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Inflammatory pathways linking obesity and ovarian dysfunction
Journal of Reproductive Immunology 88 (2011) 142–148
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Inflammatory pathways linking obesity and ovarian dysfunction Rebecca L. Robker a,∗ , Linda L.-Y. Wu a , Xing Yang b a b
School of Paediatrics and Reproductive Health, Robinson Institute, University of Adelaide, Adelaide, South Australia 5005, Australia Reproductive Medical Center, Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510275, China
a r t i c l e
i n f o
Article history: Received 13 October 2010 Received in revised form 20 December 2010 Accepted 16 January 2011
Keywords: Oxidative stress Endoplasmic reticulum stress Lipotoxicity Lipid Ovary
a b s t r a c t This review summarizes some of the recent advances in obesity research and describes how we and others have built upon these findings to better understand the impact of obesity on granulosa cells, cumulus cells and oocytes within the ovaries of obese females. Obesity is associated with lipid accumulation in non-adipose tissue cells and the induction of oxidative stress and endoplasmic reticulum stress responses that are tightly linked with systemic inflammation. Analysis of ovarian cells and fluid of obese women indicates that these same mechanisms are activated in the ovary in response to obesity. Studies in mice support this and allow further dissection of the pathways by which diet-induced obesity contributes to changes in mitochondria and the endoplasmic reticulum. These studies are in their infancy but cumulatively provide basic information about the cellular mechanisms that may lead to the impaired ovulation and reduced oocyte developmental potential that is observed in obese females. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Inflammatory basis of metabolic disease
Obesity is prevalent in many countries around the world and is a major risk factor for chronic diseases, particularly the constellation known as the metabolic syndrome. This has precipitated a resurgence in obesity research and important new findings about the etiology of this disease, above all the understanding that obesity is an inflammatory response. This now changes how we think about the onset of obesity complications including one that is typically neglected-female infertility. This short mini-review briefly summarizes some of the most recent advances in obesity research and then focuses on findings from our group, as well as others, which indicate obesity causes changes in the ovary similar to its effects in other tissues. Our intent is to highlight that little is known in this area, yet emerging evidence indicates that obesity impacts granulosa cells, cumulus cells and oocytes within the ovaries of obese females.
Adipose tissue is now recognized as far more than a storage depot for triglyceride produced during excess nutrition, but rather as a dynamic endocrine organ (Rajala and Scherer, 2003). However, as adipose tissue expands during obesity its physiology changes dramatically, initiating a chronic inflammatory response that ultimately extends to and influences the entire body.
∗ Corresponding author. E-mail address: [email protected] (R.L. Robker). 0165-0378/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2011.01.008
2.1. Immune cells in adipose tissue Adipose tissue contains a remarkable number and diverse array of immune cells. Immune cell types identified in adipose tissue include neutrophils (Elgazar-Carmon et al., 2008), T cells (Robker et al., 2004; Kintscher et al., 2008), NK cells (Ohmura et al., 2010) and even dendritic cells (Brake et al., 2006); with each cell type shown to be altered in either number or activation status by diet-induced obesity. Macrophage infiltration into adipose tissue was the first change to be clearly associated with obesity (Weisberg et al., 2003; Xu et al., 2003) and since
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then a large number of studies have demonstrated the mechanisms by which macrophages are recruited into adipose tissue and how they contribute to the adipose tissue inflammation associated with obesity (reviewed in Suganami and Ogawa, 2010). Briefly, as adipocytes undergo excessive hypertrophy in order to store surplus fat they produce increased levels of adipose-specific cytokines or adipokines and begin to exhibit hypoxia, oxidative stress, and endoplasmic reticulum stress. These changes lead to increased angiogenesis and an infiltration of immune cells, which are recruited into adipose tissue in response to specific adipokines such as MCP-1. Then bidirectional communication involving adipocyte-mediated release of free fatty acids and macrophage release of cytokines triggers further inflammatory responses. Stress responses in adipocytes also contribute to the release of endogenous ‘danger signals’ which include degraded extracellular matrix, modified LDLs and many other specific proteins that further activate macrophages via Toll-like receptors (TLRs), scavenger receptors and others. The end result of these paracrine loops is that in obesity, adipose tissue is filled with immune cells and secretes high levels of free fatty acids and inflammatory cytokines (Suganami and Ogawa, 2010). The systemic milieu produced by this inflamed adipose tissue contributes to cellular stress responses in many other tissues including liver and endothelial cells, leading to the dangerous complications of obesity such as insulin resistance, diabetes, hypertension and stroke. 2.2. Obesity-induced lipotoxicity responses: oxidative stress and endoplasmic reticulum stress In addition to adipose tissue, chronic overnutrition and obesity cause intracellular lipid accumulation in many other tissues, eliciting lipotoxicity responses that activate stress pathways that can culminate in apoptosis (Brookheart et al., 2009). Initially it is the elevated circulating triglycerides and free fatty acids that cause lipid accumulation in cells; in particular long chain fatty-acids, such as palmitate, induce insulin resistance and lipotoxicity pathways that result in cell death in multiple organ systems (Van Herpen and Schrauwen-Hinderling, 2008). Oxidative stress is one of the hallmark responses to intracellular lipid overload and is linked to the cellular dysfunctions that result from obesity-induced metabolic syndrome (reviewed in Schrauwen and Hesselink, 2004; Roberts and Sindhu, 2009). High levels of intracellular free fatty acids impact the mitochondrial membrane structure causing the release of reactive oxygen species (ROS), particularly O− 2 . ROS are highly cytotoxic and if not inactivated by endogenous anti-oxidant enzymes, i.e. catalase, superoxide dismutases and/or glutathione peroxidase, react with cellular macromolecules, oxidizing proteins and lipids, damaging intracellular membranes and DNA. Oxidative stress is known to have detrimental effects in many tissues including the female reproductive tract (Agarwal et al., 2003, 2008). In addition to oxidative stress, endoplasmic reticulum (ER) stress is becoming more widely recognized as a mediator of perturbed cellular function arising from
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obesity-induced lipid accumulation in the cells of many tissues. Mechanisms of ER stress are still emerging. Briefly, high levels of intracellular free fatty acids damage the endoplasmic reticulum membrane integrity, compromising ER functions such as protein folding, lipid processing and Ca2+ homeostasis (see Schroder and Kaufman, 2005; Rutkowski and Kaufman, 2007 for reviews). The accumulation of unfolded proteins in the lumen of the ER triggers the Unfolded Protein Response initiated by the release and activation of 3 ER membrane-associated proteins. PKR-like eukaryotic initiation factor 2a kinase (PERK) phosphorylates the translation factor eIF2a which suppresses global protein translation but increases expression of the transcription factor ATF4. Release of inositol requiring enzyme 1 (IRE1) from the ER membrane allows it to splice X-box binding protein (Xbp1) mRNA into an alternative form (Xbp-1s) which acts as a highly active transcription factor. Release of activating transcription factor 6 (ATF-6) enables it to be proteolytically cleaved and translocate to the nucleus. This cohort of transcription factors: ATF4, Xbp1 and ATF6; activated by the changing ER status, induces the expression of an array of molecular chaperones and heat shock proteins, as well as increases protein degradation and clearance of mis-folded proteins, in an attempt to normalize ER functional capacities. This tripartite response has been shown to be activated in response to obesity in many tissues, including adipose tissue, liver, pancreas and brain (Ozcan et al., 2004, 2009), as cells attempt to cope with excessive demands on ER capacity. If cellular homeostasis cannot be restored additional pathways such as Ca2+ release and caspase activation are induced to initiate apoptosis (Breckenridge et al., 2003). Oxidative stress and endoplasmic reticulum stress are clearly linked, with each able to exacerbate the other (Cullinan and Diehl, 2006; Malhotra and Kaufman, 2007) and both are intertwined with inflammatory pathways (Gregor and Hotamisligil, 2007; Qatanani and Lazar, 2007; De Ferranti and Mozaffarian, 2008; Hotamisligil, 2010). For instance eIF2a-mediated inhibition of protein translation suppresses inhibitory I≡ levels which results in activation of NF≡ (Jiang et al., 2003), a major transcriptional regulator of inflammatory cytokine production. Another example is that the ATF6-related protein CREBH can be proteolytically activated in response to inflammatory cytokines, and induces the production of C-reactive protein by the liver (Zhang et al., 2006). This co-ordination of mitochondrial and endoplasmic reticulum stress responses with the induction of inflammatory mediators, particularly cytokines, is one mechanism by which systemic inflammation induced by obesity is intensified. 3. Obesity is associated with female infertility One of the neglected complications of obesity is female infertility. With obesity prevalent in young women (Ogden et al., 2010) it has become essential to better understand the effects of obesity on all aspects of the female reproductive system in order to alleviate their detrimental consequences on fertility as well as on fetal health. The ovary contains and nurtures oocytes within dynamic tissue structures called ovarian follicles. These
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consist of vascularised steroidogenic theca cells separated by a basement membrane from the avascular estrogenic granulosa cell layer which surrounds a fluid-filled central cavity. Through this separation from the vasculature it has been supposed that the environment of oocytes is uniquely controlled, however, the composition of ovarian follicle fluid is generally reflective of maternal circulation, including metabolite levels (Sutton et al., 2003). At the center of the follicle the growing oocyte is surrounded by specialized granulosa cells known as cumulus cells, which provide the oocyte with maternal nutrition via gap junctions. Maternal hormonal and nutritional signals regulate the growth of the oocyte and the timing of its release/ovulation, as well as endow it with ‘developmental competence’ or the ability to become an embryo if fertilized. Accumulating evidence indicates that obesity impacts these functions of the ovary, diminishing female reproductive potential and impairing the earliest phases of embryo formation. 3.1. Obesity decreases conception rates A woman’s chance of a natural pregnancy is decreased with obesity even if she appears to be ovulating normally and otherwise healthy (Zaadstra et al., 1993; Jensen et al., 1999; Gesink Law et al., 2007; Ramlau-Hansen et al., 2007; Van Der Steeg et al., 2008; Wise et al., 2010). For example, in a large cohort of women in the USA obese women had an 18% reduction in the probability of conceiving in any given month, which translates to an increased time to pregnancy of approximately 2 months longer for obese women to conceive compared to non-obese women (Gesink Law et al., 2007). This delayed time to pregnancy may be due in large part to anovulatory infertility which is increased with obesity (Grodstein et al., 1994; Rich-Edwards et al., 2002). Further, meta-analysis shows that anovulation in obese women is less likely to be ameliorated by treatment with gonadotropin hormones (Mulders et al., 2003). These findings are supported by studies in mice which have found that the incidence of anovulation is increased in mice fed high fat diet (Minge et al., 2008; Wu et al., 2010) even when treated with gonadotropins (Wu et al., 2010). 3.2. Oocyte quality is affected by obesity The effects of obesity on oocyte quality may be investigated in women undergoing assisted reproduction using IVF or ICSI (reviewed in Robker, 2008) where oocytes are aspirated from the ovary and fertilized and monitored in vitro. The few studies which evaluated oocyte maturity observed that it is decreased in women with obesity (Wittemer et al., 2000; Dokras et al., 2006; Esinler et al., 2008). Fertilization rates have also been reported to be decreased with obesity (Salha et al., 2001; Van Swieten et al., 2005; Orvieto et al., 2009; Ferreira et al., 2010; Zhang et al., 2010), yet at least as many studies have also reported no difference. Most studies do not document embryo quality but two have reported that this aspect of oocyte developmental competence is impaired in oocytes from obese women (Carrell et al., 2001; Metwally et al., 2007).
Mouse models allow more in-depth study and show clearly that diet-induced obesity is detrimental to oocytes. Mice fed obesogenic high fat diets have oocytes that exhibit delayed maturation (Jungheim et al., 2010), decreased in vivo fertilization rates (Wu et al., 2010) and decreased developmental competence, assessed as abnormal development to the blastocyst stage (Minge et al., 2008; Igosheva et al., 2010; Jungheim et al., 2010). Thus, dietinduced obesity in mice and women impacts ovarian functions, namely oocyte development and ovulation; however, the mechanisms by which this occurs are not yet understood. 3.3. Obesity-induced changes in ovarian cells and oocytes With increasing rates of obesity in young women and increasing prevalence of obesity-associated anovulation and infertility, a number of laboratories have begun to investigate how obesity impacts critical functions of the ovary. These studies have for the most part analyzed cellular functions and biomarkers known to be dysregulated by obesity in other tissues. The findings to date are summarized in Fig. 1. Although this field of study is in its infancy, the emerging results indicate that the ovary is affected in similar ways as other tissues, with obesity causing lipid accumulation, oxidative and endoplasmic reticulum stress and the activation of inflammatory pathways. 3.4. Increased lipids in ovarian cells with obesity A hallmark of excess nutrition is the accumulation of lipid in non-adipose cells and the ovary is not exempt from such a response. In ovaries from mice fed a high fat diet for 4 weeks both cumulus cells and oocytes contained markedly increased levels of neutral lipid (Wu et al., 2010). In women obesity is associated with a dramatic increase in follicular fluid triglyceride levels (Robker et al., 2009) that are tightly correlated with increased levels of free fatty acids (Robker, unpublished data). Increased lipid levels are known to be a key initiating factor in the lipotoxicity responses that occur in response to obesity and thus it is important to understand (1) what types of lipid moieties accumulate in which ovarian cell types; (2) what nutritional and hormonal circumstances lead to lipid accumulation, and (3) whether in ovarian cells, as in other cells, increased intracellular lipid is the trigger for subsequent inflammatory and stress responses. 3.5. Markers of inflammation in follicles of obese women C-Reactive protein (CRP) levels are dramatically increased in follicular fluid of obese women (Robker et al., 2009). CRP levels in follicle fluid are relatively similar to those in serum (Orvieto et al., 2004) and there is a known significant relationship between CRP levels and body mass index (BMI) (Tchernof et al., 2002). How increased CRP during obesity might impact the ovarian follicle and maturing oocyte is not known and is an important area for future investigation.
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Fig. 1. Mechanisms by which obesity may lead to ovarian dysfunction. As detailed in the text, diet-induced obesity in mice and increased body mass index (BMI) in women activates multiple aspects of lipotoxicity: intracellular lipid accumulation, inflammatory responses, endoplasmic reticulum (ER) stress and oxidative stress, each of which has been assessed using the listed markers. GPx: glutathione peroxidase.
A number of cytokines are elevated in circulation during obesity, for instance TNF␣ (Hotamisligil et al., 1993) and IL-6 (Bastard et al., 2002), as well as the adipokine leptin which also exhibits immune modulating functions (Fernandez-Riejos et al., 2010). Cytokine levels in follicle fluid of obese women have not been investigated with the exception of leptin and IL-18. Leptin in follicle fluid is increased with BMI and adiposity (Mantzoros et al., 2000; Hill et al., 2007) and correlated with serum levels (Butzow et al., 1999; Mantzoros et al., 2000; Hill et al., 2007). In one study (Mantzoros et al., 2000) but not another (Hill et al., 2007) low follicular fluid leptin levels predicted pregnancy success. No differences were found in IL-18 levels in follicular fluid of obese women compared to non-obese women (Kilic et al., 2009); however, perhaps because of the hormonal stimulation protocol or the small number of participants, this study also did not see the expected difference in serum IL-18 levels in obese women reported by others (Esposito et al., 2002). Our laboratory analyzed a panel of cytokines whose levels in circulation are known to be altered by obesity, including adiponectin, IL-6, IL10, MCP-1, TNF␣ and others, in the follicle fluid of women with varying BMIs. We found that some cytokines were significantly increased in follicle fluid from obese women yet they were not strictly correlated with BMI (Robker, unpublished data). Thus future studies are needed to determine why follicle fluid cytokine levels are dysregulated by obesity, but only in some contexts. Alterations in ovarian cytokine production might also be predicted to impact the normal functions of immune cells resident in the ovary (Brannstrom and Enskog, 2002; Wu et al., 2004). As yet there has been no investigation of whether immune cells, which are known to be increased in adipose tissue in response to obesity, are altered in number or activation status in the ovaries of obese women or in animal models of obesity.
3.6. Oxidative stress in the ovary in response to obesity Lipotoxicity responses include oxidative stress and the production of ROS which, if not properly detoxified, are detrimental to cells due to their ability to oxidize other proteins, for instance apolipoprotein complexes such as LDL. Oxidized LDL (oxLDL) is elevated in serum of obese women (Mutlu-Turkoglu et al., 2003) and recently this marker of oxidative stress has been shown to be elevated in the follicle fluid of obese women as well (Bausenwein et al., 2010). LOX1 is a scavenger receptor for oxLDL which when bound activates NF≡ signalling and inflammatory cytokine production (Cominacini et al., 2000). LOX-1 expression is increased in granulosa cells of obese women compared to non-obese women, but this difference is seen only in women treated with relatively low doses of FSH as part of their infertility treatment (Vilser et al., 2010). Another scavenger receptor, SRBI, was also observed to be modestly increased in granulosa cells of obese women (Robker et al., 2009). Inflammatory ROS production also leads to the generation of H2 O2 which is detoxified by catalase and glutathione peroxidase enzymes. Obese women have increased circulating levels of catalase and glutathione peroxidase enzyme activity suggesting an upregulation of these anti-oxidant systems (Bausenwein et al., 2010). Similarly, in the ovarian follicles of obese women this anti-oxidant system appears to be amplified since both catalase and glutathione peroxidase enzyme activity were significantly higher in follicle fluids from obese women (Bausenwein et al., 2010) than non-obese women. Surprisingly, superoxide dismutase (SOD) activity which is the earlier step in ROS detoxification (converting O− 2 to oxygen and H2 O2 ) was not altered in follicle fluid with obesity. Direct measures of ROS production in ovarian cells or fluid from obese compared to non-obese women have not been reported, however, results in mice
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Table 1 Alterations in ovarian granulosa cells (GC) and follicle fluid (FF) of obese women. Increases (↑), decreases (↓) or no difference (ND) in each of the markers assessed in obese women is indicated. Marker analyzed
Cellular function
Effect of obesity
Reference
Triglyceride C-Reactive protein Leptin IL-18 oxLDL LOX-1 SRBI Catalase Glutathione peroxidase ATF4
Neutral lipid Inflammatory marker Adipo/cytokine Cytokine Oxidized lipoprotein Scavenger receptor Scavenger receptor Anti-oxidant enzyme Anti-oxidant enzyme ER stress response
↑ In FF ↑ In FF ↑ In FF ND ↑ In FF ↑ mRNA in GC ↑ mRNA in GC ↑ In FF ↑ In FF ↑ mRNA in GC
Robker et al. (2009) Robker et al. (2009) Mantzoros et al. (2000) Kilic et al. (2009) Bausenwein et al. (2010) Vilser et al. (2010) Robker et al. (2009) Bausenwein et al. (2010) Bausenwein et al. (2010) Wu et al. (2010)
clearly demonstrate that a high fat diet for 6 weeks results in increased ROS production, an oxidized redox state and glutathione depletion in both preovulatory oocytes and zygotes from female mice (Igosheva et al., 2010). 3.7. Endoplasmic reticulum stress in the ovary in response to obesity Endoplasmic reticulum (ER) stress is emerging as a key response to lipotoxicity and inflammatory responses that occur in response to obesity. In mouse ovarian cells (cumulus–oocyte complexes and granulosa cells) the ER stress marker genes ATF4, GRP78, ATF6 and Xbp-1s were increased in expression (Wu et al., 2010; Wu, unpublished data). ATF4 mRNA was also increased in granulosa cells of obese women compared to granulosa cells of non-obese women (Wu et al., 2010). Both ER stress and oxidative stress are associated with alterations in mitochondrial function (Malhotra and Kaufman, 2007). In oocytes of mice fed obesogenic diets for 4 weeks or 6 weeks mitochondrial membrane potential is dramatically different from that of mice fed control diet (Igosheva et al., 2010; Wu et al., 2010). Oocyte mitochondrial function is important for oocyte developmental competence (Van Blerkom, 2009) and thus such alterations may contribute to the defects in oocyte maturation (Jungheim et al., 2010), fertilization (Wu et al., 2010) and embryo and fetal development seen in mice fed high fat diet (Minge et al., 2008; Igosheva et al., 2010; Jungheim et al., 2010). Oxidative stress and ER stress can also both culminate in cellular apoptosis if not resolved (Breckenridge et al., 2003). In mice fed high fat diet for 4 weeks (Wu et al., 2010) or 16 weeks (Jungheim et al., 2010) there is increased apoptosis in ovarian cells, further indicating that lipotoxicity pathways are induced in the ovary in response to high fat diet-induced obesity. Similar mechanisms appear to be activated in ovaries of obese women since increased numbers of dead cells were observed in follicle fluid of obese women compared to non-obese (Vilser et al., 2010).
linked with systemic inflammation. Analysis of ovarian cells and fluid of obese women indicates that these same mechanisms are activated in the human ovary in response to obesity (Table 1). Studies in mice support this and are able to further dissect the cellular mechanisms by which diet-induced obesity is contributing to changes in mitochondria and the endoplasmic reticulum. That obesity leads to female sub-fertility has long been known; however, very recent work is the first elucidating specific cellular and molecular mechanisms by which this occurs (Fig. 1). Studies in animal models and the translation of the results to humans demonstrate that diet-induced obesity induces multiple components of the lipotoxicity response in ovarian cells and oocytes. Excess lipid is present in follicle fluid of obese women and in the cumulus cells and oocytes of obese mice. Secondly there is an altered inflammatory profile within the ovary during obesity with increased levels of CRP, adipokines and cytokines, potentially due to higher levels in circulation or induction by LOX-1. Oxidative stress responses are clearly activated in the ovary during obesity since there are increased levels of oxLDL and ROS, skewed expression of anti-oxidant enzymes and profound changes in mitochondrial membrane potential. Lastly, we have identified activation of the endoplasmic reticulum stress pathway in ovarian cells of obese mice and women, a response that is likely due to increased lipid content of cells and intricately linked with oxidative stress responses. How each of these components may affect each other and contribute to the overall lipotoxicity responses in ovarian cells is still unclear, but cumulatively this emerging research provides some starting information about the cellular mechanisms that may lead to impaired ovulation and reduced oocyte developmental potential in obese women. Acknowledgements This work was supported by the National Health and Medical Research Council (to R.L.R.) and the Channel 7 Children’s Research Fund (to L.L.W., R.L.R.).
4. Summary
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Rapidly evolving research clearly demonstrates that obesity is associated with lipid accumulation in nonadipose tissue cells and the induction of oxidative and endoplasmic reticulum stress responses that are tightly
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Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Inflammatory pathways linking obesity and ovarian dysfunction Rebecca L. Robker a,∗ , Linda L.-Y. Wu a , Xing Yang b a b
School of Paediatrics and Reproductive Health, Robinson Institute, University of Adelaide, Adelaide, South Australia 5005, Australia Reproductive Medical Center, Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510275, China
a r t i c l e
i n f o
Article history: Received 13 October 2010 Received in revised form 20 December 2010 Accepted 16 January 2011
Keywords: Oxidative stress Endoplasmic reticulum stress Lipotoxicity Lipid Ovary
a b s t r a c t This review summarizes some of the recent advances in obesity research and describes how we and others have built upon these findings to better understand the impact of obesity on granulosa cells, cumulus cells and oocytes within the ovaries of obese females. Obesity is associated with lipid accumulation in non-adipose tissue cells and the induction of oxidative stress and endoplasmic reticulum stress responses that are tightly linked with systemic inflammation. Analysis of ovarian cells and fluid of obese women indicates that these same mechanisms are activated in the ovary in response to obesity. Studies in mice support this and allow further dissection of the pathways by which diet-induced obesity contributes to changes in mitochondria and the endoplasmic reticulum. These studies are in their infancy but cumulatively provide basic information about the cellular mechanisms that may lead to the impaired ovulation and reduced oocyte developmental potential that is observed in obese females. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Inflammatory basis of metabolic disease
Obesity is prevalent in many countries around the world and is a major risk factor for chronic diseases, particularly the constellation known as the metabolic syndrome. This has precipitated a resurgence in obesity research and important new findings about the etiology of this disease, above all the understanding that obesity is an inflammatory response. This now changes how we think about the onset of obesity complications including one that is typically neglected-female infertility. This short mini-review briefly summarizes some of the most recent advances in obesity research and then focuses on findings from our group, as well as others, which indicate obesity causes changes in the ovary similar to its effects in other tissues. Our intent is to highlight that little is known in this area, yet emerging evidence indicates that obesity impacts granulosa cells, cumulus cells and oocytes within the ovaries of obese females.
Adipose tissue is now recognized as far more than a storage depot for triglyceride produced during excess nutrition, but rather as a dynamic endocrine organ (Rajala and Scherer, 2003). However, as adipose tissue expands during obesity its physiology changes dramatically, initiating a chronic inflammatory response that ultimately extends to and influences the entire body.
∗ Corresponding author. E-mail address: [email protected] (R.L. Robker). 0165-0378/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2011.01.008
2.1. Immune cells in adipose tissue Adipose tissue contains a remarkable number and diverse array of immune cells. Immune cell types identified in adipose tissue include neutrophils (Elgazar-Carmon et al., 2008), T cells (Robker et al., 2004; Kintscher et al., 2008), NK cells (Ohmura et al., 2010) and even dendritic cells (Brake et al., 2006); with each cell type shown to be altered in either number or activation status by diet-induced obesity. Macrophage infiltration into adipose tissue was the first change to be clearly associated with obesity (Weisberg et al., 2003; Xu et al., 2003) and since
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then a large number of studies have demonstrated the mechanisms by which macrophages are recruited into adipose tissue and how they contribute to the adipose tissue inflammation associated with obesity (reviewed in Suganami and Ogawa, 2010). Briefly, as adipocytes undergo excessive hypertrophy in order to store surplus fat they produce increased levels of adipose-specific cytokines or adipokines and begin to exhibit hypoxia, oxidative stress, and endoplasmic reticulum stress. These changes lead to increased angiogenesis and an infiltration of immune cells, which are recruited into adipose tissue in response to specific adipokines such as MCP-1. Then bidirectional communication involving adipocyte-mediated release of free fatty acids and macrophage release of cytokines triggers further inflammatory responses. Stress responses in adipocytes also contribute to the release of endogenous ‘danger signals’ which include degraded extracellular matrix, modified LDLs and many other specific proteins that further activate macrophages via Toll-like receptors (TLRs), scavenger receptors and others. The end result of these paracrine loops is that in obesity, adipose tissue is filled with immune cells and secretes high levels of free fatty acids and inflammatory cytokines (Suganami and Ogawa, 2010). The systemic milieu produced by this inflamed adipose tissue contributes to cellular stress responses in many other tissues including liver and endothelial cells, leading to the dangerous complications of obesity such as insulin resistance, diabetes, hypertension and stroke. 2.2. Obesity-induced lipotoxicity responses: oxidative stress and endoplasmic reticulum stress In addition to adipose tissue, chronic overnutrition and obesity cause intracellular lipid accumulation in many other tissues, eliciting lipotoxicity responses that activate stress pathways that can culminate in apoptosis (Brookheart et al., 2009). Initially it is the elevated circulating triglycerides and free fatty acids that cause lipid accumulation in cells; in particular long chain fatty-acids, such as palmitate, induce insulin resistance and lipotoxicity pathways that result in cell death in multiple organ systems (Van Herpen and Schrauwen-Hinderling, 2008). Oxidative stress is one of the hallmark responses to intracellular lipid overload and is linked to the cellular dysfunctions that result from obesity-induced metabolic syndrome (reviewed in Schrauwen and Hesselink, 2004; Roberts and Sindhu, 2009). High levels of intracellular free fatty acids impact the mitochondrial membrane structure causing the release of reactive oxygen species (ROS), particularly O− 2 . ROS are highly cytotoxic and if not inactivated by endogenous anti-oxidant enzymes, i.e. catalase, superoxide dismutases and/or glutathione peroxidase, react with cellular macromolecules, oxidizing proteins and lipids, damaging intracellular membranes and DNA. Oxidative stress is known to have detrimental effects in many tissues including the female reproductive tract (Agarwal et al., 2003, 2008). In addition to oxidative stress, endoplasmic reticulum (ER) stress is becoming more widely recognized as a mediator of perturbed cellular function arising from
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obesity-induced lipid accumulation in the cells of many tissues. Mechanisms of ER stress are still emerging. Briefly, high levels of intracellular free fatty acids damage the endoplasmic reticulum membrane integrity, compromising ER functions such as protein folding, lipid processing and Ca2+ homeostasis (see Schroder and Kaufman, 2005; Rutkowski and Kaufman, 2007 for reviews). The accumulation of unfolded proteins in the lumen of the ER triggers the Unfolded Protein Response initiated by the release and activation of 3 ER membrane-associated proteins. PKR-like eukaryotic initiation factor 2a kinase (PERK) phosphorylates the translation factor eIF2a which suppresses global protein translation but increases expression of the transcription factor ATF4. Release of inositol requiring enzyme 1 (IRE1) from the ER membrane allows it to splice X-box binding protein (Xbp1) mRNA into an alternative form (Xbp-1s) which acts as a highly active transcription factor. Release of activating transcription factor 6 (ATF-6) enables it to be proteolytically cleaved and translocate to the nucleus. This cohort of transcription factors: ATF4, Xbp1 and ATF6; activated by the changing ER status, induces the expression of an array of molecular chaperones and heat shock proteins, as well as increases protein degradation and clearance of mis-folded proteins, in an attempt to normalize ER functional capacities. This tripartite response has been shown to be activated in response to obesity in many tissues, including adipose tissue, liver, pancreas and brain (Ozcan et al., 2004, 2009), as cells attempt to cope with excessive demands on ER capacity. If cellular homeostasis cannot be restored additional pathways such as Ca2+ release and caspase activation are induced to initiate apoptosis (Breckenridge et al., 2003). Oxidative stress and endoplasmic reticulum stress are clearly linked, with each able to exacerbate the other (Cullinan and Diehl, 2006; Malhotra and Kaufman, 2007) and both are intertwined with inflammatory pathways (Gregor and Hotamisligil, 2007; Qatanani and Lazar, 2007; De Ferranti and Mozaffarian, 2008; Hotamisligil, 2010). For instance eIF2a-mediated inhibition of protein translation suppresses inhibitory I≡ levels which results in activation of NF≡ (Jiang et al., 2003), a major transcriptional regulator of inflammatory cytokine production. Another example is that the ATF6-related protein CREBH can be proteolytically activated in response to inflammatory cytokines, and induces the production of C-reactive protein by the liver (Zhang et al., 2006). This co-ordination of mitochondrial and endoplasmic reticulum stress responses with the induction of inflammatory mediators, particularly cytokines, is one mechanism by which systemic inflammation induced by obesity is intensified. 3. Obesity is associated with female infertility One of the neglected complications of obesity is female infertility. With obesity prevalent in young women (Ogden et al., 2010) it has become essential to better understand the effects of obesity on all aspects of the female reproductive system in order to alleviate their detrimental consequences on fertility as well as on fetal health. The ovary contains and nurtures oocytes within dynamic tissue structures called ovarian follicles. These
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consist of vascularised steroidogenic theca cells separated by a basement membrane from the avascular estrogenic granulosa cell layer which surrounds a fluid-filled central cavity. Through this separation from the vasculature it has been supposed that the environment of oocytes is uniquely controlled, however, the composition of ovarian follicle fluid is generally reflective of maternal circulation, including metabolite levels (Sutton et al., 2003). At the center of the follicle the growing oocyte is surrounded by specialized granulosa cells known as cumulus cells, which provide the oocyte with maternal nutrition via gap junctions. Maternal hormonal and nutritional signals regulate the growth of the oocyte and the timing of its release/ovulation, as well as endow it with ‘developmental competence’ or the ability to become an embryo if fertilized. Accumulating evidence indicates that obesity impacts these functions of the ovary, diminishing female reproductive potential and impairing the earliest phases of embryo formation. 3.1. Obesity decreases conception rates A woman’s chance of a natural pregnancy is decreased with obesity even if she appears to be ovulating normally and otherwise healthy (Zaadstra et al., 1993; Jensen et al., 1999; Gesink Law et al., 2007; Ramlau-Hansen et al., 2007; Van Der Steeg et al., 2008; Wise et al., 2010). For example, in a large cohort of women in the USA obese women had an 18% reduction in the probability of conceiving in any given month, which translates to an increased time to pregnancy of approximately 2 months longer for obese women to conceive compared to non-obese women (Gesink Law et al., 2007). This delayed time to pregnancy may be due in large part to anovulatory infertility which is increased with obesity (Grodstein et al., 1994; Rich-Edwards et al., 2002). Further, meta-analysis shows that anovulation in obese women is less likely to be ameliorated by treatment with gonadotropin hormones (Mulders et al., 2003). These findings are supported by studies in mice which have found that the incidence of anovulation is increased in mice fed high fat diet (Minge et al., 2008; Wu et al., 2010) even when treated with gonadotropins (Wu et al., 2010). 3.2. Oocyte quality is affected by obesity The effects of obesity on oocyte quality may be investigated in women undergoing assisted reproduction using IVF or ICSI (reviewed in Robker, 2008) where oocytes are aspirated from the ovary and fertilized and monitored in vitro. The few studies which evaluated oocyte maturity observed that it is decreased in women with obesity (Wittemer et al., 2000; Dokras et al., 2006; Esinler et al., 2008). Fertilization rates have also been reported to be decreased with obesity (Salha et al., 2001; Van Swieten et al., 2005; Orvieto et al., 2009; Ferreira et al., 2010; Zhang et al., 2010), yet at least as many studies have also reported no difference. Most studies do not document embryo quality but two have reported that this aspect of oocyte developmental competence is impaired in oocytes from obese women (Carrell et al., 2001; Metwally et al., 2007).
Mouse models allow more in-depth study and show clearly that diet-induced obesity is detrimental to oocytes. Mice fed obesogenic high fat diets have oocytes that exhibit delayed maturation (Jungheim et al., 2010), decreased in vivo fertilization rates (Wu et al., 2010) and decreased developmental competence, assessed as abnormal development to the blastocyst stage (Minge et al., 2008; Igosheva et al., 2010; Jungheim et al., 2010). Thus, dietinduced obesity in mice and women impacts ovarian functions, namely oocyte development and ovulation; however, the mechanisms by which this occurs are not yet understood. 3.3. Obesity-induced changes in ovarian cells and oocytes With increasing rates of obesity in young women and increasing prevalence of obesity-associated anovulation and infertility, a number of laboratories have begun to investigate how obesity impacts critical functions of the ovary. These studies have for the most part analyzed cellular functions and biomarkers known to be dysregulated by obesity in other tissues. The findings to date are summarized in Fig. 1. Although this field of study is in its infancy, the emerging results indicate that the ovary is affected in similar ways as other tissues, with obesity causing lipid accumulation, oxidative and endoplasmic reticulum stress and the activation of inflammatory pathways. 3.4. Increased lipids in ovarian cells with obesity A hallmark of excess nutrition is the accumulation of lipid in non-adipose cells and the ovary is not exempt from such a response. In ovaries from mice fed a high fat diet for 4 weeks both cumulus cells and oocytes contained markedly increased levels of neutral lipid (Wu et al., 2010). In women obesity is associated with a dramatic increase in follicular fluid triglyceride levels (Robker et al., 2009) that are tightly correlated with increased levels of free fatty acids (Robker, unpublished data). Increased lipid levels are known to be a key initiating factor in the lipotoxicity responses that occur in response to obesity and thus it is important to understand (1) what types of lipid moieties accumulate in which ovarian cell types; (2) what nutritional and hormonal circumstances lead to lipid accumulation, and (3) whether in ovarian cells, as in other cells, increased intracellular lipid is the trigger for subsequent inflammatory and stress responses. 3.5. Markers of inflammation in follicles of obese women C-Reactive protein (CRP) levels are dramatically increased in follicular fluid of obese women (Robker et al., 2009). CRP levels in follicle fluid are relatively similar to those in serum (Orvieto et al., 2004) and there is a known significant relationship between CRP levels and body mass index (BMI) (Tchernof et al., 2002). How increased CRP during obesity might impact the ovarian follicle and maturing oocyte is not known and is an important area for future investigation.
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Fig. 1. Mechanisms by which obesity may lead to ovarian dysfunction. As detailed in the text, diet-induced obesity in mice and increased body mass index (BMI) in women activates multiple aspects of lipotoxicity: intracellular lipid accumulation, inflammatory responses, endoplasmic reticulum (ER) stress and oxidative stress, each of which has been assessed using the listed markers. GPx: glutathione peroxidase.
A number of cytokines are elevated in circulation during obesity, for instance TNF␣ (Hotamisligil et al., 1993) and IL-6 (Bastard et al., 2002), as well as the adipokine leptin which also exhibits immune modulating functions (Fernandez-Riejos et al., 2010). Cytokine levels in follicle fluid of obese women have not been investigated with the exception of leptin and IL-18. Leptin in follicle fluid is increased with BMI and adiposity (Mantzoros et al., 2000; Hill et al., 2007) and correlated with serum levels (Butzow et al., 1999; Mantzoros et al., 2000; Hill et al., 2007). In one study (Mantzoros et al., 2000) but not another (Hill et al., 2007) low follicular fluid leptin levels predicted pregnancy success. No differences were found in IL-18 levels in follicular fluid of obese women compared to non-obese women (Kilic et al., 2009); however, perhaps because of the hormonal stimulation protocol or the small number of participants, this study also did not see the expected difference in serum IL-18 levels in obese women reported by others (Esposito et al., 2002). Our laboratory analyzed a panel of cytokines whose levels in circulation are known to be altered by obesity, including adiponectin, IL-6, IL10, MCP-1, TNF␣ and others, in the follicle fluid of women with varying BMIs. We found that some cytokines were significantly increased in follicle fluid from obese women yet they were not strictly correlated with BMI (Robker, unpublished data). Thus future studies are needed to determine why follicle fluid cytokine levels are dysregulated by obesity, but only in some contexts. Alterations in ovarian cytokine production might also be predicted to impact the normal functions of immune cells resident in the ovary (Brannstrom and Enskog, 2002; Wu et al., 2004). As yet there has been no investigation of whether immune cells, which are known to be increased in adipose tissue in response to obesity, are altered in number or activation status in the ovaries of obese women or in animal models of obesity.
3.6. Oxidative stress in the ovary in response to obesity Lipotoxicity responses include oxidative stress and the production of ROS which, if not properly detoxified, are detrimental to cells due to their ability to oxidize other proteins, for instance apolipoprotein complexes such as LDL. Oxidized LDL (oxLDL) is elevated in serum of obese women (Mutlu-Turkoglu et al., 2003) and recently this marker of oxidative stress has been shown to be elevated in the follicle fluid of obese women as well (Bausenwein et al., 2010). LOX1 is a scavenger receptor for oxLDL which when bound activates NF≡ signalling and inflammatory cytokine production (Cominacini et al., 2000). LOX-1 expression is increased in granulosa cells of obese women compared to non-obese women, but this difference is seen only in women treated with relatively low doses of FSH as part of their infertility treatment (Vilser et al., 2010). Another scavenger receptor, SRBI, was also observed to be modestly increased in granulosa cells of obese women (Robker et al., 2009). Inflammatory ROS production also leads to the generation of H2 O2 which is detoxified by catalase and glutathione peroxidase enzymes. Obese women have increased circulating levels of catalase and glutathione peroxidase enzyme activity suggesting an upregulation of these anti-oxidant systems (Bausenwein et al., 2010). Similarly, in the ovarian follicles of obese women this anti-oxidant system appears to be amplified since both catalase and glutathione peroxidase enzyme activity were significantly higher in follicle fluids from obese women (Bausenwein et al., 2010) than non-obese women. Surprisingly, superoxide dismutase (SOD) activity which is the earlier step in ROS detoxification (converting O− 2 to oxygen and H2 O2 ) was not altered in follicle fluid with obesity. Direct measures of ROS production in ovarian cells or fluid from obese compared to non-obese women have not been reported, however, results in mice
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Table 1 Alterations in ovarian granulosa cells (GC) and follicle fluid (FF) of obese women. Increases (↑), decreases (↓) or no difference (ND) in each of the markers assessed in obese women is indicated. Marker analyzed
Cellular function
Effect of obesity
Reference
Triglyceride C-Reactive protein Leptin IL-18 oxLDL LOX-1 SRBI Catalase Glutathione peroxidase ATF4
Neutral lipid Inflammatory marker Adipo/cytokine Cytokine Oxidized lipoprotein Scavenger receptor Scavenger receptor Anti-oxidant enzyme Anti-oxidant enzyme ER stress response
↑ In FF ↑ In FF ↑ In FF ND ↑ In FF ↑ mRNA in GC ↑ mRNA in GC ↑ In FF ↑ In FF ↑ mRNA in GC
Robker et al. (2009) Robker et al. (2009) Mantzoros et al. (2000) Kilic et al. (2009) Bausenwein et al. (2010) Vilser et al. (2010) Robker et al. (2009) Bausenwein et al. (2010) Bausenwein et al. (2010) Wu et al. (2010)
clearly demonstrate that a high fat diet for 6 weeks results in increased ROS production, an oxidized redox state and glutathione depletion in both preovulatory oocytes and zygotes from female mice (Igosheva et al., 2010). 3.7. Endoplasmic reticulum stress in the ovary in response to obesity Endoplasmic reticulum (ER) stress is emerging as a key response to lipotoxicity and inflammatory responses that occur in response to obesity. In mouse ovarian cells (cumulus–oocyte complexes and granulosa cells) the ER stress marker genes ATF4, GRP78, ATF6 and Xbp-1s were increased in expression (Wu et al., 2010; Wu, unpublished data). ATF4 mRNA was also increased in granulosa cells of obese women compared to granulosa cells of non-obese women (Wu et al., 2010). Both ER stress and oxidative stress are associated with alterations in mitochondrial function (Malhotra and Kaufman, 2007). In oocytes of mice fed obesogenic diets for 4 weeks or 6 weeks mitochondrial membrane potential is dramatically different from that of mice fed control diet (Igosheva et al., 2010; Wu et al., 2010). Oocyte mitochondrial function is important for oocyte developmental competence (Van Blerkom, 2009) and thus such alterations may contribute to the defects in oocyte maturation (Jungheim et al., 2010), fertilization (Wu et al., 2010) and embryo and fetal development seen in mice fed high fat diet (Minge et al., 2008; Igosheva et al., 2010; Jungheim et al., 2010). Oxidative stress and ER stress can also both culminate in cellular apoptosis if not resolved (Breckenridge et al., 2003). In mice fed high fat diet for 4 weeks (Wu et al., 2010) or 16 weeks (Jungheim et al., 2010) there is increased apoptosis in ovarian cells, further indicating that lipotoxicity pathways are induced in the ovary in response to high fat diet-induced obesity. Similar mechanisms appear to be activated in ovaries of obese women since increased numbers of dead cells were observed in follicle fluid of obese women compared to non-obese (Vilser et al., 2010).
linked with systemic inflammation. Analysis of ovarian cells and fluid of obese women indicates that these same mechanisms are activated in the human ovary in response to obesity (Table 1). Studies in mice support this and are able to further dissect the cellular mechanisms by which diet-induced obesity is contributing to changes in mitochondria and the endoplasmic reticulum. That obesity leads to female sub-fertility has long been known; however, very recent work is the first elucidating specific cellular and molecular mechanisms by which this occurs (Fig. 1). Studies in animal models and the translation of the results to humans demonstrate that diet-induced obesity induces multiple components of the lipotoxicity response in ovarian cells and oocytes. Excess lipid is present in follicle fluid of obese women and in the cumulus cells and oocytes of obese mice. Secondly there is an altered inflammatory profile within the ovary during obesity with increased levels of CRP, adipokines and cytokines, potentially due to higher levels in circulation or induction by LOX-1. Oxidative stress responses are clearly activated in the ovary during obesity since there are increased levels of oxLDL and ROS, skewed expression of anti-oxidant enzymes and profound changes in mitochondrial membrane potential. Lastly, we have identified activation of the endoplasmic reticulum stress pathway in ovarian cells of obese mice and women, a response that is likely due to increased lipid content of cells and intricately linked with oxidative stress responses. How each of these components may affect each other and contribute to the overall lipotoxicity responses in ovarian cells is still unclear, but cumulatively this emerging research provides some starting information about the cellular mechanisms that may lead to impaired ovulation and reduced oocyte developmental potential in obese women. Acknowledgements This work was supported by the National Health and Medical Research Council (to R.L.R.) and the Channel 7 Children’s Research Fund (to L.L.W., R.L.R.).
4. Summary
References
Rapidly evolving research clearly demonstrates that obesity is associated with lipid accumulation in nonadipose tissue cells and the induction of oxidative and endoplasmic reticulum stress responses that are tightly
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