Review 74 www.thelancet.com Vol 366 July 2, 2005 Introduction In 1994, the discovery of leptin fundamentally changed our perspective of adipose tissue from that of an inert energy store to a true endocrine organ that secretes metabolically active hormones. Leptin was identi?ed as the hormone whose absence resulted in morbid obesity in the ob/ob mouse, 1 thus acquiring its name from the Greek word ?leptos? (thin). Although initially hopes were high that leptin would prove important in the pathophysiology and thus treatment of human obesity, early studies quickly showed that human obesity is generally not associated with leptin de?ciency due to a leptin gene defect, 2 a ?nding not altogether surprising in view of the complex, multifactorial nature of obesity. It was also soon realised, however, that leptin might be more important at the other end of the energy homoeostasis spectrum?ie, energy deprivation rather than obesity. In this context, studies in mice 3 and also in people 4 have shown that leptin has a role in the neuroendocrine adaptation to starvation, which includes changes in hormone concentrations that probably have a protective effect. These ?ndings are clinically relevant for common disease states associated with low leptin concentrations and neuroendocrine abnormalities?ie, energy-de?cient states such as exercise-induced amenorrhoea, non- athletic forms of hypothalamic amenorrhoea, and anorexia nervosa. We review the role of leptin in neuroendocrine function, the reproductive and other neuroendocrine abnormalities associated with these energy-de?cient conditions, and the evidence that low leptin concentrations could play a part in their pathophysiology and potentially their treatment. We also discuss the clinical relevance of these syndromes with respect to effect on fertility and skeletal health. Leptin physiology Leptin is a 167 aminoacid protein product of the obgene that was discovered in 1994 through positional cloning in the ob/ob obese mouse, a model of morbid obesity resulting from absence of leptin due to a gene mutation. 1 The tertiary structure of leptin suggests that it belongs to the cytokine family. Leptin is expressed mainly in white adipose tissue, but also in stomach, placenta, and the mammary gland. 5 Leptin circulates in the serum in a free form or bound to leptin-binding proteins, and the sum of free and bound leptin (ie, total leptin) is the generally accepted standard of measurement. Like other hormones, leptin is secreted in a pulsatile way and has a substantial diurnal variation with an increase of about 50% in the late evening and early morning hours that might be related to an intrinsic circadian component, meal timing, and the sleep-wake cycle. 6?8 Leptin concentrations correlate with the amount of fat mass, with higher amounts in more obese people (?gure 1). 2 Although the amount of fat is an important determinant of leptin concentrations, other factors are also relevant, including sex, adipose tissue-speci?c factors such as adipocyte size and visceral versus subcutaneous fat distribution, other hormones (eg, insulin, glucocorticoids) and cytokines (eg, tumour necrosis factor H9251, interleukin 1). 6,9?11 Importantly, women have higher leptin concentrations than men even after Lancet 2005; 366: 74?85 Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Stoneman 816, Boston, MA 02215, USA (JLChanMD, ChristosSMantzorosMD) Correspondence to: DrChristosSMantzoros email@example.com Role of leptin in energy-deprivation states: normal human physiology and clinical implications for hypothalamic amenorrhoea and anorexia nervosa Jean L Chan, Christos S Mantzoros Leptin is an adipocyte-secreted hormone that plays a key part in energy homoeostasis. Advances in leptin physiology have established that the main role of this hormone is to signal energy availability in energy-de?cient states. Studies in animals and human beings have shown that low concentrations of leptin are fully or partly responsible for starvation-induced changes in neuroendocrine axes, including low reproductive, thyroid, and insulin-like growth factor (IGF) hormones. Disease states such as exercise-induced hypothalamic amenorrhoea and anorexia nervosa are also associated with low concentrations of leptin and a similar spectrum of neuroendocrine abnormalities. We have recently shown in an interventional, proof-of-concept study that leptin can restore ovulatory menstrual cycles and improve reproductive, thyroid, and IGF hormones and bone markers in hypothalamic amenorrhoea. Further studies are warranted to establish the safety and effectiveness of leptin for the infertility and osteoporosis associated with hypothalamic amenorrhoea, and to clarify its role in anorexia nervosa. Search strategy and selection criteria We searched MEDLINE for articles relating ?leptin? to ?hypothalamic-pituitary-peripheral axes?, ?neuroendocrine function?, ?hypothalamic amenorrhea?, ?exercise-induced amenorrhea?, and ?anorexia nervosa?, with emphasis on articles on human beings. Review www.thelancet.com Vol 366 July 2, 2005 75 adjusting for body-mass index, which may be due to differential body-fat distribution or the effects of sex steroids. 12,13 Finally, acute caloric deprivation for 2?3days results in a large decrease in leptin concentrations to roughly 20?30% of baseline, before major changes in bodyweight or fat mass have arisen. 4,9 Leptin: hormone for feast or famine? The neuroendocrine perspective Obesity: a high-leptin state Leptin acts as a signal from the periphery to the brain, conveying information about the amount of energy available in adipose tissue or acute changes in energy availability (or both). Although leptin was originally discovered as an anti-obesity hormone, because congenital absence of leptin causes morbid obesity, the hope that leptin de?ciency would play a part in the cause of common obesity in people has not been borne out. Rare cases of functional leptin de?ciency due to mutations in the leptin or leptin receptor genes have been reported in human beings; children with leptin de?ciency due to a leptin gene mutation are healthy at birth but develop morbid obesity in early childhood and are responsive to leptin treatment. 14?16 It has also been proposed that heterozygotes for leptin gene mutations have relatively low concentrations of leptin (relative to their amount of fat mass) as the main cause of their obesity, 17 but further studies are needed to determine whether such individuals could bene?t from exogenous leptin administration and to clarify the clinical relevance of this genetic variation in several populations of obese people. Almost all human obesity, however, is associated with apparent resistance to leptin, with high circulating concentrations of leptin in more obese subjects. 2 The notion was supported by a clinical trial of recombinant human leptin (r-metHuLeptin) for obesity 18 in which weight loss was only moderate (an average of 0·7?7·1 kg at 24 weeks depending on dose) in response to administration of r-metHuLeptin. Since there was considerable intersubject variability in the weight loss in this trial, 18 and since obesity in general might be associated with relative, rather than absolute, resistance to leptin, further studies are warranted to clarify the subset of obese individuals for whom r-metHuLeptin treatment would achieve the greatest weight loss. Energy deprivation: a low-leptin state By contrast with the ?nding that leptin concentrations increase gradually over time with increases in fat mass, such concentrations are remarkably sensitive to acute energy deprivation. 4 Serum leptin concentrations fall rapidly in response to complete fasting and out of proportion to changes in fat mass, 4,9 suggesting that this hormone plays a more important part in the physiology of energy de?cit rather than energy excess (ie, obesity). Starvation also elicits changes in several neuroendocrine axes that can be thought of as protective from a teleological standpoint: decrease in reproductive hormones to limit procreation; fall in thyroid hormones to conserve metabolism; increase in stress hormones (cortisol) to mobilise needed energy stores; and rise in growth hormone with a decrease in insulin-like growth factor-1 (IGF-1)?ie, a state of decreased energy expenditure for growth-related processes while enabling growth hormone to increase use of alternative fuels through lipolysis. These neuroendocrine alterations have adaptive value by mobilising needed energy stores and diverting limited resources towards important physiological processes and away from energy- consuming processes that are not so essential for immediate survival (eg, reproductive function). Because energy deprivation results in changes in neuroendocrine axes as well as low leptin concentrations, we postulated that falling leptin concentrations might mediate the neuroendocrine response to fasting. We have tested this hypothesis in mice and people by measuring hormone concentrations in response to fasting alone or fasting with administration of replacement-dose leptin to restore serum concentrations of this hormone and comparing these responses to a control fed state. These studies 3,4 indicate that low leptin concentrations are important in signalling energy de?cit to the hypothalamic-pituitary axes, whereas high leptin concentrations in obesity are associated with resistance to the catabolic effect of leptin to suppress appetite and heighten energy expenditure (?gure1). Leptin ? ? +/? + + ? Reproductive hormones Thyroid hormone IGF-1 Adrenal Leptin Energy homoeostasis Neuroendocrine function Energy deficiency Energy excess GnRH TRH CRH NPY/AGRP MCH H9251-MSH ? Figure 1: Physiology of central effects of leptin in regulating neuroendocrine function and energy homoeostasis in energy de?ciency and energy excess GnRH=gonadotropin releasing hormone. TRH=thyrotropin releasing hormone. CRH=corticotropin releasing hormone. NPY=neuropeptide Y. AGRP=agouti-related protein. MCH=melanin-concentrating hormone. H9251-MSH=H9251-melanocyte-stimulating hormone. Review 76 www.thelancet.com Vol 366 July 2, 2005 Leptin and the hypothalamic-pituitary- peripheral endocrine axes Hypothalamic-pituitary-gonadal axis Leptin acts by binding to speci?c leptin receptor isoforms (one long and several short), which are widely distributed in many tissues. 5 The long isoform is expressed abundantly in the hypothalamus and activates mainly the Janus kinase signal transducer and activator of transcription system to change the expression of hypothalamic neuropeptides and thus regulate energy homoeostasis. 19,20 Leptin receptors have also been identi?ed at all levels of this axis, including anterior pituitary, 21 ovary, 22 and endometrium. 23 Converging lines of evidence from in-vitro studies and models of leptin de?ciency in animals and man suggest that leptin has an important role in reproduction and regulation of the hypothalamic- pituitary-gonadal axis. 24 In vitro, leptin stimulates gonadotropin-releasing hormone pulsatility and release. 25,26 Leptin-de?cient ob/ob mice are not only morbidly obese but also sterile, and leptin treatment (but not weight loss alone) corrects the sterility of these mice. 27,28 Food deprivation reduces testosterone concentrations in normal male mice and prolongs the onset of vaginal oestrus in female mice, whereas exogenous leptin administration restores the decline in testosterone and luteinising hormone (LH). 3 Rare cases of functional leptin de?ciency in people due to mutations in the leptin or leptin receptor gene have also provided valuable insights. These cases include two adult women with amenorrhoea, an adult man who had never entered puberty, 14,16,29 and three adolescent sisters with low gonadotropin concentrations and no evidence of pubertal development. 30 In a 9-year-old leptin- de?cient child, leptin-replacement therapy for 12 months resulted not only in substantial loss of fat mass, but also the development of a pulsatile nocturnal pattern of gonadotropin secretion consistent with early puberty 15 that progressed to normal LH and follicle- stimulating hormone (FSH) pulsatility with continued replacement therapy. 31 In another report, 32 long-term physiological leptin replacement for 18months in three leptin-de?cient, hypogonadal adults resulted in clinical signs of puberty and increases in LH pulsatility and testosterone levels in a 27-year-old man and development of ovulatory menstrual cycles in two adult women with luteal phase defects. 32 Similarly, in healthy women, ultradian ?uctuations in leptin concentrations are synchronous with both LH and oestradiol ?uctuations. 33 Our observational studies in boys have shown a rise in leptin concentrations before onset of puberty. 34 Moreover, in healthy men, short-term fasting for 2?5 days signi?cantly decreased mean LH concen- trations, LH pulse frequency, and serum testosterone concentrations, 35,36 an effect that is probably due to decreased hypothalamic pulses of gonadotropin- releasing hormone. 37 To establish whether these fasting-associated falls in reproductive hormone concentrations are mediated by low leptin concentrations, we studied lean men in three conditions: baseline fed state, fasting alone, or fasting with administration of replacement-dose r- metHuLeptin designed to restore serum leptin to physiological fed-state concentrations. 4 Administration of replacement-dose r-metHuLeptin during fasting fully prevented the starvation-induced decrease in LH pulsatility and testosterone concentrations in these men. 4 These ?ndings indicate that leptin has an important role in the physiology of reproduction and that falling concentrations during short-term energy deprivation in healthy men are responsible for the food deprivation-induced decline in reproductive hormones. Hypothalamic-pituitary-thyroid axis An important interaction between leptin and this axis has also been shown. Leptin increases release of thyrotropin-releasing hormone in hypothalamic explants from fasted rats. 38 Leptin administration in rodents prevents the fasting-induced suppression of prothyrotropin-releasing hormone mRNA in para- ventricular nucleus neurons, 39 reverses the inhibitory effect of food deprivation on spontaneous pulsatile thyroid-stimulating hormone (TSH) secretion, 40 and partly blunts the decline in thyroxine concentrations associated with 2-day starvation. 3 In healthy men, leptin and TSH rhythms show a similar 24-h pattern of variability with substantial pattern synchrony of ultradian ?uctuations, a pattern that is impaired in leptin-de?cient individuals. 41 Interestingly, people with a mutation in the leptin receptor and functional leptin de?ciency have evidence of hypothalamic hypothyroidism with low thyroxine, normal basal TSH, and sustained TSH response to thyrotropin-releasing hormone. 30 Leptin administration increased free thyroxine (T4) and free tri-iodothyronine (T3) concentrations in three leptin- de?cient children, 31 and reversed the decreased T4 and T3 concentrations in four healthy individuals on a long- term hypocaloric diet. 42 Finally, in healthy lean men, leptin replacement during acute leptin de?ciency induced by fasting signi?cantly partly reversed the fasting-induced decrease in TSH pulsatility and increased free T4 concentrations within the normal range, indicating that suppression of TSH associated with fasting is mediated by leptin and that leptin could have effects on free thyroxine concentrations. 4 Hypothalamic-pituitary-adrenal axis In mice, acute starvation 3 and stress induced by hind leg restraint increases corticosterone and adrenocorti- cotropin concentrations, whereas exogenous leptin administration reverses the activation of this axis. 3,43 This effect appears to be mediated by inhibition of hypothalamic corticotropin releasing hormone based on Review www.thelancet.com Vol 366 July 2, 2005 77 studies in rat hypothalamic explants and cultured pituitary cells. 43 In human beings, an inverse relation has been established between ?uctuations in leptin, adrenocorticotropin, and cortisol independent of glucocorticoid effects on leptin, 6 but people with mutations in the leptin or leptin receptor gene seem to have normal adrenal function. By contrast with ?ndings in mice, fasting-induced changes in the hypothalamic- pituitary-adrenal axis as well as the renin-aldosterone axes might be independent of leptin in humans. 4 Leptin- de?cient individuals had elevated basal cortisol and adrenocorticotropin concentrations, but normal urinary free cortisol and response to dexamethasone suppression. 16 Similarly, investigation of the hypothalamic-pituitary-adrenal axis in those with functional leptin de?ciency due to a mutation in the leptin receptor gene did not reveal any abnormalities. 30 Finally, in an uncontrolled study of leptin administration for 18 months to three leptin-de?cient adults, leptin resulted in increased 24-h mean cortisol concentrations, 32 by contrast with the hormone?s failure to lower cortisol concentrations during acute fasting in lean men. 4 Since these discrepancies might re?ect differences in study duration (acute vs chronic leptin de?ciency) or possible confounding by exogenous factors in the long-term uncontrolled study on leptin- de?cient adults, more detailed and controlled studies are needed to fully explore this topic. Hypothalamic-pituitary-growth hormone-IGF-1 axis Leptin receptors are present in normal pituitary tissue and pituitary adenomas, 44 and in vitro leptin increases stimulated secretion of growth hormone from pituitary cells. 45,46 Exogenous administration of leptin to fasted mice fully prevents suppression of both growth hormone and IGF-1 concentrations and partly corrects the fasting-induced suppression of growth hormone releasing hormone mRNA expression. 47 Leptin-de?cient people had decreased growth hormone response to insulin-induced hypoglycaemia and exercise tests, but normal ?nal heights, 16 whereas long-term leptin replacement increased concentrations of IGF-binding protein-1 (IGFBP-1) and IGBGP-2 (but not IGF-1 or IGFBP-3) in a small, uncontrolled study. 32 In addition to decreased secretion of growth hormone and low IGF-1 and IGFBP-3 concentrations, individuals with functional leptin de?ciency due to a leptin receptor mutation had a mild but substantial growth delay during early childhood. 30 In healthy lean men, leptin replacement during acute fasting partly prevented the fall in total IGF-1 but not free IGF-1 concentrations during fasting, with no apparent effect of leptin on IGFBP 1, 2, or 3. 4 These discrepancies, which need to be assessed in more detail, could again re?ect differences in study duration or the uncontrolled nature of the study on leptin- de?cient adults, which found improvement in IGF binding proteins. 32 Pathophysiology of leptin in energy deprivation states Exercise-induced amenorrhoea and other neuroendocrine abnormalities Menstrual abnormalities arise commonly in association with intensive exercise and athletic sports. The reported frequency of amenorrhoea in female athletes varies widely from a few percent up to 50%, with a positive correlation with training intensity. 48?50 Additionally, independent of training intensity, the type of activity is important with long-distance runners, gymnasts, and ballet dancers (sports that favour a leaner physique) having a particularly high rate of amenorrhoea (40?50%) compared with sports such as swimming (12%). 49,51?54 These differences could be attributable to the higher percentage of body fat in swimmers compared with runners and ballet dancers, 52 which lends support to the hypothesis proposed by Frisch 55 that integrity of menstrual function depends critically on a certain percentage of body fat (roughly 22%). Even athletes with so-called regular menstrual cycles can have impaired integrity of the hypothalamic-pituitary-gonadal axis, with reduced pulsatile LH frequency and amplitude and luteal phase defects. 56?58 Hypothalamic amenorrhoea The amenorrhoea that occurs in association with exercise in the absence of organic disease or ovarian failure is called hypothalamic amenorrhoea. This condition arises when impaired secretion of gonadotropin-releasing hormone (low amplitude, low frequency, or most commonly both) leads to low or normal gonadotropin concentrations, low oestrogen concentrations, and subsequently the absence of menstrual cycles. 59 Interestingly, the derangement in pulse secretion of gonadotropin-releasing hormone is often associated with night-time augmentation typical of prepuberty or peripuberty. 60,61 Thus, hypothalamic amenorrhoea can be deemed to represent a regression to a prepubertal or peripubertal pattern of gonadotropin secretion (?gure2). Compelling evidence suggests that relative energy de?cit (chronic low energy availability related to high levels of energy expenditure, LH FSH FSH LH Gonads GnRH Prepuberty Adulthood Hypothalamic amenorrhoea Figure 2: LH and FSH pulsatility in prepuberty, adulthood, and hypothalamic amenorrhoea GnRH=gonadotropin-releasing hormone. Review 78 www.thelancet.com Vol 366 July 2, 2005 insuf?cient nutritional intake, or both) plays an important part in the cause of deranged gonadotropin- releasing hormone secretion and resulting reproductive dysfunction, 52,62 but the factor responsible for this abnormality remained elusive until it was postulated that lower leptin concentrations might be responsible for initiation of an adaptive response to an energy-deprived state. 3 Other neuroendocrine abnormalities In addition to disturbances in the hypothalamic- pituitary-gonadal axis, women with exercise-induced amenorrhoea have abnormalities in other neuroendocrine systems, speci?cally thyroid, cortisol, and IGF hormones. 61,63?65 Amenorrhoeic athletes have lower T4 and T3 concentrations than women who cycle regularly and were similarly athletic, 66,67 and their leptin concentrations were signi?cantly correlated with total T4 and T3 concentrations. 66 The hypothalamic- pituitary-adrenal axis is also altered in this setting. Not unexpectedly, athletic women with hypothalamic amenorrhoea have hypersecretion of cortisol with elevated 24-h urinary cortisol and serum cortisol concentrations. 54,56,68?71 The relative hypersecretion of cortisol is associated with normal adrenocorticotropin secretion and a lessened response to corticotropin releasing hormone, suggesting an increased adrenal sensitivity to adrenocorticotropin and activation of the hypothalamic-pituitary-adrenal axis. 56,72 Finally, exercise-induced amenorrhoea is also associated with alterations in the growth hormone?IGF-1 axis, including increased secretion of growth hormone and decreased IGF-1. 63,65,73,74 These ?ndings are consistent with resistance to growth hormone, which is typical of an energy-de?cient state, and might affect bone health and metabolism, since IGF-1 is a nutritionally regulated bone trophic hormone. Non-athletic functional hypothalamic amenorrhoea Hypothalamic amenorrhoea also arises in weight- stable, non-athletic women (often termed functional hypothalamic amenorrhoea), and has generally been thought of as psychogenic in origin with an important contribution from stress. 52,61 The frequency of this condition is reported to range from 8·5% in women aged 13?18 years (with 3 months of amenorrhoea) to 7·6% in women aged 15?24 years, 3·0% in women aged 25?34 years, and 3·8% in women aged 35?44 years. 75 A common perception is that women with this condition tend to be highly motivated, intelligent, and involved in high-stress occupations. 76 It has also been proposed that functional hypothalamic amenorrhoea might develop in the setting of stressful life events or increased vulnerability to stress, or both. 77 Small studies have reported certain speci?c attitudes (rigid, perfectionistic) in women with this condition compared with women with organic amenorrhoea or eumenorrhoeic controls, 78 but data are con?icting with regard to depressive symptoms. 65,78 Finally, no inciting factors can be identi?ed in many cases. 61 Although women with non-athletic forms of hypothalamic amenorrhoea might have normal bodyweight and body-mass index, several studies, 65,73,79 but not all, 80 have shown that these women have a lower percentage of body fat than regularly cycling control women?ie, lower than the normal range for women. 81 Furthermore, it is increasingly recognised that relative energy de?cit due to subclinical nutritional de?ciencies can play a part in the genesis of this disorder. Although overt eating disorders or excessive exercise may not be present, women with hypothalamic amenorrhoea score higher on eating disorder questionnaires and have decreased caloric (particularly fat) intake, increased ?bre intake, higher aerobic activity, or a combination of these characteristics. 65,80,82 These women might not meet the criteria for anorexia nervosa with psychiatric pathology of disturbed body image and fear of eating (see below), but this type of restrictive eating pattern, particularly fat restriction (albeit less severe than anorexia nervosa), can result in subclinical nutritional de?cits that are relevant to the low fat mass and, especially, the low leptin concentrations associated with this condition. Thus, these women can be judged to have energy de?cit as well, which is related to decreased energy intake with or without increased energy expenditure. Consistent with this notion, non-athletic women with this condition have an array of neuroendocrine and metabolic abnormalities similar to women with exercise-induced amenorrhoea, including decreased gonadotropin-releasing hormone pulsatility and oestradiol concentrations, lower thyroid hormone concentrations, increased secretion of growth hormone with decreased IGF-1, and higher cortisol concentrations. 61,64,65,69,73,79,83 Body-mass index (kg/m 2 ) 12 0 5 10 15 20 25 30 14 16 18 20 22 24 26 Leptin ( H9262 g/L) Hypothalamic amenorrhoea (n=14) Anorexia nervosa (n=11) Normal control women (n=34) Figure 3: Serum leptin concentrations in relation to body-mass index in normal controls, women with hypothalamic amenorrhoea, and women with anorexia nervosa at baseline and after nutritional treatment for weight restoration 91,106 Review www.thelancet.com Vol 366 July 2, 2005 79 Anorexia nervosa Hypothalamic amenorrhoea is also one of the hallmarks of anorexia nervosa, a disorder characterised by dis- turbed body image, intense fear of obesity, and severe restriction of food intake resulting in loss of bodyweight to less than 85% of that expected for age and height. 84 The prevalence of anorexia nervosa has been reported to range from 0·5 to 2 per 1000 women. 84,85 This condition is essentially a self-imposed state of starvation and thus leads to neuroendocrine disturbances that are characteristic of energy de?cit (reduced oestrogen and thyroid hormones, increased growth hormone, reduced IGF-1, and increased cortisol) but are more severely abnormal. 84,86 Not surprisingly, this condition is associated with signi?cant morbidity and mortality, with an estimated mortality rate of 2?10% over 5?10years. 84 Role of leptin in hypothalamic amenorrhoea and anorexia nervosa Amenorrhoeic female athletes have lower serum leptin concentrations than weight-matched controls 87 and eumenorrhoeic athletes, 66 as well as a striking absence of the normal diurnal pattern of leptin concentrations in athletes with normal menstrual cycles (?gure 3). 68 By contrast, the diurnal pattern of leptin concentrations is preserved in women with functional hypothalamic amenorrhoea. 65 The reduced leptin cannot be fully accounted for by differences in percentage body fat, 68 and non-athletic women with the condition also have lower leptin concentrations than regularly cycling women matched for bodyweight and fat mass. 73,79,80 Similarly, serum leptin concentrations in women with anorexia nervosa are lower than those of normal-weight controls as a result of decreased bodyweight and fat mass, 88?101 with a strikingly reduced diurnal variation of leptin concentrations. 95,96 Additionally, patients with anorexia nervosa have lower concentrations of leptin in cerebrospinal ?uid and a higher ratio of cerebrospinal ?uid to plasma leptin than healthy controls. 91 Interestingly, concentrations of the soluble leptin receptor, which are the main binding protein for leptin, are also raised in patients with anorexia, 102?105 resulting in an even lower free leptin index and suggesting a role for leptin binding proteins in the regulation of energy homoeostasis. Soluble leptin receptor concentrations increase in response to refeeding in some studies 104,105 but not all. 103 In view of the ?nding that leptin concentrations are low in hypothalamic amenorrhoea, the accompanying neuroendocrine abnormalities characteristic of a low- leptin state, and the research supporting an important role for leptin in reproductive and neuroendocrine function, we tested the hypothesis that low leptin concentrations could be directly responsible for these reproductive and hormonal abnormalities by administering r-metHuLeptin at replacement doses to women with hypothalamic amenorrhoea related to strenuous exercise or low weight. 106 We measured the effect of returning serum leptin concentrations to normal on ovulation, follicle growth, gonadotropin- Maximum f o l licle diamet er (mm) 0 0·5 1·0 1·5 2·0 2·5 3·0 3·5 Baseline (-1 mo) Baseline (0) Leptin Rx Number of dominant f o l licles * * Ov arian v olume ( c m 3 ) Baseline (-1 mo) Endometrial thicknes s (mm) * Baseline (0) Leptin Rx * 0 5 10 15 20 25 30 0 1 2 3 4 5 6 7 8 9 0 5 10 15 20 25 Figure 4: Improvement of ovarian and endometrial morphology in women with hypothalamic amenorrhoea (n=8) during treatment with r-metHuLeptin for up to 3 months Adapted from ref 106. Error bars=SE. Baseline (?1 month)=time point just before beginning of baseline observation period and 1 month before initiation of r-metHuLeptin treatment. Baseline (0)=time point just before initiation of r-metHuLeptin. Leptin Rx=maximum value during r-metHuLeptin treatment. *pH110210·05 vsbaseline (0). Review 80 www.thelancet.com Vol 366 July 2, 2005 releasing hormone pulsatility, hormone concentrations (including reproductive, thyroid, IGF-1, and cortisol), and markers of bone formation and turnover. We noted that r-metHuLeptin replacement improved LH pulse frequency and mean concentrations and resulted in ovulation in three of eight women and preovulatory follicle growth and withdrawal bleed in an additional two, with overall improvement in ovarian parameters (including ovarian volume and number of dominant follicles) in all individuals (?gure 4). Figure 5 shows hormone concentrations, ovulation, and menses in a representative woman with hypothalamic amenorrhoea of 6 years? duration who ovulated after 35 days of r-metHuLeptin treatment. This treatment also improved reproductive, thyroid, and IGF hormones and increased markers of bone formation but did not change cortisol, adrenocorticotropin, or bone resorption. 106 The ?ndings from this study suggest that leptin is a peripheral signal of adequate energy stores necessary for normal reproductive and neuroendocrine function. Since low leptin concentrations have an important role in the pathophysiology of hypothalamic amenorrhoea, leptin replacement could become a potential new therapeutic option. Similarly to other hormone-de?cient states such as hypothyroidism or adrenal insuf?ciency, leptin replacement to correct the underlying leptin de?ciency might represent a more physiological treatment for hypothalamic amenorrhoea by correcting not only the oestrogen de?ciency resulting from low gonadotropins, but also other related neuroendocrine defects such as low concentrations of thyroid and IGF-1. Although r-metHuLeptin administration at low physiological doses was not associated with more weight loss than that expected with placebo injections, use of a higher pharmacological dose in the initial proof-of- concept study was associated with a small but signi?cant weight loss of about 2 kg. 106 Thus, additional studies are warranted to establish the ideal dose and duration of r-metHuLeptin necessary to restore reproductive and other neuroendocrine function without inducing an undesirable degree of weight loss in women who are already lean. Dietary treatment for anorexia nervosa results in an increase in serum leptin concentrations as weight recovers. 89,107?112 Concentrations of leptin in cerebrospinal ?uid also increase to normal with dietary treatment but this rise occurs before bodyweight has returned to normal, suggesting a possible mechanism for resistance to weight gain. 91 The rise in leptin with dietary therapy correlates substantially with increasing gonadotropins until gonadotropins peak, indicating that increasing leptin in response to weight gain could activate the hypothalamic- pituitary-gonadal axis. 108 Although patients with anorexia nervosa can recover weight with therapy, this weight recovery is not always associated with resumption of menses. 93 An increase in the free leptin index, however, is associated with resumption of menstrual function but not with weight recovery alone. 104 Since leptin concentrations might be similar in amenorrhoeic and eumenorrhoeic women with anorexia nervosa, it has been suggested that leptin is a necessary but not suf?cient signal for recovery of menstrual function in this condition. 93 Several observational studies have proposed a critical threshold level for leptin of roughly 2 H9262g/L that might be necessary for an increase in LH and thus menses to resume, but this remains to be con?rmed by interventional studies. 93,108,113,114 Therefore, leptin could represent a so-called metabolic gate to gonadotropin secretion. 114 The main treatments for women with anorexia nervosa remain nutritional therapy to return body-weight and body-mass index to normal, which should result in an increase of endogenous leptin concentrations, and oestrogen administration. The time needed to recover bodyweight after nutritional treatment can be substantial, however, and the long-term effectiveness of this method remains to be proven. Additionally, those patients with anorexia nervosa who do recover bodyweight might remain amenorrhoeic for some time and can have dif?culty maintaining the recovered weight. Further studies are needed to determine whether r- metHuLeptin treatment with careful dose titration to avoid weight loss in women with anorexia nervosa who have recovered weight but remain amenorrhoeic will have similar bene?ts to those in exercise-induced or functional hypothalamic amenorrhoea. Clinical relevance: infertility and osteoporosis Hypothalamic amenorrhoea is associated with substantial morbidity in terms of fertility and skeletal health. In developed countries, infertility affects 10?20% of couples. 115 Infertility is attributable to the female partner in 37% of couples and to both partners in 35%. 52 0102030405060708090 Leptin ( H9262 g/L), LH (IU/L), progesterone ( H9262 g/L) 0 50 100 150 200 250 Oestradiol ng/L) Leptin LH Progesterone E2 r-metHuLeptin treatment Ovulation Menses Menses Days after starting r-metHuLeptin treatment 0 10 20 30 40 50 60 Figure 5: Changes in reproductive hormones and occurrence of ovulation and menses in a representative woman with hypothalamic amenorrhoea treated with r-metHuLeptin for 2 months Ovulation occurred on day 36 of treatment (21·4 mm follicle), followed by menses on day 51 and again 2 weeks after end of treatment. 106 Review www.thelancet.com Vol 366 July 2, 2005 81 Ovulatory disorders account for 25% of the disease states causing infertility in women, with the most common cause of anovulation being hypothalamic dysfunction (38%). 52 At the moment, treatment with injectable gonadotropins is expensive, carries a high risk of multiple gestation, and does not address the entire spectrum of pathology. R-metHuLeptin might prove to be a more effective treatment by returning the defects in the hypothalamic-pituitary-gonadal axis to normal and thus correcting the amenorrhoea and infertility. Prolonged amenorrhoea also has a deleterious effect on the skeletal system. 116 Bone density in women with hypothalamic amenorrhoea is greatly reduced at several skeletal sites, including trabecular and cortical bone at both axial and appendicular weight-bearing sites, 117?120 and is associated with an increased risk for developing stress fractures. 116,117,121?125 Bone does not mineralise properly with mechanical stress in this condition, 54 and extra weight-bearing activity does not seem to completely compensate for the effect of amenorrhoea and low oestrogen concentrations on bone loss. 122 Furthermore, amenorrhoea during important years for bone accretion can result in failure to achieve peak bone mass, possibly leading to a life-long increased risk of fractures. 54 In accordance with this notion, osteoporosis is most severe when amenorrhoea begins in adolescence and during prolonged amenorrhoea. 119,120 Osteoporosis is a very common complication of anorexia nervosa, affecting more than 50% of female patients with this condition. 126 Bone density is greatly reduced, especially at the lumbar spine but also at the proximal femur and distal radius. 127,128 This loss can lead to diminished growth and increased risk of kyphosis and fractures as late as 20 to 40 years after diagnosis of anorexia nervosa, 129 since bone density might not return to the normal range for years after recovery. 126 The cause of bone loss in women with hypothalamic amenorrhoea and anorexia nervosa might be related not only to oestrogen de?ciency, but also to other hormonal abnormalities (eg, low serum free testosterone concentrations, higher cortisol concentrations, low IGF-1 levels, low dehydroepiandrosterone concentrations, low leptin concentrations, or some combination). 116,126,130?132 Several studies have shown that leptin directly stimulates bone growth in vitro and increases bone density in leptin- de?cient animals, 133?138 raising the possibility that low leptin contributes to bone loss in hypothalamic amenorrhoea. Central administration of leptin causes bone loss in mice through the sympathetic nervous system, 139,140 a ?nding that might not be directly applicable to human beings since leptin does not activate this system. 4 Observational studies 141?149 and a few anecdotal reports of leptin treatment in people with congenital leptin de?ciency 31 and lipoatrophy 150 have reported con?icting results on the relation between leptin and bone density. Thus, future interventional leptin studies are needed to clarify the role of leptin in bone metabolism in people. Oestrogen replacement is commonly prescribed for patients with hypothalamic amenorrhoea to prevent bone loss, 151 but evidence to support this indication has not been consistent. Although in some studies, oestrogen improved lumbar spine, femoral neck, and/or total bone density, 152,153 low bone density might not be completely reversible by resumption of menses or oestrogen therapy. 154,155 In women with anorexia nervosa and amenorrhoea, oestrogen replacement did not prevent progressive osteopenia, 156 although the combination of IGF-1 and oestrogen resulted in a 2·8% improvement in bone density at the spine compared with placebo in a similar population. 130 Since the pathophysiology of bone loss in hypothalamic amenorrhoea might be different than that for post- menopausal osteoporosis due to the associated neuro- endocrine abnormalities (eg, low IGF-1), or low leptin levels (or both), r-metHuLeptin administration could be a more appropriate and bene?cial treatment for low bone density by correcting the underlying patho- physiology (low oestrogen as well as neuroendocrine abnormalities or possible direct effects of leptin on bone, or both). Conclusion and future directions Much research on leptin, stimulated by intense interest since its discovery in 1994, has transformed and re?ned our understanding of this molecule. From the initial simplistic view of leptin as an adipocyte-derived hormone that acts in a negative feedback loop with the brain to decrease appetite, we now have a far greater although still incomplete understanding of the pleiotropic nature of this hormone, which affects physiological processes as diverse as neuroendocrine, metabolic, immune, and haematopoietic function. Leptin has emerged as the key hormonal mediator of the adaptation to starvation, and in this context regulates several neuroendocrine axes. These important advances in our understanding of leptin physiology have direct clinical relevance for disease states associated with low leptin concentrations and neuroendocrine abnormalities. Hypothalamic amenorrhoea is a common condition associated with relative leptin de?ciency as well as reproductive and neuroendocrine abnormalities typical of a low-leptin state. Results from an interventional leptin study in women with hypothalamic amenorrhoea 106 suggest that low concentrations of leptin, a peripheral signal that energy stores are adequate, is the unifying link underlying these reproductive and neuroendocrine de?cits. Findings from this initial proof-of-concept study not only help to elucidate the pathophysiology of hypothalamic amenorrhoea but may also have important implications for treatment of this condition. The only treatment presently available for women who are not interested in fertility is oestrogen, which has side-effects and does not address other neuroendocrine abnormalities or the underlying infertility. Review 82 www.thelancet.com Vol 366 July 2, 2005 Future larger-scale studies will need to determine the safety and effectiveness of r-metHuLeptin for restoring reproductive function and improving bone metabolism in women with hypothalamic amenorrhoea and also to establish the role of r-metHuLeptin in other low-leptin states such as anorexia nervosa. If future studies con?rm the initial data, r-metHuLeptin could prove a better treatment for hypothalamic amenorrhoea by directly addressing the underlying pathophysiology. An enormous amount of progress has been made in understanding leptin physiology since it was ?rst identi?ed 10 years ago (from in-vitro, animal, and toxicology studies to human physiology and proof-of- concept treatment studies)?an achievement that tends to be the exception rather than the rule in medical research and that highlights the potential for even greater advances in scienti?c knowledge and development of treatment in the upcoming years. 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