Mapping leptin's link to reproduction

Activation of leptin receptor signaling via tyrosine phosphorylation of Y1077 provides an important link between control of energy homeostasis and the reproductive axis. In this issue, Patterson and colleagues used transgenic technology to assess the physiological role of LepR-b Tyr₁₀₇₇-dependent signaling in vivo, and defined a role for Tyr₁₀₇₇-dependent signaling in the regulation of endocrine function [1].

Adipose tissue plays a major role in the regulation of energy homeostasis, not only by storing energy in form of fat, but also by functioning as a major, dynamic endocrine organ, secreting several factors essential for maintaining metabolic homeostasis. Among those factors, whose local and circulating levels are affected by the degree of body adiposity, is the adipocyte hormone leptin. More than 15 years after its discovery, there are numerous studies highlighting the important role of leptin in the regulation of energy homeostasis and neuroendocrine function [2]. Leptin is secreted proportional to fat content and communicates to the central nervous system (CNS) the repletion of peripheral energy stores, suppressing feeding and permitting energy expenditure via activation of a variety of neuroendocrine and autonomic processes.

Lack of leptin action due to null mutations of either leptin or the leptin receptor results in increased appetite, decreased metabolic rate, infertility, immune dysfunction, and insulin resistance both in humans and rodents. Consistent with a major role for leptin action in the CNS, mice with a neuron-specific deletion of the leptin receptor closely resemble the phenotype of mice that lack the leptin receptor in the whole body (db/db) [3]. On the other hand, expression of LepR-b in the brain of db/db mice rescues obesity, diabetes, and completely restores fertility in males and partially in female mice [4].

The long form of the leptin receptor (LepR-b) is a type I cytokine receptor that consists of a single membrane-spanning domain. Binding of leptin to the extracellular domain induces a conformational change that subsequently activates the associated tyrosine kinase Janus kinase 2 (JAK2). Activation of JAK2 stimulates the phosphorylation of multiple residues (Tyrosine 985, Tyrosine 1138, and Tyrosine 1077) on the intracellular domain of LepR-b (Fig. 1). Phosphorylation of each of these residues leads to the recruitment of a distinct set of downstream signaling molecules.

Fig. 1.

Intracellular leptin receptor signaling. Binding of leptin to its receptor (LepR-b) mediates a conformational change and subsequent activation of the tyrosine kinase Janus kinase (JAK)2, resulting in phosphorylation of three LepR-b tyrosine residues (Tyr₉₈₅, Tyr₁₀₇₇, Tyr₁₁₃₈). Phosphorylation of Tyr₉₈₅ recruits SHP2, leading to the activation of the ERK signaling cascade. Phosphorylated Tyr₉₈₅ also binds to suppressor of cytokine signaling (SOCS)3, a negative regulator of LepR-b signaling. Phosphorylation of Tyr₁₀₇₇ recruits and activates the transcription factor signal transducer and activator of transcription (STAT)5. Phosphorylation of Tyr₁₁₃₈ mediates activation of STAT3 and STAT5. Activated STAT3 translocates to the nucleus and mediates expression of target genes including SOCS3. SOCS3 acts as a negative feedback inhibitor of leptin signaling, in part, by binding to tyrosine residue 985.

In vitro studies have shown that phosphorylated Tyr₉₈₅ recruits the SH2-containing tyrosine phosphatase-2 (SHP2), which presents the first step in the activation of the extracellular signal-regulated kinase (ERK) cascade [5]. Additionally, phosphorylated Tyr₉₈₅ also recruits SOCS3, a negative regulator of leptin action. Phosphorylation of tyrosine residue 1138 mainly recruits the latent transcription factor signal transducer and activator of transcription 3 (STAT3), which upon subsequent, JAK-2-dependent, phosphorylation translocates to the nucleus to regulate gene expression, e.g. of SOCS3 to inhibit leptin signaling in a negative feedback loop [5]. Finally, cell culture studies have shown that phosphorylation of Tyr₁₀₇₇ promotes the recruitment and transcriptional activation of STAT5 [5] (Fig. 1). Additionally, leptin initiates several other intracellular signaling pathways, including activation of phosphatidylinositol kinase-3 (PI3K) and the mammalian target of Rapamycin (mTOR) and inhibition of the AMP-dependent protein kinase (AMPK) [6].

By using an advanced targeted knock-in approach in which the endogenous Lepr gene is replaced by sequences encoding a substitution mutant of the LepR-b phosphorylation site Tyr₁₀₇₇ (Y1077F), Patterson and colleagues directly addressed a potential physiological function of LepR-b Tyr₁₀₇₇-dependent signaling in vivo. In this issue of Molecular Metabolism, they report that abrogation of Tyr₁₀₇₇-dependent signaling in vivo causes mild obesity and regulates female reproduction [1].

Previous elegant studies by the same group have already addressed the individual physiological roles of the other two known LepR-b tyrosine residues, Tyr₁₁₃₈ and Tyr₉₈₅, using the same knock-in approach in vivo. Animals in which the gene encoding the leptin receptor had been replaced with an allele coding for a replacement of Tyr₁₁₃₈ in LepR-b with a serine residue that specifically disrupts LepR-b STAT3 signaling are hyperphagic and obese [7]. However, these animals are fertile and less hyperglycemic than db/db mice, suggesting that additional LepR-b signals regulate fertility and glucose homeostasis.

On the other hand, mutation of LepR-b tyrosine residue 985 reduces food intake, decreases expression of orexigenic neuropeptides, and protects from high-fat diet-induced obesity in female mice along with a normal functioning neuroendocrine system [8]. Thus, leptin signaling via LepR-b Tyr₉₈₅ most likely attenuates leptin action especially in females by inducing autoinhibitory signals, presumably via the SOCS3-mediated feedback loop.

The role of LepR-b Tyr₁₀₇₇-dependent signaling in mediating leptin's effects, however, was as yet not well defined. Patterson and colleagues report that, while on a normal diet only female mice with abrogated Tyr₁₀₇₇-dependent signaling show a significant increase in body weight, both sexes show a slight increase in body weight when challenged with a high-fat diet. This mild obesity is most likely due to a 5–10% increase in cumulative food intake. These metabolic alterations develop in the absence of significant changes in circulating plasma leptin/insulin concentrations, changes in expression of orexigenic/anorexigenic hypothalamic neuropeptides or altered glucose tolerance [1].

Interestingly, female mice carrying the mutation of Tyr₁₀₇₇ exhibit impaired reproductive function, as they progress to a second estrus cycle less frequently than controls and show increased intervals between cycles. Collectively, these results suggest that LepR-b Tyr₁₀₇₇-dependent signaling only plays a modest role in energy balance but is required for ongoing appropriate function of the reproductive system [1].

Leptin controls reproduction depending on the energy state of the body, and sufficient levels of leptin are a prerequisite for the maintenance of reproductive capacity. In times of abundance, leptin communicates the sufficiency of the body's energy stores to the CNS and permits energy expenditure on energy-consuming processes such as reproduction. Conversely, under conditions of metabolic stress such as anorexia or excessive exercise, decreased leptin levels promote feeding and energy conservation in order to ensure survival of the organism [9].

Gonadotropin-releasing hormone (GnRH) cells of the hypothalamus are the primary regulators of the reproductive axis, regulating puberty and ovulation. Most of the GnRH-producing cells in the brain reside in the preoptic area (POA) of the hypothalamus. GnRH is secreted into the hypophyseal portal blood vessels and controls secretion of the pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Leptin regulates gonadotropin secretion via the regulation of GnRH function [9]. However, GnRH neurons in the POA do not express LepR-b, indicating that leptin indirectly regulates these cells by acting on interneurons upstream of GnRH neurons.

Furthermore, recent studies have suggested that leptin action modulates kisspeptin 1 (Kiss1)-expressing cells in the hypothalamus [9]. Kiss1 is an important modulator of GnRH neurons and is expressed in several hypothalamic nuclei, including the anteroventral periventricular nucleus (AVPV), a site known to regulate secretion of gonadotropins, and also the arcuate nucleus (ARC). The Kiss1 receptor, the G protein-coupled receptor GPR54, is expressed on GnRH neurons, and its mutation results in dysfunction of the reproductive system. In the ARC, the majority of Kiss1-positive cells also co-express neurokinin B (tachykinin 2, Tac2) and dynorphin A. However, no AVPV Kiss1 neurons and only a few ARC Kiss1 neurons express the LepR-b, indicating that leptin regulation of Kiss1 neurons is also likely to be indirect.

The results of the study by Patterson and colleagues provide direct evidence that leptin signaling via LepR-b Tyr₁₀₇₇ might serve as an important mechanism by which leptin modulates endocrine function, linking body adiposity and the reproductive axis. While Tyr₁₀₇₇ signaling plays only a modest role in the control of metabolism and is not required for the onset of fertility, it is required for maintaining female reproductive function [1]. These findings complement previous studies on the role of leptin in the regulation of the neuroendocrine system, providing a missing link in leptin's regulation of the reproductive axis. While reproductive function is only modestly impaired in obese, hyperphagic Tyr₁₁₃₈ mutant mice, reproduction is severely impaired in mice mutant for all three intracellular tyrosine residues, similar to db/db mice, and these mice also exhibit an impaired energy balance [10]. These studies already suggested that a LepR-b tyrosine phosphorylation site other than Tyr₁₁₃₈ is required in the control of reproduction by LepR-b.

However, future studies have to resolve the question of how leptin Tyr₁₀₇₇-Stat5 signaling controls reproduction. Mutant Tyr₁₀₇₇ mice do not exhibit detectable changes in hypothalamic mRNA expression of known neuroendocrine regulators of reproduction (i.e. Kiss1, Tac2). The effect of LepR-b Tyr₁₀₇₇ signaling on reproduction must therefore be mediated independently of alterations in expression of these neuropeptides. Furthermore, since previous studies have shown no overlap between LepR-b-expressing and GnRH-containing neurons, and only a few Kiss1 neurons express the LepR-b, leptin exerts its Tyr₁₀₇₇-mediated metabolic and reproductive effects most likely via indirect modulation of GnRH neuron activity by acting at multiple sites in the CNS.

Future studies will therefore have to clarify the location of these leptin-responsive cells involved in reproductive control and how leptin, via Tyr₁₀₇₇ STAT5-dependent signaling, mediates its effects without altering hypothalamic mRNA expression of known regulators of the reproductive axis. Through the identification that LepR-b Tyr₁₀₇₇ is the major phosphorylation site mediating leptin's effects on reproduction, the results of this study provide essential information on the link between control of energy homeostasis and reproduction. These findings therefore represent an important step to inform future investigations on leptin's role in the regulation of endocrine function and energy balance.