by Susan M Gale PhD, V Daniel Castracane PhD, and Christos S Mantzoros MD DSc
Understanding the clinical relevance of leptin as a moderator of reproductive function in obese women
Obesity and the regulation of energy balance have always been highly topical areas of interest, but the recent epidemic of obesity in Western countries has provoked a higher level of research activity amongst the scientific and medical communities worldwide. Obesity is a well recognized risk factor for many health conditions and now ranks as a major factor of causes of death in the U.S. The list of related health disorders is extensive: heart disease, diabetes, cancer, asthma, sleep apnea, arthritis, reproductive complications and psychological disturbances are included as possible health consequences. Sadly, this growing concern is not restricted to the adult population; childhood obesity is increasing at an alarming rate, placing affected children at an increased risk of adult obesity and its attendant health risks. The U.S. Department of Health and Human Services has projected that obesity may replace cigarette smoking as a major health risk in the United States in a few decades.
Figure 1: Leptin is secreted by adipose tissue in proportion to the adipose tissue mass. Leptin circulates in the serum either in the free form or bound to specific binding proteins, the most abundant being the soluble form of the leptin receptor. Leptin acts by binding to the long form of the leptin receptor (mainly in the hypothalamus but also in the ovaries) and is cleared by the short form of leptin receptor which is mainly expressed in the kidneys.
Impaired reproductive function is one of the many possible complications of obesity. For decades, scientists have proposed a link between nutritional status, fat stores and fertility. Studies to date indicate that both over- and under-nutritional states interfere with reproductive function but do so only when extreme. In western societies, obesity appears to be one of the major yet neglected causes of reproductive disorders amongst young women. There is a higher incidence of cycle irregularity, anovulation, infertility, spontaneous miscarriage and dystocia in obese women. While the mechanism through which obesity impairs ovulation and fertility is largely still unknown, it is well established that there is a decreased concentration of sex hormone binding globulin and increased androgen and insulin secretion in overweight individuals. Chronically elevated concentrations of circulating insulin are associated with insulin resistance, and the combination of high androgen and insulin levels to the abnormal localization of body fat. Preliminary clinical trials have demonstrated that drugs that increase insulin sensitivity together with adequate dietary control and weight loss have improved spontaneous ovulation and fertility in obese women. Conversely, women who are extremely underweight also suffer from reproductive dysfunction in addition to osteoporosis and other conditions related to reduced estrogen status.
Introducing leptin: a cytokine with multiple physiological roles
Many years ago it was demonstrated that obesity was regulated at least to some degree by a humoral factor. It took many years to establish that this circulating substance was a hormone produced by adipose tissue, now known as leptin. In 1994, leptin was isolated, purified and its sequence determined by Friedman and his group at Rockefeller Center.
Leptin is a 16kDa protein with the tertiary structure of a cytokine that is highly conserved among mammalian species. It is encoded by the ob gene and is expressed predominantly by adipocytes, although low levels have been detected in the hypothalamus, pituitary, placenta, skeletal muscle, and gastric and mammary epithelia under certain conditions. Leptin circulates either as a free hormone or bound to specific binding proteins, the most abundant of which is the soluble extracellular portion of the long form of the leptin receptor. The long form of the leptin receptor is expressed mainly in the hypothalamus, but leptin receptors have also been identified in gonadotrope cells of the anterior pituitary, endometrium, Leydig cells of the testis, and the granulosa, theca and interstitial cells of the ovary. The short form of the leptin receptor is expressed mainly in the kidneys and lungs, and is responsible for leptin clearance from the circulation (see Figure 1).
As mentioned above, the soluble circulating form of the leptin receptor serves as the circulating binding protein for leptin, a situation analogous to the growth hormone binding protein. Both the leptin and growth hormone binding protein represent the extracellular domain of the membrane receptor, which circulate in the serum and bind leptin and growth hormone respectively. The leptin soluble receptor seems to be the major if not exclusive circulating binding protein for leptin, and therefore an accurate understanding of its function and circulating levels is required to better evaluate the relevance of this binding protein and leptin bioavailability in various physiological and disease states.
Leptin and energy status
Adipose tissue and plasma leptin concentrations correlate with the amount of energy stored as fat, and obese individuals express higher levels of leptin than do lean individuals. Not surprisingly, a positive correlation between leptin and body mass index (BMI) has been well documented by numerous scientific groups around the world. In addition, leptin levels are dependent on the state of energy balance, such that fasting results in a decrease in leptin while overfeeding increases leptin within hours in rodents and within days in humans.
While leptin functions as an anti-obesity hormone in rodents, it is clear that elevated leptin levels in humans do not prevent diet-induced obesity. Commonly, most cases of obesity are characterized by increased adipocyte leptin content and high circulating leptin levels, and the administration of exogenous leptin has generally failed to reduce adiposity significantly in humans. Thus, it has been suggested that the lack of response to high levels of leptin in obesity represents a state of leptin resistance. The pathogenesis of leptin resistance is currently under intense investigation.
Leptin: the “unexpected” reproductive hormone
Early studies designed to investigate the role of leptin in obesity and appetite regulation also gave rise to an early appreciation of the relationship between reproductive function and body energy reserves. It is now suggested that leptin may serve as the critical link between the body’s adipose tissue and the hypothalamo-pituitary axis, indicating whether adequate energy stores are sufficient to support normal reproductive function and fecundity.
The obese ob/ob genetic mouse model identified in the 1950s is known to be deficient in leptin. The phenotype is significantly obese due to uncontrollable feeding behaviour, and reproductive function is greatly impaired. Males and females are sterile, although on occasions, the ob/ob male can reproduce if maintained on a restricted diet. Notably, the sterility of females cannot be reversed with dietary control, but may be restored with the administration of exogenous leptin ). This latter observation in the 1990’s was the first indication of a broader role for leptin in normal reproductive function, and paved the way for further studies examining the relationship between obesity and compromised reproductive function.
Following these exciting initial findings in the ob/ob mouse strain, it was found that leptin administration could enhance the timing of the onset of puberty in a number of animal species. While the role of leptin in sexual development has not been fully defined, current evidence from human and rodent studies support the hypothesis that leptin plays a permissive rather than causal role in the timing of puberty. It would appear that when body energy reserves rise above a critical level, circulating leptin increases to a threshold concentration signaling the central nervous system that the body can support sexual function and procreation. In humans, the observation that obese girls reach menarche at an earlier age compared to normal weight girls is consistent with this hypothesis, and underlines the importance of leptin in normal reproductive development and function). Further studies have shown that increasing circulating leptin levels are observed immediately before a child enters puberty, as evidenced by increasing gonadal hormones.
In periods of starvation when fat stores are depleted, it is proposed that low leptin levels cause the reduction in pituitary LH, resulting in an anovulatory state. Amenorrhea may occur in anorexia nervosa, bulimia nervosa, and in elite women athletes. In all of these conditions, serum leptin levels have been found to be low and correlate with body weight and percentage body fat
At the other extreme of body weight, obesity is also associated with reproductive dysfunction.
It has also been shown that increasing obesity is associated with increasing frequency of anovulatory cycles and increased numbers of atretic follicles. These findings are consistent with the observed direct inhibitory action of high leptin levels on ovarian steroidogenesis leading to ineffective follicular maturation. It has been proposed that the serum leptin levels of obese women may contribute to reproductive function when still in the physiological range but may result in reproductive dysfunction when at supraphysiological levels. Leptin regulates reproduction by acting at different levels of the hypothalamic-pituitary-gonadal axis: (1) a central effect of increasing leptin levels (i.e. physiological levels of leptin signal the onset of early menarche, whereas supraphysiological leptin levels are accompanied by resistance to the response of the gonadotropes to GnRH); (2) a peripheral effect (i.e. higher leptin levels have an inhibitory effect on ovarian function leading to anovulation) (Moschos et al, 2002).
Leptin: clinical relevance in reproductive medicine
The clinical relevance of leptin in reproductive medicine is only just becoming apparent, and thus further research is required before this “new” reproductive hormone achieves routine clinical status in reproductive endocrinology.
There are a number of clinical conditions for which exogenous recombinant leptin is being considered as a potential therapeutic treatment. In states of negative energy balance such as anorexia nervosa, leptin replacement therapy is being evaluated as a potential therapeutic to normalize various neuroendocrine processes including reproductive function. It has recently been shown that the administration of leptin replacement therapy to normal subjects who have been fasted for three days restores the starvation induced changes of LH pulsatility and testosterone levels. Administration of exogenous leptin to other low leptin states are also being investigated for potential pharmaceutical value.
There are several potential clinical situations in which the measurement of leptin and its soluble extracellular receptor may offer diagnostic value in the future. Brzechffa and colleagues in 1996 reported that leptin is increased in women with polycystic ovarian syndrome (PCOS), and this finding was later confirmed by Vicennati and colleagues in 1998. To assess the potential contribution of leptin to the pathogenesis of PCOS, Mantzoros and colleagues in 1997 measured the leptin levels in 24 obese women with PCOS and 12 weight- and age-matched controls. Leptin concentrations were not significantly different between PCOS patients and controls. As obesity is a predisposing factor to PCOS, it would appear that the elevation in leptin in PCOS patients is merely related to increased adiposity and not due to an important process in the pathogenesis of the disease.
Leptin has been studied as a diagnostic tool for IVF applications. Brannian and colleagues in 2001 reported that maternal circulating leptin concentrations, particulary relative to BMI, was highly predictive of IVF outcomes. In a study of one hundred and seventy-two IVF cycles, IVF outcomes such as fertilization, embryo development, implantation and pregnancy were analyzed relative to baseline (i.e., the day gonadotrophin stimulation was initiated) non-fasting serum leptin concentrations and BMI. It was found that serum leptin correlated with BMI as expected, and that serum leptin negatively correlated with pregnancy success whereas BMI did not correlate with pregnancy outcome. Furthermore, Brannian found that women with a low leptin:BMI ratio had significantly more superior quality embryos on day 3 post-retrieval and a greater implantation rate than women with a high leptin:BMI ratio.
Studies in women with PCOS undergoing IVF treatment also indicate that leptin may prove to be a valuable predictive marker of IVF outcomes. In a study undertaken by Mantzoros and colleagues, leptin concentrations were measured in women who succeeded in becoming pregnant within three cycles of in-vitro fertilization (IVF) or gamete intra-fallopian transfer, in women who failed to become pregnant within three cycles, and in women with polycystic ovarian syndrome (PCOS). After adjustment for age and BMI, women with PCOS who became pregnant tended to have lower mean follicular fluid leptin concentrations than women with PCOS who did not succeed at becoming pregnant. Further studies are needed to assess whether the prognostic role for follicular fluid leptin in human reproduction is independent of other factors, and to elucidate the underlying mechanisms.
New hormones, new horizons
The explosion of interest in obesity and a new respect for adipose tissue as an “endocrine organ” have resulted in the identification of a variety of new hormones related to energy homeostasis. Several hormones have been found to be expressed and secreted predominantly by adipose tissue, including adiponectin and resistin.
Adiponectin is a hormone whose expression and secretion are decreased in obesity and/or type 2 diabetes. It has been found that adiponectin improves insulin resistance mainly by decreasing triglyceride content in muscle and liver in obese mice and this effect results from increased expression of molecules involved in both fatty-acid combustion and energy dissipation in muscle. In a separate study, a single injection of adiponectin was found to lower blood glucose levels in obese and diabetic mice, and this effect was not achieved through increasing insulin levels. Taken together, these results indicate that the replenishment of adiponectin might prove a novel treatment modality for insulin resistance and type 2 diabetes.
Adiponectin has recently been evaluated as a potential risk assessment biomarker for endometrial cancer. Given that obesity is an established risk factor for endometrial cancer and that adiponectin is a hormone secreted exclusively by adipocytes, Petridou and colleagues investigated the association of adiponectin with the occurrence of endometrial cancer. Among women younger than 65 years of age, a significant inverse association was found between circulating adiponectin levels and endometrial cancer risk. This study is the first of its kind to directly link insulin resistance with a reproductive disorder, and thus future studies should be undertaken to establish the role of insulin resistance and related hormonal regulators in the pathogenesis of various diseases of the reproductive system.
Resistin is yet another peptide hormone that has been implicated in the development of insulin resistance. Studies in laboratory rodents indicate that resistin causes tissues to become less sensitive to the action of insulin, which is the hallmark of type 2 diabetes. Studies on circulating resistin in human subjects have not yet been published and consequently a role in human energy homeostasis remains to be determined. It is interesting that adiponectin and resistin have also been demonstrated in the placenta, so that again a link between energy balance and reproductive function has been established.
A discussion regarding energy homeostasis would not be complete with mention of Ghrelin. Produced mainly by the fundus of the stomach, Ghrelin is a known growth hormone secretagogue, and also one of the most important stimulants of appetite. It has been described as the “couch potato” hormone, stimulating metabolic effects opposite to those of leptin. Ghrelin stimulates food intake, enhances the use of carbohydrates and reduces fat utilization, increases gastric motility and acid secretion, and reduces locomotor activity. Analogous with the other hormones discussed above, ghrelin is also expressed in the placenta indicating a potential role in reproductive function.
Undoubtedly, there is an endocrinological basis to obesity. As it would appear that the regulation of appetite and energy expenditure has an impact on reproductive function, it is possible that the newly identified energy homeostasis panel of hormones will prove to be clinically useful in the diagnosis and management of various reproductive disorders.
Susan M Gale PhD, and V Daniel Castracane PhD are affiliated with diagnostic Systems Laboratories in Webster, Tex.; Christos S Mantzoros MD DSc is at the Beth Israel Deaconess Hospital in Boston, Mass.
