Studies in animal models have provided substantial evidence that the starvation-induced fall in leptin levels plays a central role in regulating the neuroendocrine adaptation to starvation (8
), suggesting that the suppression of leptin levels with starvation or food restriction may be of more critical importance than the increases in leptin that occur with overfeeding and obesity (9
). In humans, as in mice, congenital absence of leptin or functional leptin deficiency due to inactivating mutations of the leptin receptor causes severe obesity accompanied by neuroendocrine abnormalities (2
). The nature of the neuroendocrine defects in leptin-deficient rodents and in leptin-deficient or leptin-resistant humans differ in some important respects, however, suggesting that the role of leptin in mediating the neuroendocrine response to starvation may be different in humans versus rodents (2
Although observational studies have reported associations of circulating leptin levels with the levels of several neuroendocrine hormones in healthy humans (13
), to our knowledge no prior interventional studies have been performed to assess the role of changing leptin levels on neuroendocrine function in lean individuals. Our data suggest that a reduction of leptin levels in lean men regulates the acute fasting-induced changes in the hypothalamic-pituitary-gonadal (HPG) axis and, in part, changes in the hypothalamic-pituitary-thyroid (HPT) axis and IGF-1 binding capacity, but it is not responsible for changes in the HPA, renin-aldosterone, and GH-IGF-1 axes associated with acute fasting. Finally, suppression of leptin does not appear to be responsible for the changes in fuel utilization or energy expenditure observed during short-term fasting and tends to contribute to the increased number of calories ingested in the immediate post-fasting period.
In healthy men, fasting significantly decreases serum testosterone levels through changes in pulsatile LH secretion (14
), an effect that may be mediated by decreased hypothalamic GnRH pulses (25
). In this study, we found that leptin replacement during fasting had the most significant effect on the HPG axis, with full restoration of LH pulsatility characteristics and testosterone levels. This indicates that the fall in testosterone with decreased leptin is a result of inadequate LH stimulation of testosterone secretion and is consistent with the hypothesis that a threshold leptin level between 0.5 and 2 ng/ml may be necessary for normal LH secretion (26
). This effect of low leptin on LH pulsatility is most likely secondary to effects on the hypothalamus to influence pulsatile GnRH release, on the basis of in vitro and animal data (27
), although an additional direct effect of leptin on the pituitary cannot be excluded.
Our findings lend further credence to converging lines of evidence from animal models and observational studies in humans indicating an important role of leptin in reproduction (28
). Food deprivation decreases testosterone levels in male mice and prolongs the onset of vaginal estrus in female mice, whereas exogenous leptin administration restores the decline in testosterone and LH levels in fasted normal mice (8
) and corrects the sterility of leptin-deficient ob/ob
). In healthy women, ultradian fluctuations in leptin levels are synchronous with both LH and estradiol fluctuations (24
). Rises in leptin may be associated with the onset of puberty in boys (31
), although the literature is divided on this point (32
). In addition, reproductive dysfunction occurs in humans with leptin deficiency or resistance due to mutations in the leptin gene or the leptin receptor gene, respectively. These rare cases have included two adult leptin-deficient women with amenorrhea, an adult leptin-deficient man who had never entered puberty (3
), and three leptin-resistant adolescent sisters with low gonadotropin levels and no evidence of pubertal development (22
). In one case, intervention with leptin replacement therapy for 1 year in a 9-year-old leptin-deficient child resulted not only in marked loss of fat mass but the development of a pulsatile nocturnal pattern of gonadotropin secretion consistent with early puberty (6
) that progressed to normal LH and FSH pulsatility with continued leptin replacement (34
). Thus, these data are consistent with a role for leptin as a “gate” for normal reproductive function when adequate energy stores are achieved and suggest that leptin may have a role for treating the reproductive dysfunction seen in low leptin states, such as hypothalamic amenorrhea and eating disorders, and may also have therapeutic applications in conditions such as delayed puberty, which is associated with decreased leptin levels (35
We then examined whether r-metHuLeptin administration regulates the fasting-induced changes in thyroid function. A complex sequence of alterations in serum TSH and thyroid hormone levels has been observed in humans undergoing a short-term fast (37
). These include a decrease in TSH pulse amplitude (39
), a decrease of serum T3 levels, and an increase of rT3 (the less biologically potent hormone), whereas T4 levels remain unchanged due to its longer half-life. This suggests that fasting shifts T4 conversion from T3 to rT3 (16
), and it has been proposed that decreased T3 and thyroid receptor protein levels are of teleological importance in limiting energy expenditure and catabolism during a fast (16
Our data confirm the expected changes of thyroid hormone levels in response to short-term fasting, are consistent with findings reported in leptin-deficient children (4
), and demonstrate for the first time that r-metHuLeptin administration prevents the fasting-induced changes of TSH secretion and results in a slight increase of FT4, as previously described in leptin-deficient subjects (34
). Whether leptin regulates hypothalamic TRH release and/or pituitary TSH secretion needs to be studied further. Moreover, since r-metHuLeptin did not alter the fasting-induced changes in T3 and rT3, our findings provide no evidence for an effect of leptin on expression and/or activity of deiodinases, which most likely mediate the short-term starvation-induced changes in T3 and rT3 (16
) and may be influenced by changes of other metabolic signals such as FFA. Finally, since TBG levels were not differentially affected by either fasting or r-metHuLeptin administration, one could propose that the observed changes in T3 and T4 also reflect free T3 and free T4 changes. In fact, measured free T4 was unchanged in the fasting state but increased in response to r-metHuLeptin administration, although the magnitude of the change was small.
It has previously been shown that leptin administration in rodents reverses the inhibitory effect of food deprivation on spontaneous pulsatile TSH secretion (40
) and prevents the suppression of pro-TRH mRNA in paraventricular nucleus neurons that occurs with fasting (41
). In healthy men, we have found that leptin and TSH rhythms exhibit a similar 24-hour pattern of variability with significant pattern synchrony of ultradian fluctuations, a pattern that is impaired in leptin-deficient subjects (23
). Interestingly, leptin-resistant subjects with a mutation in the leptin receptor had evidence of hypothalamic hypothyroidism with low T4, normal basal TSH, and sustained TSH response to TRH (22
). Although r-metHuLeptin administration has been found to increase FT4 and FT3 levels in leptin-deficient children (34
) and to reverse the decreased T3 and T4 levels in four subjects on a long-term hypocaloric diet (42
), r-metHuLeptin did not alter the changes in thyroid hormone levels in this acute fasting paradigm with the exception of a slight increase of FT4, despite preventing TSH pulsatility alterations. This was most likely due to the long half-life of thyroid hormones and the short duration of the fast in this study. Thus, further studies are required to study the effect of r-metHuLeptin administration on all components of the HPT axis in response to more prolonged fasting or long-term hypocaloric diets. Findings of these studies will have not only physiologic but also therapeutic importance in the context of the plateauing weight loss seen in response to dieting or use of antiobesity medications.
During early starvation, serum GH levels rise, and pulsatile GH secretion increases with increased pulse frequency and 24-hour integrated GH concentrations (43
). Although GH is the main regulator of IGF-1 synthesis in the liver and peripheral tissues, fasting decreases IGF-1 levels despite elevated GH levels, most likely because of changes in other determinants of its secretion, such as hormones (insulin) and nutritional status per se as well as GH resistance in the liver (18
). The combination of increased GH and decreased IGF-1 levels may have adaptive value by diminishing the energy expenditure necessary for growth-related processes while enabling GH to promote the mobilization of alternative fuels through lipolysis.
It has previously been shown that the exogenous administration of leptin to fasted mice fully prevents the suppression of both GH and IGF-1 levels and corrects in part the fasting-induced suppression of growth hormone–releasing hormone mRNA expression (46
). Leptin-deficient subjects have decreased GH response to insulin-induced hypoglycemia and exercise tests but normal heights (4
), whereas leptin-resistant subjects have a mild but significant growth delay during early childhood in addition to decreased GH secretion and low IGF-1 and IGFBP-3 levels (22
). We found that replacement-dose r-metHuLeptin does not prevent the fasting-induced changes in GH pulsatility or free IGF-1 levels, but on the basis of partial restoration of total IGF-1 levels and the lack of a specific effect on IGFBP-1, -2, and -3, it may have an effect on one or more of IGFBP-4, -5, or -6. Thus, the role of leptin in regulating insulin-like growth factors and their binding proteins will require further investigation.
Fasting for 5 days has been shown to alter pulsatile and rhythmic cortisol release with a modest elevation of glucocorticoid levels in healthy men (47
). In mice, acute starvation increases corticosterone and ACTH levels, whereas exogenous leptin administration reverses the activation of the HPA axis (8
). A significant inverse relationship between fluctuations in leptin, ACTH, and cortisol has been demonstrated in humans, independent of glucocorticoid effects on leptin (13
), but unlike mice, human subjects with mutations in the leptin or leptin receptor gene appear to have normal adrenal function. Leptin-deficient subjects had elevated basal cortisol and ACTH levels but normal urinary free cortisol and response to dexamethasone suppression (4
). Similarly, evaluation of the HPA axis in the leptin-resistant subjects did not reveal any abnormalities (22
). In this study, we found evidence for mild activation of the HPA axis with acute fasting but no effect of replacement r-metHuLeptin, suggesting species-specific differences in leptin regulation of the HPA axis as compared with rodents (8
). Thus, the synchronous but inverse relationship of cortisol and leptin fluctuations reported previously (13
) does not appear to be causal but is probably due to a third factor. In addition, the paradigm used in this study does not exclude the possibility that leptin replacement during fasting needs to be administered in a pulsatile fashion for regulation of the HPA axis. Finally, it remains unknown whether leptin administration may alter ACTH pulsatility or affect β-hydroxysteroid dehydrogenase activity and thus cortisol levels in peripheral tissues.
Fasting with minimal sodium intake is associated with an initial natriuresis phase and negative sodium balance followed by avid sodium retention (48
). In this study, subjects received a much smaller dose of NaCl during fasting than in the fed state (500 versus 3,768 mg per day), which may account for the fact that urinary sodium tended to but did not decrease significantly with fasting. Fasting did significantly increase PRA and aldosterone, however, but r-metHuLeptin did not alter these fasting-induced changes in the mineralocorticoid axis. These findings are in contrast to the effect of leptin administration on increase natriuresis (49
) and sympathetic activity (51
) in normal, nonobese leptin-resistant Zucker rats (51
). Our findings are in agreement with a previous study in nonobese men on an isocaloric diet in whom leptin administration (0.3 mg/kg per day) for 6 days had no effect on autonomic activity or urinary catecholamines (52
). We found that urine epinephrine levels increased in response to fasting, but replacement-dose r-metHuLeptin had only a moderate effect in partially preventing the rise in urine epinephrine levels and no effect on urine norepinephrine levels. Thus in humans, leptin may regulate epinephrine synthesis in the adrenal medulla but has less effect on peripheral SNS activity as reflected by norepinephrine levels.
Since r-metHuLeptin administration did not alter any fasting-induced metabolic changes (17
), we propose that the fall of leptin levels with starvation is not required for the shift in fuel utilization brought about by fasting, which may be due to the fasting-induced suppression of insulin (53
). These results are consistent with the finding that leptin replacement in fasting mice had no effect on ketone levels (8
). Despite the lack of a significant effect on metabolic parameters and hunger questionnaires (most likely a less sensitive and precise assessment of appetite than caloric measurement), subjects given replacement-dose r-metHuLeptin tended to consume fewer calories in the postfasting state (although this did not reach statistical significance). This raises the possibility that, similar to the results seen in fasted rodents (8
), the fall of leptin may contribute to the increased appetite seen after fasting. Interestingly, a recent relatively small, randomized, double-blind clinical trial demonstrated that administration of high, pharmacologic doses of pegylated r-metHuLeptin in addition to a hypocaloric diet produced significant suppression of appetite as measured by eating/hunger questionnaires (54
), but this was not seen in response to the physiologic doses used in this study. The potential effect of leptin to regulate appetite and food intake after fasting requires further investigation in larger studies, but if confirmed, these data in association with the role of leptin in normalizing neuroendocrine changes may have implications for the role of leptin in the treatment of obesity.
Leptin binds to the leptin receptor (ObR) in the hypothalamus, activating the signal transducer and activator of transcription-3 (STAT3) signaling pathway, which mediates the metabolic effects of leptin through changes in melanocortin production and energy homeostasis. Recent evidence from transgenic animal models suggests that, in addition to the STAT3 signaling system, a distinct and parallel signal transduction pathway exists that regulates hypothalamic neuropeptide Y (NPY) and controls fertility, most likely through activation of extracellular signal-related kinase (ERK) and PI3K kinase (55
). Although the thyroid axis has not been studied in detail in these transgenic animal models, it is well known that NPY also regulates the HPT axis (56
). Thus, our data in lean men are consistent with a clear effect of exogenously administered leptin to regulate the ObR–ERK/PI-3 kinase–NPY pathway but not the ObR-STAT3-melanocortin pathway for signal transduction in the hypothalamus of lean men. Elucidation of the factors that inactivate the ObR–STAT3 system in humans is of major physiologic and therapeutic importance.
In summary this study represents, to our knowledge, the first interventional study to assess the role of leptin in the physiology of acute fasting in humans and demonstrates that its role differs in several respects from that in rodents. We found that replacement-dose r-metHuLeptin administered during an acute fasting state prevents key changes in the HPG and HPT axes and, in part, changes of total IGF-1, demonstrating that the fall in leptin with fasting may be both necessary and sufficient for these physiologic adaptations in normal men. In contrast to findings in rodents, fasting-induced changes in the HPA, renin-aldosterone, and GH/IGF-1 axes as well as fuel utilization are independent of leptin in the setting of acute leptin deficiency in humans. These findings suggest that decreased leptin levels may be responsible for several neuroendocrine abnormalities seen in low leptin states, such as anorexia nervosa, hypothalamic amenorrhea, and lipoatrophy. Thus, interventional studies involving leptin administration are required to fully clarify the physiologic and potentially therapeutic role of leptin in these specific disease states. These data may also explain the development of compensatory neuroendocrine changes underlying the plateauing effect of hypocaloric diets prescribed for weight loss in overweight patients. In this regard, further studies are needed to establish whether leptin also regulates neuroendocrine function in overweight, leptin-resistant subjects and in women, who have higher endogenous leptin levels. In addition, more prolonged fasting studies are required to evaluate the effect of chronic leptin administration on neuroendocrine axes, particularly those axes in which a longer time frame may be required for fasting-induced changes to be evident. Finally, although the HPG and HPT axes appear to only require leptin levels above a certain threshold for activation, it remains possible that regulation of other neuroendocrine axes, such as the HPA axis, may require leptin pulsatility and thus administration of leptin in a pulsatile fashion in order to be evident. Ongoing and future studies will fully elucidate these important issues in human leptin physiology.