydroepiandrosterone in mice.Reprod Fertil Dev. 2006; 18: 533-544Crossref PubMed Scopus (30) Google Scholar, 11Luchetti C.G. Mikó E. Szekeres-Bartho J. Paz D.A. Motta A.B. Dehydroepiandrosterone and metformin modulate progesterone induced blocking factor (PIBF), cyclooxygenase 2 (COX2) and cytokines in early pregnant mice.J Steroid Biochem Mol Biol. 2008; 111: 200-207Crossref PubMed Scopus (23) Google Scholar) consisted of female prepubertal (25-day-old) mice of the BALB/c strain. The dehydroepiandrosterone (DHEA) group consisted of animals injected daily with DHEA (6 mg/100 g body weight, dissolved in 0.10 mL sesame oil) for 20 consecutive days, and the DHEA + M group consisted of animals injected with DHEA and given metformin orally (50 mg/100 g body weight in 0.05 mL of water, given orally with a cannula) for 20 days. The controls consisted of two groups: [1] animals injected with oil (0.1 mL) and given water orally (0.05 mL) for 20 consecutive days (C group) and [2] the metformin-alone group which consisted of mice treated orally with 50 mg metformin/kg body weight in 0.05 mL of water for 20 days (M group). Mice (10 per group) were housed under controlled temperature (22°C) and illumination (14 hours light, 10 hours dark, with lights on at 05:00 hours), and they were allowed free access to Purina rat chow and water. All the procedures involving animals were approved by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) according to the Animal Care and Use Committee Statement of CONICET, 1996. After 20 days of treatment, the animals were killed by cervical dislocation, and freshly dissected uteri from each group were immediately frozen at –70°C until both the messenger RNA (mRNA) and protein determinations.The content of proteins corresponding to the PPAR γ1 and γ2 isoforms were evaluated in uterine tissue by Western blot analysis. Each sample was applied to 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and separated proteins were transferred onto nitrocellulose membranes. After blocking, the membranes were incubated with rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against PPARγ using β-actin (1:500) as an internal control. Rat skeletal muscle extract was used as a positive control for PPARγ. Negative controls were performed in the absence of the primary antibody. Individual bands were quantified directly from membranes by densitometry by use of ImageJ software (http://rsbweb.nih.gov/ij/).We found that both PPAR γ1 and γ2 isoforms were present in uterine tissue. We also found that neither DHEA nor DHEA + M treatment modified the expression of PPARγ protein when compared with controls (Table 1). To study whether the gene expression of PPARγ1 and PPARγ2 isoforms was modified by DHEA or DHEA + M treatments, the mRNA contents were measured by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Total mRNA from each group of uterine tissue was extracted using TriReagent (Invitrogen, Buenos Aires, Argentina). The products were separated on 2% agarose and visualized with ethidium bromide staining. The 18S protein gene was used as an internal control. The analysis was performed by densitometry scanning by use of an ImageQuant RT ECL (GE Life Sciences, Piscataway, NJ). Bands were compared with internal control by use of ImageJ. For amplification of PPARγ cDNA, the primers were sense 5′-TGA CAC AGA GAT GCC ATT CTG G 3′ and antisense 5′ GAG CTA GAC CCA ATG GTT GCT GAT TAC 3′.Table 1Protein and messenger-RNA expression of uterine peroxisome proliferator-activated receptors type γ 1 and 2, 12-lipoxygenase and 15-lipoxygenase from control mice and mice treated with metformin and dehydroepiandrosterone with and without metformin.GroupPPARγ1/β actin(arbitrary units)PPARγ2/β actin(arbitrary units)PPAR/18S(arbitrary units)12-LOX/β actin(arbitrary units)12-LOX/18S(arbitrary units)15-LOX/β actin(arbitrary units)15-LOX/18S(arbitrary units)Control0.74 ± 0.060.51 ± 0.09425 ± 801.009 ± 0.0393,030 ± 5300.986 ± 0.1131,882 ± 435Metformin0.77 ± 0.080.63 ± 0.091,655 ± 140aP< 0.001.0.674 ± 0.0822,910 ± 2801.091 ± 0.1141,912 ± 75DHEA0.78 ± 0.040.66 ± 0.08415 ± 400.437 ± 0.088bP<0.05.1,210 ± 80cP< 0.01 by analysis of variance between control, metformin, DHEA, and DHEA+ metformin groups.1.084 ± 0.2992,287 ± 435DHEA + metformin0.92 ± 0.070.65 ± 0.03615 ± 1800.723 ± 0.0331,150 ± 60cP< 0.01 by analysis of variance between control, metformin, DHEA, and DHEA+ metformin groups.0.836 ± 0.2031,312 ± 390Note: Each value is expressed in arbitrary units and represents the mean ± standard error of the mean of 10 measurements from different animals. DHEA = dehydroepiandrosterone; LOX = lipoxygenase; PPAR = peroxisome proliferator-activated receptor.a P< 0.001.b P<0.05.c P< 0.01 by analysis of variance between control, metformin, DHEA, and DHEA+ metformin groups. Open table in a new tab We found that PPARγ mRNA levels were detectable in uterine tissue and that neither DHEA nor DHEA + M treatment modified the expression of PPARγ mRNA when compared with controls (Table 1). In view of these results, we were interested in determining whether hyperandrogenism and the treatment with metformin were able to modulate the expression of 12-LOX and 15-LOX, enzymes responsible for the synthesis of PPAR ligands. The protein content of uterine 12-LOX and 15-LOX were evaluated by Western blot analysis. The protocol was similar to that described previously, with polyclonal antibodies (Santa Cruz Biotechnology) against 12-LOX (1:100) and 15-LOX (1:100).The DHEA decreased the protein expression of 12-LOX when compared with controls (see Table 1), whereas metformin prevented this effect (see Table 1). We also found that neither DHEA nor DHEA + M modified the protein expression of 15-LOX when compared with controls (see Table 1). To determine whether the effect of the treatments in the 12-LOX and 15-LOX proteins was a reflection of 12-LOX and 15-LOX gene expression, the mRNA levels corresponding to both enzymes were measured by RT-PCR. Using the same protocol as that described earlier, for amplification of 12-LOX complementary DNA (cDNA) the primers were sense 5′-TGATCAGGTAGTGAGCACAGGT-3′ and antisense 5′-CCTTCACATACCTGGCAGTGA-3′. For amplification of 15-LOX, the primers were sense 5′-TAGCCATCCAGCTCGAACTG-3 and antisense 5′-GGTGTAGAGTAGGTGAGGAACTA-3′.Table 1 shows that DHEA decreased the gene expression of 12-LOX when compared with controls and that metformin was not able to prevent this effect. We also found that neither DHEA nor metformin modified the gene expression of 15-LOX (see Table 1 and Supplementary Fig. 1 [available online]).The detrimental effects of the excess of androgens in the endometrial function contribute to the infertility of women with PCOS (2Okon M.A. Laird S.M. Tuckerman E.M. Li T.C. Serum androgen levels in women who have recurrent miscarriages and their correlation with markers of endometrial function.Fertil Steril. 1998; 69: 682-690Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 3Tuckerman E.M. Okon M.A. Li T. Laird S.M. Do androgens have a direct effect on endometrial function? An in vitro study.Fertil Steril. 2000; 74: 771-779Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 4Sir-Petermann T. Maliqueo M. Angel B. Lara H.E. Perez-Bravo F. Recabarren S.E. Maternal serum androgens in pregnant women with polycystic ovary syndrome: possible implications in prenatal androgenization.Hum Reprod. 2002; 17: 2573-2579Crossref PubMed Scopus (301) Google Scholar). We had previously reported that the excess of androgens induced the development of uterine structures closely related to the development of precancerous structures (7Elia E. Vighi S. Lombardi E. Motta A.B. Detrimental effects of hyperandrogenism on uterine functions.Int Immunopharmacol. 2008; 8: 1827-1834Crossref PubMed Scopus (12) Google Scholar, 8Elia E.M. Belgorosky D. Faut M. Vighi S. Pustovrh C. Devoto L. et al.The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.Mol Hum Reprod. 2009; 15: 421-432Crossref PubMed Scopus (20) Google Scholar). These adverse effects are mediated by a proinflammatory status characterized by an increased production of prostaglandins by the uterus (7Elia E. Vighi S. Lombardi E. Motta A.B. Detrimental effects of hyperandrogenism on uterine functions.Int Immunopharmacol. 2008; 8: 1827-1834Crossref PubMed Scopus (12) Google Scholar, 8Elia E.M. Belgorosky D. Faut M. Vighi S. Pustovrh C. Devoto L. et al.The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.Mol Hum Reprod. 2009; 15: 421-432Crossref PubMed Scopus (20) Google Scholar). We also found that metformin partially prevented these adverse effects (7Elia E. Vighi S. Lombardi E. Motta A.B. Detrimental effects of hyperandrogenism on uterine functions.Int Immunopharmacol. 2008; 8: 1827-1834Crossref PubMed Scopus (12) Google Scholar, 8Elia E.M. Belgorosky D. Faut M. Vighi S. Pustovrh C. Devoto L. et al.The effects of metformin on uterine tissue of hyperandrogenized BALB/c mice.Mol Hum Reprod. 2009; 15: 421-432Crossref PubMed Scopus (20) Google Scholar). These findings, together with the fact that the activation of the PPAR system is related to the uterine function (26Li Q. Cheon Y.P. Kannan A. Shanker S. Bagchi I.C. Bagchi M.K. A novel pathway involving progesterone receptor, 12/15-lipoxygenase-derived eicosanoids, and peroxisome proliferator-activated receptor gamma regulates implantation in mice.J Biol Chem. 2004; 279: 11570-11581Crossref PubMed Scopus (77) Google Scholar, 29Sung B. Park S. Yu B.P. Chung H.Y. Amelioration of age-related inflammation and oxidative stress by PPARγ activator: suppression of NF-κB by 2,4-thiazolidinedione.Exp Gerontol. 2006; 41: 590-599Crossref PubMed Scopus (77) Google Scholar), the synergistic action of metformin + PPAR treatment described in PCOS (27Pan Q.R. Li W.H. Wang H. Sun Q. Xiao X.H. Brock B. et al.Glucose, metformin, and AICAR regulate the expression of G protein-coupled receptor members in INS-1 beta cell.Horm Metab Res. 2009; 41: 799-804Crossref PubMed Scopus (29) Google Scholar, 28Prabhakar P.K. Doble M. Synergistic effect of phytochemicals in combination with hypoglycemic drugs on glucose uptake in myotubes.Phytomedicine. 2009; 16: 1119-1122Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 30Glueck C.J. Moriera A. Goldenberg N. Sieve L. Wang P. Pioglitazone and metformin in obese women with polycystic ovary syndrome not optimally responsive to metformin.Hum Reprod. 2003; 18: 1618-1625Crossref PubMed Scopus (98) Google Scholar, 31Legro R.S. Pregnancy considerations in women with polycystic ovary syndrome.Clin Obstet Gynecol. 2007; 50: 295-304Crossref PubMed Scopus (28) Google Scholar), and the role of PPAR in regulating the prostaglandin pathway (32Subbaramaiah K. Lin D.T. Hart J.C. Dannenberg A.J. Peroxisome proliferator-activated receptor gamma ligands suppress the transcriptional activation of cyclooxygenase-2: evidence for involvement of activator protein-1 and CREB-binding protein/p300.J Biol Chem. 2001; 276: 12440-12448Crossref PubMed Scopus (273) Google Scholar), led us to evaluate the action of metformin and the PPARγ system on hyperandrogenized uteri.We found that both PPAR γ1 and γ2 are expressed in uterine tissue from prepubertal mice and that neither protein nor gene expression of PPARγ are regulated by the excess of androgen and metformin. The fact that metformin alone increased PPARγ mRNA levels might be due to the fact that metformin is capable of acting in basal conditions even in the absence of stimulus (33Sander V.A. Facorro G.B. Piehl L. Rubín de Celis E. Motta A.B. Effect of dehydroepiandrosterone and metformin on corpus luteum in mice.Reproduction. 2009; 138: 571-579Crossref PubMed Scopus (9) Google Scholar). However, experiments are being designed to clarify this point.Because the expression of PPARγ was not affected by hyperandrogenism, we studied whether hyperandrogenism altered the ligands of PPARγ and consequently the activation of PPARγ. Here we demonstrated for the first time that protein and mRNA corresponding to the enzymes 12-LOX and 15-LOX, which are responsible for synthesizing ligands of PPARγ (26Li Q. Cheon Y.P. Kannan A. Shanker S. Bagchi I.C. Bagchi M.K. A novel pathway involving progesterone receptor, 12/15-lipoxygenase-derived eicosanoids, and peroxisome proliferator-activated receptor gamma regulates implantation in mice.J Biol Chem. 2004; 279: 11570-11581Crossref PubMed Scopus (77) Google Scholar, 34Huang J.T. Welch J.S. Ricote M. Binder C.J. Willson T.M. Kelly C. et al.Interleukin-4-dependent production of PPAR-γ ligands in macrophages by 12/15-lipoxygenase.Nature. 1999; 400: 378-382Crossref PubMed Scopus (766) Google Scholar, 35Schild R.L. Schaiff W.T. Carlson M.G. Cronbach E.J. Nelson D.M. Sadovsky Y. The activity of PPAR γ in primary human trophoblasts is enhanced by oxidized lipids.J Clin Endocrinol Metab. 2002; 87: 1105-1110Crossref PubMed Scopus (80) Google Scholar), are present in uterine tissue from prepubertal mice. In fact, this metabolic pathway of PPARγ activation has been described in other systems, such as monocytes (36Nagy L. Tontonoz P. Alvarez J.G. Chen H. Evans R.M. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARγ.Cell. 1998; 93: 229-240Abstract Full Text Full Text PDF PubMed Scopus (1574) Google Scholar), macrophages (34Huang J.T. Welch J.S. Ricote M. Binder C.J. Willson T.M. Kelly C. et al.Interleukin-4-dependent production of PPAR-γ ligands in macrophages by 12/15-lipoxygenase.Nature. 1999; 400: 378-382Crossref PubMed Scopus (766) Google Scholar), fibroblasts (35Schild R.L. Schaiff W.T. Carlson M.G. Cronbach E.J. Nelson D.M. Sadovsky Y. The activity of PPAR γ in primary human trophoblasts is enhanced by oxidized lipids.J Clin Endocrinol Metab. 2002; 87: 1105-1110Crossref PubMed Scopus (80) Google Scholar), and uteri (26Li Q. Cheon Y.P. Kannan A. Shanker S. Bagchi I.C. Bagchi M.K. A novel pathway involving progesterone receptor, 12/15-lipoxygenase-derived eicosanoids, and peroxisome proliferator-activated receptor gamma regulates implantation in mice.J Biol Chem. 2004; 279: 11570-11581Crossref PubMed Scopus (77) Google Scholar). However, only the 12-LOX enzyme was susceptible to the adverse action of hyperandrogenism and responded to metformin treatment, thus suggesting that 15-LOX could be a constitutive enzyme in the synthesis of PPARγ ligands. The excess of androgens decreased both the gene and protein expression of 12-LOX, but metformin was able to prevent the decrease in the protein expression, although not the gene expression, of 12-LOX. These findings suggest that metformin might be contributing with a posttranscriptional mechanism in the regulation of 12-LOX expression. However, experiments are being addressed to clarify this point.The fact that rosiglitazone (synthetic PPARγ ligand) activates the PPARγ system by increasing PPARγ mRNA levels (37Chen Q. Sun X. Chen J. Cheng L. Wang J. Wang Y. Sun Z. Direct rosiglitazone action on steroidogenesis and proinflammatory factor production in human granulose-lutein cells.Reprod Biol Endocrinol. 2009; 7: 147-152Crossref PubMed Scopus (33) Google Scholar) together with data presented here that metformin can modulate the enzyme that synthesizes PPARγ ligand could explain why the combined treatment with glitazones and metformin is more effective in preventing abortions than each separate treatment (28Prabhakar P.K. Doble M. Synergistic effect of phytochemicals in combination with hypoglycemic drugs on glucose uptake in myotubes.Phytomedicine. 2009; 16: 1119-1122Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 30Glueck C.J. Moriera A. Goldenberg N. Sieve L. Wang P. Pioglitazone and metformin in obese women with polycystic ovary syndrome not optimally responsive to metformin.Hum Reprod. 2003; 18: 1618-1625Crossref PubMed Scopus (98) Google Scholar, 31Legro R.S. Pregnancy considerations in women with polycystic ovary syndrome.Clin Obstet Gynecol. 2007; 50: 295-304Crossref PubMed Scopus (28) Google Scholar). It appears that the direct relationship between 12-LOX activity and implantation (26Li Q. Cheon Y.P. Kannan A. Shanker S. Bagchi I.C. Bagchi M.K. 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