Leptin and Leptin Receptor
Leptin [LEP; Leptin; Leptin (murine obesity homolog); Leptin (obesity homolog, mouse); OB; Obese protein; Obese, mouse, homolog of; Obesity factor; OBS].
Leptin receptor [CD295; DB; HuB219; LEP-R; LEPR; Leptin receptor; OB receptor; OB-R; OBR].
People long believed that obesity is entirely behavioral (e.g., lack of willpower) and not physiological. This dogma was first challenged with the identification of two obese mouse strains. The ob/ob mouse had been discovered in 1950 and characterized as the first model for massive obesity, marked hyperphagia, and mild transient diabetes. In 1965, a new obesity mutant had been discovered: the db/db mouse. Besides the marked obesity and hyperphagia, and unlike the ob/ob mutant, these animals develop severe, life-shortening diabetes. The phenotypes suggested that the genes are involved in a common metabolic pathway. When a wild type mouse was parabioticaly paired (surgically joining of the bloodstream of two animals) to a db/db mouse, the former animal died due to starvation. Similar experiments with ob/ob mice showed that db/db mouse overexpressed a strong, circulating satiety factor to which they cannot respond themselves and that is absent in ob/ob mice (reviewed in Coleman 2010). Forty years later, Friedman and colleagues identified this factor, product of the ob gene, and called it leptin after the Greek “leptos” meaning “thin” (Zhang et al. 1994). The leptin receptor (LEPR) was cloned 1 year later as the product of the db gene (Tartaglia et al. 1995).
Leptin and the Leptin Receptor
Leptin, the product of the ob gene, is a hormone mainly, but not exclusively, produced by adipocytes in the white adipose tissue and its serum levels positively correspond with the energy stored in the fat mass (Halaas et al. 1995). Other sites of (lower) expression include: brown adipose tissue, placenta, ovaries, skeletal muscle, stomach, mammary epithelial cells, and bone marrow. Mature leptin is a nonglycosylated 16 kDa protein of 146 amino acids. The crystal structure at 2.4 Å resolution showed a typical 4-α-helical cytokine structure with four antiparallel α-helices in an up-up-down-down arrangement, and an intramolecular disulphide bridge.
The leptin receptor (LEPR), product of the db gene, is a highly glycosylated single-membrane spanning class I cytokine receptor. Members of this family are hallmarked by the presence of cytokine receptor homology domains. The LEPR contains two such domains (CRH1 and CRH2), and additional immunoglobulin-like (IGD) and fibronectin type III (FNIII) domains. Alternative mRNA splicing and/or ectodomain shedding (only observed in humans) results in the expression of six LEPR isoforms: one long, 4 short, and one soluble form. The long form is the only form capable of efficient signaling and is highly expressed in the specific nuclei of the hypothalamus, a region in the brain important in the regulation of body weight (Mercer et al. 1996), but expression can also be detected in other, peripheral cell types (see further). Short and soluble forms have a broader expression pattern and are thought to play a role in transport, renal clearance, and/or modulation of the bioavailability of leptin.
LEPR Activation and Signaling
Class I cytokine receptors lack intrinsic kinase activity and use cytoplasmic associated JAKs for signaling. In the case of the LEPR, JAK2 becomes activated by cross-phosphorylation, and further rapidly phosphorylates three conserved tyrosine residues in the cytoplasmic domain, thereby providing docking sites for signaling molecules, and initiation of downstream signaling via the JAK/STAT (signal transducers and activators of transcription), MAPK (mitogen-activated protein kinase), and PI3K (phosphatidylinositol 3-kinase) as the most important pathways.
In a well-accepted model for JAK/STAT signaling, STAT molecules are recruited to the phosphorylated receptor tyrosine residues and become a substrate for the JAK activity. Upon phosphorylation, STATs form homo- and/or heterodimers, translocate to the nucleus, and modulate transcription of target genes. STAT3 is essential for leptin-regulated energy metabolism (Vaisse et al. 1996) but also activation of STAT1, STAT5, and STAT6 could be shown. The SH2-containing protein tyrosine phosphatase 2 (SHP2) allows LEPR-induced activation of the MAPK pathway, leading to the upregulation of the immediate early genes egr-1 and c-fos in the hypothalamus. LEPR activation leads to phosphorylation of several members of the insulin-receptor substrate (IRS) family, recruitment and activation of PI3K, and accumulation of its product phosphatidylinositol 3,4,5-triphosphate (PIP3). Downstream effects are the activation of 3-phosphoinositide-dependent protein kinase 1 (PDK1), Akt, and cyclic nucleotide phosphodiesterase 3B (PDE3B) (Park and Ahima 2014).
Like all cytokines, leptin actions are under tight control of multiple negative feedback mechanisms that terminate signaling and prevent overactivation. Cytokine stimulation induces the expression of members of the SOCS (suppressor of cytokine signaling) family. These SOCS proteins dampen signaling by two mechanisms: competition binding to the activated receptors and/or inhibiting activation of JAK kinases. SOCS3 is the major inhibitor of leptin’s metabolic regulation (Reed et al. 2010), but also SOCS2 and CIS (cytokine-inducible SH2-containing protein) contribute to dampening of LEPR signaling. Additional negative regulation mechanisms include the PTP1B (protein tyrosine phosphatase 1B) driven dephosphorylation of JAK2, and the control of LEPR expression on the cellular surface (reviewed in Wauman and Tavernier 2011).
Biological Functions of Leptin
Loss-of-function mutations in the leptin or LEPR genes (like the ob and db mutation; see above) not only cause severe obesity but also abnormalities in hematopoiesis, immunity, reproduction, bone metabolism, and blood pressure. This lead to the concept that leptin can act as a “metabolic switch” that links the body’s energy stores to these high energy-demanding processes.
The role of leptin in the control of human immunity was first postulated based on the observation that obese members of a Turkish family with congenital leptin deficiency died during childhood because of infections (Ozata et al. 1999). Since then, leptin emerged as a regulator of both the innate and adaptive responses. In innate immunity, leptin promotes secretion of inflammatory cytokines and the activation of dendritic cells, macrophages, neutrophils, monocytes, and natural killer cells (Naylor and Petri 2016). In adaptive immunity, low leptin levels due to starvation or malnutrition, or loss-of-function leptin or LEPR mutations, have a severe negative impact on the numbers and the differentiation of B and T cells in the thymus and spleen. Other functions in adaptive immunity include naïve CD4+ cell proliferation, promotion of T helper 1 (TH1) responses, suppression of CD4+CD25high regulatory T cells, and activation of TH17 cells (Naylor and Petri 2016).
In most mammals, many aspects of the reproductive function (e.g., puberty, gestation, lactation) require high amounts of energy to proceed. The association between leptin and reproduction originates from the observations that humans and rodents with congenital leptin deficiency fail to undergo puberty, that extreme leanness (anorexia) is associated with a delay in sexual maturation, while conversely, obesity is linked to premature puberty. Furthermore, leptin administration can correct the sterility of ob/ob mice and initiate reproductive functioning in normal female mice. The hormone affects reproduction both indirectly via gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and kisspeptins release, or directly on the ovaries (the LEPR is expressed in preovulatory follicles, ovarian theca cells, granulosa cells, and oocytes) (Catteau et al. 2015).
A role for leptin in bone metabolism was first observed in leptin and LEPR deficient mice as these animals display significantly longer vertebral length and higher bone mass. Intracerebroventricular infusion of leptin causes bone loss in leptin deficient and wild type mice, indicating that leptin regulation of bone metabolism includes central pathways. Indeed, leptin activates neurons in the ventromedial hypothalamus and signals via the sympathetic nervous system to suppress osteoblast proliferation while osteoclast resorption is promoted. In contrast to this central bone loss, leptin also increases bone formation via a more direct, peripheral way by activation of bone marrow mesenchymal stem cells, osteoblasts, osteoclasts, and chondrocytes. This activation includes proliferation, survival, differentiation (into osteoblastic lineage), and enhanced synthesis of collagen and extracellular matrix proteins. Finally, it has been shown that leptin has different effects on different parts of the skeleton in the body, and that there were significant differences between the axial and appendicular skeleton (Chen and Yang 2015).
Leptin modulates the sympathetic nerve activity that contributes to the regulation of blood pressure (BP). Indeed, it causes chronic increase in BP at physiological concentrations and may contribute to obesity related hypertension. POMC neurons in the arcuate nucleus of the hypothalamus and melanocortin 4 receptors (MC4R) signaling are critical in this regulation. Of note, leptin-induced increase in BP is partially counteracted by its metabolic actions: decreased appetite and increased energy expenditure tend to reduce BP (Abel and Sweeney 2012).
Leptin and Disease
Obesity, a major public health problem worldwide, is a complex medical condition in which accumulation of excess body fat leads to negative effects on health, including type 2 diabetes, heart disease, obstructive sleep apnea, cancer, and joint disease. Obesity is the result of the complex interplay between environmental and (epi)genetic factors. Environmental factors include increased caloric intake, reduced physical activity, and a more sedentary lifestyle in the Western world. Genetically, loss-of-function mutations in leptin or LEPR genes, but also genetic interference with leptin’s hypothalamic signaling gives rise to a very pronounced obese phenotype. Nowadays, it is estimated that not less than 50 genes are related to an increased obesity risk in humans and rodents (Farooqi and O’Rahilly 2014).
The paradoxically elevated levels of (biological active) leptin observed in most obese individuals is called “leptin resistance” and explains why these subjects do not respond to leptin treatment and why leptin mostly “failed” in the clinic. Possible mechanisms underlying this leptin resistance include a defective leptin transport over the blood-brain-barrier, impaired LEPR expression, and/or defects in signaling (Balland and Cowley 2015).
Its proinflammatory characteristics may link leptin to the onset and progression of several autoimmune diseases like multiple sclerosis (MS), autoimmune arthritis, hepatitis, and colitis. The role of leptin especially in MS is well documented: (i) Leptin levels are significantly increased in both serum and cerebrospinal fluid of relapsing-remitting MS patients. (ii) Serum leptin expression is increased before the clinical onset of experimental autoimmune encephalomyelitis (EAE) in mice, correlating with disease susceptibility. (iii) Acute starvation representing lower leptin levels, delayed disease onset, and attenuated clinical symptoms. (iv) Leptin-deficient ob/ob mice are resistant to EAE induction, while recombinant leptin replacement restores their susceptibility. (v) Leptin or LEPR antagonists slow disease progression and reduce disease relapses (Matarese et al. 2010).
Numerous clinical reports show that obesity is a risk factor for several cancers, such as prostate, breast, colorectal cancer, multiple myeloma, and melanoma. Etiological causes include insulin resistance, chronic hyperinsulinemia, local inflammation, and secretion of adipokines like leptin. Leptin promotes the survival (expression of not anti-apoptotic genes) and proliferation of several cancer cell lines. Effects on the tumor environment include the secretion of metalloproteinases that favor invasion and metastasis, while upregulation of E-cadherin and overexpression of extracellular matrix proteins promote adhesion of cancer cells. Finally, leptin promotes angiogenesis through the expression of vascular endothelial growth factor and its receptor (García-Robles et al. 2013).
Two decades ago, leptin and its receptor have been identified as key players in the control of body weight and metabolism. However, obesity is only in rare cases a consequence of loss-of-function mutations in the leptin gene. Treatment with the hormone is efficient in inducing weight loss and restoring metabolism in these patients. In contrast, the majority of obese subjects have high leptin levels, but they cannot respond to it, a paradox called leptin resistance.
Leptin is more than an antiobesity hormone, but more a pleiotropic cytokine with both beneficial and undesired effects. Under healthy conditions, it plays a role in a series of (high energy demanding) processes like immunity, reproduction, and bone metabolism. In pathological situations like uncontrolled immune responses in autoimmune diseases, tumorigenesis, elevated blood pressure, and certain cardiovascular diseases, it is desirable to interfere with leptin’s actions. More profound insights in the structure, signaling, and complex biology of the leptin-LEPR system will be beneficial in understanding the pathogenesis of some diseases and ultimately in the identification and/or optimization of clinical intervention strategies.
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