Leptin Is a Hormone That Tends to Reduce Food Intake. It Is Produced by What Kind of Cells?

Leptin, the protein product of the obese (ob or Lep) gene, is a hormone synthesized by adipocytes that signals available energy reserves to the brain, and thereby influences development, growth, metabolism and reproduction. In mammals, leptin functions as an adiposity signal: circulating leptin fluctuates in proportion to fat mass, and information technology acts on the hypothalamus to suppress food intake. Orthologs of mammalian Lep genes were recently isolated from several fish and two amphibian species, and here we study the identification of two Lep genes in a reptile, the cadger Anolis carolinensis. While vertebrate leptins show large divergence in their primary amino acid sequence, they form similar tertiary structures, and may accept similar potencies when tested in vitro on heterologous leptin receptors (LepRs). Leptin binds to LepRs on the plasma membrane, activating several intracellular signaling pathways. Vertebrate LepRs betoken via the Janus kinase (Jak) and bespeak transducer and activator of transcription (STAT) pathway. Three tyrosine residues located inside the LepR cytoplasmic domain are phosphorylated past Jak2 and are required for activation of SH2-containing tyrosine phosphatase-2, STAT5 and STAT3 signaling. These tyrosines are conserved from fishes to mammals, demonstrating their critical role in signaling past the LepR. Leptin is anorexigenic in representatives of all vertebrate classes, suggesting that its role in energy balance is ancient and has been evolutionarily conserved. In addition to its integral office as a regulator of appetite and energy balance, leptin exerts pleiotropic deportment in development, physiology and beliefs.

© 2011 South. Karger AG, Basel

Introduction

Two mouse strains discovered at the Jackson Laboratory in 1950 and 1965 had identical phenotypes: morbid obesity, insulin resistance, infertility and lethargy [1]. The two strains were designated obese (ob/ob) and diabetic (db/db), and were found to be due to single cistron deficiencies. A serial of elegant parabiosis experiments conducted by Douglas Coleman showed that the ob/ob strain was deficient in a claret-borne factor, while the db/db strain was deficient in the receptor for this factor [1,2,3]. Over forty years passed before the mouse obese gene (ob or Lep) was positionally cloned by Jeffrey Friedman's group and was institute to encode a hormone that they named 'leptin' afterwards the Greek give-and-take 'leptos' for thin [4]. Leptin is a member of the type I helical cytokine family unit, and is related to growth hormone, prolactin and the interleukins [five]. The yr after leptin was identified Tartaglia et al. [six] reported the isolation of the leptin receptor gene (LepR) by expression cloning. Before long thereafter it was confirmed that the mutation in the db/db mouse was in the LepR cistron [7,8].

In mammals leptin is secreted into the bloodstream, primarily from adipocytes, and acts on the brain to regulate food intake and metabolism [four,9]. Leptin acts on the hypothalamus to signal when the trunk has sufficient energy stores, thus inhibiting appetite (i.east. it functions as an 'adipostat'). The deportment of leptin occur over both short and long time frames. In the short term, plasma leptin serves equally a satiety signal [10], and over longer periods, daily mean plasma leptin concentration communicates long-term energy status to the brain [11]. Primal leptin signaling plays a pivotal part in the regulation of metabolic activeness by peripheral tissues [12,13,14]. The rising prevalence of human obesity and blazon 2 diabetes has generated intense interest in the physiological roles that leptin plays in free energy balance and food intake regulation [fifteen,16,17,eighteen,xix].

In mammals, leptin acts on complex neural circuitry to regulate food intake and energy metabolism [20,21]. Leptin receptor expression is highest in neurons within nuclei of the basomedial hypothalamus that include the arcuate (ARC), dorsomedial hypothalamic and ventromedial hypothalamic nuclei [22,23]. Master targets for leptin action in the hypothalamus are two populations of neurons located in the ARC that projection axons to the lateral hypothalamic area [21,24]. Leptin acts on ARC proopiomelanocortin (POMC)/cocaine and amphetamine related transcript (CART) neurons to increase POMC (and CART) biosynthesis which generates an anorectic signal via alpha melanocyte-stimulating hormone (αMSH) [24,25]. Leptin too acts on ARC neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons to inhibit expression of the orexigenic signals NPY and AgRP [24,25]. Leptin receptors have also been reported in several extrahypothalamic sites that include the midbrain and brainstem [21,26]. 2nd-order neurons that synthesize thyrotropin-releasing hormone (TRH) or corticotropin-releasing factor (CRF) located in the paraventricular nucleus are regulated indirectly by leptin targets in the ARC, and thus mediate leptin's inhibitory actions on food intake, increases in thermogenesis, and increases in pituitary hormone secretion [10]. Although much less is known near the system and regulation of feeding control centers in the brains of nonmammalian species, the information support that the bones features of mammalian feeding command circuits are present in fishes and amphibians [27,28,29], and that leptin engages similar neuropeptidergic pathways in the hypothalamus/preoptic surface area (for frogs, run into [30] [C. Li and R.J. Denver, unpublished], and for fishes, encounter [31,32]).

In addition to its roles in the regulation of ambition and metabolism, leptin has pleiotropic actions in evolution and physiology. Some of the major actions of leptin uncovered in recent years include the promotion of linear growth through its influence on energy balance, the induction of mitosis of dissimilar cell types including chondrocytes of the epiphyseal growth plate, and the stimulation of secretion of pituitary growth hormone [33]. Leptin is permissive for the onset of puberty in mammals, peradventure acting via the cellular energy sensor mammalian target of rapamycin (mTOR) [34]. It plays critical roles in neural development. Ob/ob and db/db mice have reduced encephalon weight and DNA content which, in ob/ob mice, can be reversed by leptin administration [35]. The hormone has been shown to induce mitosis in unlike encephalon regions [36,37] and in the hypothalamus, leptin influences the maturation of feeding control circuitry by promoting the formation of neuronal projections among hypothalamic nuclei [38]. Leptin actions also include os development, growth and homeostasis [30,39,twoscore,41,42], as well as lung evolution and role [43], immune role [44,45], thyroid function [46] and stress response [12]. A full give-and-take of the diversity of leptin deportment is beyond the scope of this review. Encounter the references cited to a higher place for detailed discussions of leptin actions.

Molecular Evolution of Vertebrate Leptin Genes

Before long afterward the mouse Lep gene was isolated, orthologous genes were identified in man and several other mammalian species [4,47,48]. Many mammalian Lep genes have since been cloned, and molecular phylogenetic analysis shows that virtually taxa class distinct clades that largely hold with accepted mammalian orders (fig. ane). High rates of leptin evolution are credible in some seals (Halichoerus grypus and Leptonychotes weddellii), beavers (Castor canadensis), pikas (Ochotona curzoniae), and marmosets (Callithrix jacchus). In adult seals leptin is expressed in the lung, whereas lung leptin expression in other mammals appears to occur but in the fetus [49]. Hammond et al. [49] speculated on a role for leptin in pulmonary surfactant production in seals that may be related to the unique respiratory challenges associated with diving. Yang et al. [50] proposed that in pikas, which are non-hibernating mammals that live at high superlative or high latitudes, adaptive evolution in leptin was driven by physiological adaptation to extreme cold.

Fig. 1

Neighbour-joining phylogram of vertebrate leptins. The alignment was conducted using Clustal W2 and based on the BLOSSUM protein weight matrix. The neighbor-joining phylogram was based on uncorrected pair-wise sequence divergence of 229 amino acrid positions (including gaps). Bootstrap values subtend major, well-supported nodes (≥90%) and were based on i,000 pseudoreplicates. The tree was rooted with homo growth hormone. Vertical lines on the right indicate mammalian orders and nonmammalian vertebrate classes. Due to the differential rates of leptin evolution in some mammalian lineages, taxa in the mammalian orders Carnivora, Cetartiodactyla, Rodentia, and Primates were each constrained to be monophyletic prior to the neighbor-joining assay. Bootstrap values are not shown for the constrained nodes. Branch lengths reflect evolutionary departure and loftier rates of leptin evolution are apparent in some seals (Halichoerus grypus and Leptonychotes weddellii), beavers (Castor canadensis), pikas (Ochotona curzoniae), and marmosets (Callithrix jacchus).

When the mouse Lep factor was first isolated the authors ended, based on low stringency Southern hybridization of the mouse Lep probe to genomic Deoxyribonucleic acid isolated from chicken, eel and fruit fly, that Lep genes were evolutionarily conserved [4]. In the decade following the discovery of the mouse Lep gene, several laboratories attempted to isolate orthologous genes from nonmammalian species using nucleic acid hybridization (east.g. library screening) or RT-PCR with degenerate primers. These attempts were uniformly unsuccessful, except in the case of the craven, where a cDNA was isolated past RT-PCR and reported to share 95% identity to the mouse cistron [51,52]. Subsequently, a putative Lep ortholog was isolated from turkey which also had very high sequence identity to rodent Lep genes [48]. The validity of these sequences has since been questioned [53,54,55,56,57]; but run into counterpoint [58]. Searches of several EST databases and the chicken genome were unsuccessful in identifying a chicken Lep sequence [53,56], and synteny analysis showed that the unabridged chromosomal region inside which the chicken Lep gene should be found (on chromosome 1, which is homologous to human being chromosome 7, mouse chromosome 6) is missing [56]. Neighbor joining analysis of all known vertebrate Lep genes showed that the chicken sequence was phylogenetically nested amongst mammals and very closely related to rodents (fig. 1; the bird sequences are not included in the tree) [55]. Abrupt et al. [55] pointed out that, based on the estimated frequency of synonymous substitutions due to random mutation in genes, the likelihood that the reported chicken Lep gene cDNA was correct was less than one in a million.

Despite the failure to identify avian orthologs of mammalian Lep genes, chickens have a leptin receptor in their genome that is expressed, is activated to signal via the Janus kinase (Jak)/signal transducer and activator of transcription 3 (STAT3) pathway by human and frog leptins in vitro, and probable mediates the actions of administered leptin in vivo [59]. Pitel et al. [56] proposed that the gene for the ligand was lost in birds while the gene for the receptor was retained. It is possible that this occurred in some birds, but that leptin genes are retained in other avian lineages. Leptin genes were clearly present in the common antecedent of birds and squamate reptiles since the cadger, Anolis carolinensis possesses 2 Lep genes (discussed beneath). Using our predicted platypus Lep gene every bit the search sequence we were able to locate two candidate Lep genes within the lizard genome (online supplementary tables 1 and two; for all online supplementary textile, run into www.karger.com?doi=10.1159/000328435); nonetheless, similar searches through the chicken and zebra finch genomes using platypus and lizard Lep cistron sequences produced no returns. More work on other avian and reptilian species is required to determine if the Lep factor has been lost in Aves.

As discussed above, for a decade after the first isolation of Lep genes in mammals no orthologs were identified in nonmammalian vertebrates. In 2005, Kurokawa et al. [60] reported the isolation of a putative homolog of mammalian leptin in the pufferfish Takifugu rubripes. The deduced pufferfish protein is but 13% similar to human leptin (or to frog leptin – see below). Kurokawa et al. did not test whether the deduced protein product of the putative pufferfish Lep factor had biological activity commensurate with a role equally a leptin.

Presently after the publication by Kurokawa et al. [60], we reported the molecular cloning of frog (Xenopus) orthologs of mammalian Lep and Lep genes and conducted the first functional characterization of this ligand-receptor pair in a nonmammalian vertebrate [30]. The frog Lep gene encodes a predicted 16.9 kDa poly peptide (fig. 2). The chief amino acid sequence of frog leptin is 35% like to human, but only thirteen% like to pufferfish leptin [60]. Nosotros showed that recombinant frog leptin activated the frog LepR in vitro, signaling via STAT3, and frog leptin was potently anorexigenic when injected intracerebroventricularly into juvenile frogs [30]. Boswell et al. [61] isolated a cDNA for a putative Lep ortholog in the tiger salamander Ambystoma tigrinum that shares sixty% identity with frog leptin.

Fig. 2

Comparison of the amino acid sequences and structures of some tetrapod leptins. a Amino acrid alignment of frog (Xenopus laevis; AY884210), lizard (Anolis carolinensis; Lep1; online suppl. table one), mouse (Mus musculus; AAA64564) and human being (Human sapiens; AAA60470) leptin. The alignment colors are as follows: ruby grapheme on yellowish background = consensus remainder derived from a completely conserved residue at a given position; blueish character on cyan groundwork = consensus residue derived from a block of similar residues at a given position; black character on green background = consensus balance derived from the occurrence of greater than 50% of a single residuum at a given position; blackness character on white background = non-similar residues [83]. Boxed regions are the most highly conserved sequences among tetrapod leptins. The indicated helices and loop structures are based on Zhang et al. [145]. Amino acids with ii stars higher up them are residues in mouse leptin that when mutated lead to loss of biological activeness [85]. Note that these amino acids tend to exist conserved amongst tetrapod leptins. Amino acids with ane star above are residues in mouse leptin that when mutated lead to fractional loss of biological activeness [85]. In most cases these amino acids show low conservation amid tetrapod leptins. Red confined underneath the alignment indicate amino acids that when mutated cause the hormone to lose receptor activation part but to retain receptor bounden activity, thereby generating an antagonist (LDFI – mouse a.a. 38–43; ST – mouse a.a. 120, 121) [86]. The two conserved cysteines are indicated by arrows. The alignment was conducted using the Marshal X module of Vector NTI Advance xi software (Invitrogen, Carlsbad, Calif., Us). b Ribbon diagrams showing secondary and tertiary structures of mouse, lizard and frog leptins. Three-dimensional modeling was done using the ProModII program at the SWISS-MODEL automatic protein modeling server, and was based on the human leptin (1AX8.pdb) Protein Information Bank structure file. c Synteny mapping of leptin genes from mouse and lizard. Factor position on the mouse chromosome was determined by NCBI sequence viewer of Mus musculus chromosome half-dozen, reference assembly (C57BL/6J). Gene positions on the lizard scaffolds were determined using the UC Santa Cruz Genome Browser of Anolis carolinensis (Feb. 2007 AnoCar v. i.0). Simply limited gene arrangement information is available for these lizard scaffolds because of their brusk length. The vertical black bar to the left of the lizard scaffold 746 indicates a cake of neighboring genes (MALL, NPHP1 and BUB1) found on mouse chromosome two (73 cM).

Sequences orthologous to mammalian Lep genes have been described in several fish species based on genomic analyses [60] and RT-PCR cloning [31,32,57,62,63,64]. Deciphering the molecular phylogeny of fish Lep genes is complicated by the fact that multiple, and sometimes highly divergent, copies have been identified in several species. These duplicates may accept resulted from historical events such equally the whole-genome duplication that occurred early in the teleost lineage [65], or the additional genome duplication event that occurred afterward in some teleost lineages leading to tetraploidy (e.grand. salmonids [66]). Differential rates of gene loss or divergence in unlike lineages, the express sequence data now available, and the tenuous phylogenetic placement of divergent fish leptins are also challenging our understanding of the history of this gene in fishes. Some fishes (e.g. zebrafish and medaka) [62,67] have two divergent types of Lep genes (designated A and B; fig. ane) that may have arisen from the initial whole-genome duplication event. Alternatively, these may have resulted from clade-specific cistron duplications with subsequent divergence of 1 of the two copies. Multiple copies of 'A lineage' Lep genes have been isolated from Atlantic salmon, common carp, and goldfish, with minimal deviation among the paralogs and likely result from lineage-specific tetraploidizations in salmonids [62,68] and cyprinids [57,63,69,70]. Takifugu rubripes, a teleost species that has undergone desperate genome reduction, appears to have retained merely ane Lep gene [60].

We searched the genome of the cadger Anolis carolinensis and identified, for the first time, Lep and LepR genes in a reptile. Anolis has two putative Lep genes (designated Lep1 and Lep2; online suppl. table 1; [71]). The predicted mature Lep1 poly peptide of the cadger is 149 amino acids in length and shares 35.half-dozen% amino acid sequence identity with human leptin. The lizard Lep2 gene has multiple, single nucleotide deletions at the finish of exon 3, which results in a frame shift. This causes the predicted Lep2 mature poly peptide to diverge from Lep1 after position 117; the Lep2 protein is predicted to be 192 amino acids. The two predicted lizard leptin proteins are 60.7% identical in the first 117 amino acids.

We conducted synteny mapping of genes neighboring mouse Lep on chromosome 6 and this supported that the two lizard Lep genes are orthologs of mouse Lep (fig. 2c). The lizard Lep1 cistron resides in a homologous genomic region to mouse Lep. The lizard Lep2 factor resides in a genomic region homologous to mouse chromosome 2; the lizard Lep2 gene likely arose through a recombination effect (that included factor RBM28). Preliminary results propose that lizard Lep1 mRNA is expressed in several tissues just Lep2 mRNA is not expressed [71]. The cadger Lep1 tin can activate LepRs: we made recombinant lizard Lep1 in E. coli and found that it activates mouse and frog LepRs in transient transfection analysis with potency comparable to the homologous leptins [L. Lavner, A. Dziuba, G.C. Boorse and R.J. Denver, unpublished].

Prior to the isolation of nonmammalian Lep genes, several groups used mammalian reagents (recombinant mouse leptin, antibodies to mouse leptin or leptin receptor) to study leptin biological science in nonmammalian species [27,72,73,74,75,76,77]. In the goldfish and a lizard, injections of recombinant mammalian leptin reduced food intake, which was consistent with the existence of a leptin-like protein in nonmammalian species that functions in free energy balance regulation [27,77,78]. Nonetheless, mammalian leptin failed to bear upon nutrient intake in several other fish species [27]. Some investigators have used (and proceed to employ) antibodies to mouse leptin to investigate the tissue distribution and expression of leptin in nonmammalian species [27,79,lxxx,81]. Given the depression conservation of the primary structure of leptins from different vertebrate classes (fig. 2), we suggest that results obtained with heterologous immunological reagents should be interpreted with caution. On the other hand, despite low principal sequence identity, leptins from different species are agile on heterologous receptors, although the potency varies [30,59]; run into besides [82] for activity of pufferfish leptin on proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. Therefore, studies in which murine leptin was used in nonmammalian species could signal to a physiological part for the endogenous, native leptins, but this must be verified one time the homologous hormones become available.

Conserved Structural Features of Vertebrate Leptins Related to Their Function

When the crystal structure of human leptin was solved, it was found to take four α helix bundle folds, closely resembling the structures of other class I helical cytokines [v]. Like other class I helical cytokines, vertebrate leptins show significant divergence in their primary structures, but are nevertheless highly like in their predicted secondary and tertiary structures (modeling based on the crystal structure of homo leptin; fig. 2b) [57,60,63]. Natural selection tends to maintain the intrinsic stability of secondary and tertiary structures of proteins [83], and this is illustrated well by the course I helical cytokines [v].

Leptin resembles other grade I helical cytokines in that it has iv antiparallel helices designated A-D, merely information technology differs in that information technology has a pocket-sized helical segment designated helix Eastward found in the loop linking helices C and D (fig. 2b). All vertebrate leptins have a pair of conserved cysteine residues that in human leptin accept been shown to form a disulfide bridge required for full biological activeness [xxx,threescore,84] (fig. 2a). Iii receptor interacting sites on mammalian leptins have been mapped past mutational analysis [85]. Site I is located on the face up of helix D, site II is on helices A and C, and site Iii at the N-terminus of helix D. Each of these regions shows some degree of conservation of primary amino acid sequence amid vertebrate leptins (fig. 2a). Amino acid substitutions that resulted in a significant reduction in biological activity of the hormone were likely selected against, every bit these positions tend to be completely or by and large conserved across tetrapods (indicated by double stars in a higher place sequences in fig. 2a).

At that place are other regions of vertebrate leptins located outside of the three identified receptor binding sites that evidence a loftier degree of sequence identity. For example, in that location is conservation in helix B and the BC loop. The most highly conserved stretch of amino acids is the six balance sequence GLDFIP (positions 38 to 43 in human leptin); this sequence is completely conserved among tetrapods (fig. 2a). Mutation of LDFI to all alanines generates an antagonist (a leptin 'mutein') that binds to the LepR but fails to actuate it [86]. Although this sequence is not a part of the receptor binding sites, it is required for activation of the LepR and has apparently been field of study to strong stabilizing option. Elinav et al. [87] produced a PEGylated class of the LDFI mutein that has enhanced antagonist activity in vivodue to reduced clearance. We recently engineered a frog leptin LDFI mutein and plant that it also has antagonist activity on the frog LepR when tested in vitro [C. Pelletier, A. Dziuba, One thousand. Cui and R.J. Denver, unpublished]. The GLDFIP sequence is absent in fish leptins [85] and may betoken a unlike mechanism for binding to and activation of the LepR compared with tetrapods. Other amino acids that have been mutagenized to generate a leptin antagonist are the Southward and T residues at positions 120 and 121, respectively, of mouse leptin [86].

As mentioned above, in that location is considerable divergence in master amino acid sequence amongst vertebrate leptins. However, their secondary and tertiary structures, and key amino acids required for biological activeness, particularly among tetrapods, are evolutionarily conserved (it should be noted that the predicted structures of nonmammalian leptin are based on models of the crystal structure of human leptin). This conservation parallels the conservation of central structural elements within the 2 cytokine receptor homology domains (CHDs) of vertebrate LepRs [88] (discussed below; fig. 3). The significance of this conservation is highlighted by the finding that heterologous hormone-receptor pairings amidst tetrapod leptins and LepRs leads to productive receptor activation, often with similar authorisation to the homologous hormone. For case, recombinant leptins of frog and lizard activated the mouse LepR with equal potency to human leptin. Similarly, human and lizard leptins activated the frog LepR, although with lower authorisation than frog leptin [thirty] [Fifty. Lavner, A. Dziuba, G.C. Boorse and R.J. Denver, unpublished].

Fig. 3

Neighbor-joining phylogram of cytokine receptor homology domains (CHD) from group 2, class i cytokines [102]. The alignment was conducted using Clustal W2 and based on the BLOSSUM protein weight matrix. The neighbour-joining phylogram was based on uncorrected pairwise sequence divergence of 218 amino acid positions (including gaps). Bootstrap values subtend major, well-supported nodes (≥90%) and were based on i,000 pseudoreplicates. Homo sapiens (Hs), Mus muscle (Mm), and Danio rerio (Dr) were used every bit representative taxa for glycoprotein 130 (GP130), GP130-like monocyte receptor (GLMR), granulocyte-CSF (GCSFR), interleukin receptors (ILR), leukemia inhibitory factor receptor (LIFR), and oncostatin K receptor (OSMR). The 2 CHDs of leptin receptors are monophyletic. Leptin receptor taxa are the aforementioned as those used for figure iv. Just unmarried representatives were included for each of the mammalian orders. The leptin receptor CHD-1 clade is blood-red and the leptin receptor CHD-2 clade is blue.

Tissue Sites of Leptin Production

In mammals, the major sites of Lep mRNA expression are adipose tissue, stomach and liver [89,90,91,92]. Lep mRNA is expressed at lower levels in heart, placenta and fetal tissues [93,94], the pituitary gland, where leptin may attune pituitary hormone secretion [95,96,97], and in the brain [98] where the hormone tin can influence neural development [36,99,100] and cerebral function in the adult [101]. In nonmammalian species, Lep mRNA appears to be more widely expressed compared with mammals, although the major sites of expression may differ among taxa [thirty]. For instance, in the frog, Lep mRNA expression levels were highest in brain, pituitary and heart [thirty]. The Lep factor is expressed throughout the frog GI tract and in the 2 major sites of expression in mammals, liver and fat, although the levels of expression in frog liver and fat were lower than in another organs that express leptin. Widespread tissue expression of the Lep gene has also been reported in several fishes [63]. In salmon, the highest Lep mRNA expression (sLepA1) was establish in the brain, liver, white muscle and ovary [63]. Many ectothermic species limited the Lep cistron in liver [30,31,32,57,threescore,62,63,64], and so this organ may be a major source of circulating leptin, and a site for nutritional regulation of leptin product [57,63].

Molecular Development of Vertebrate Leptin Receptor Genes

The actions of leptin are mediated past hormone binding to the LepR located in the plasma membrane. The LepR belongs to the class I helical cytokine receptor family unit [102] (fig. 3). These receptors all betoken via the Jak/STAT pathway [103]; although other signaling pathways may be engaged by the LepR, as discussed beneath [104]. The Jak (Jak2) and STAT (STAT3 and STAT5) proteins of import for LepR signaling (and signaling by other hormone-activated cytokine receptors) are highly conserved across vertebrate taxa, much more and then than Jaks or STATs involved with allowed signaling [105]. In mammals, six isoforms of the LepR (LepRa-f) generated by alternating splicing of transcripts derived from a single LepR gene have been identified [12]. All LepR isoforms have a common extracellular ligand binding domain. The long course of the LepR (LepRb) has an approximately 300 amino acid cytoplasmic tail that mediates intracellular signaling upon leptin binding. The LepRa, -c, -d and -f take brusque (∼30–40) amino acid cytoplasmic extensions, while the LepRe lacks transmembrane and cytoplasmic domains and may function as a secreted leptin binding poly peptide. The LepRb is the merely isoform that contains intracellular tyrosine residues necessary for signaling; the physiological functions, if any, for other LepR isoforms are unknown.

The leptin receptor has been isolated by molecular cloning, or predicted based on genome sequence in nine mammals, 3 birds, a reptile (lizard, Anolis carolinensis), an amphibian (frog, Silurana (Xenopus) tropicalis) and iv fishes, although fractional sequence data is available for other species (fig. 4; tabular array 1; online suppl. table 2). Phylogenetic analysis of LepR tracks accepted vertebrate phylogeny well with respect to the monophyly of the classes and the relationships among them (fig. 4). The rate of LepR evolution (averaged across the whole gene) appears to be abiding in different vertebrate lineages. LepR is a group ii, grade 1, helical cytokine receptor [102]. This is based partly on the structure of its CHDs. Our phylogeny based on CHDs from a diversity of vertebrae LepRs shows that the two CHDs of LepR (CHD1 and CHD2) are each monophyletic, and are sis clades with respect to other group 2, form ane, helical cytokine receptors (fig. 3). This indicates that the CHD duplication in LepR occurred afterwards this cistron was distinct from other cytokine receptors, but prior to the difference of fishes and tetrapods. The craven [106] and frog [S. Grommen and R.J. Denver, unpublished] may produce truncated LepR isoforms. Atlantic salmon have 5 LepR isoforms, one that is similar to mammalian LepRb, ane that possesses the transmembrane domain but lacks well-nigh of the cytoplasmic tail, and iii that may exist secreted forms [63]. More data are needed to assess the production and variation of LepR isoforms among vertebrates and to make up one's mind their biological roles.

Table ane

Species, common proper name, accession number, and length of vertebrate leptin (Lep) and leptin receptor (LepR) proteins used in phylogenetic analyses

Fig. 4

Neighbor-joining phylogram of vertebrate leptin receptors. The alignment was conducted using Clustal W2 and based on the BLOSSUM protein weight matrix. The neighbour-joining phylogram was based on uncorrected pairwise sequence divergence of 1,242 amino acrid positions (including gaps). Bootstrap values subtend major, well-supported nodes (≥90%) and were based on 1,000 pseudoreplicates. Vertical lines on the right indicate mammalian orders and nonmammalian vertebrate classes.

The structural features of the extracellular ligand binding domain that govern interactions between leptin and the LepR have been and continue to exist investigated due to the potential to develop selective LepR agonists and antagonists of therapeutic value [88]. Rønnestad et al. [63] recently compared the structures of the LepR extracellular domains of several vertebrate species from fish to mammal. Here we focus on structural features of the cytoplasmic domain of the LepR that are necessary for intracellular signaling.

Jak2 is constitutively associated with the mouse LepRb at membrane-proximal residues located within the cytoplasmic domain [107] (fig. five). The location of these sites has been mapped by deletion assay [107,108]. Jak2 recruitment to LepRb depends on cytoplasmic domain amino acids PXP (a.a. 14–16) located within a region chosen Box 1 that shares features with other form I cytokine receptors [102,109]. The Box one homology motif is highly conserved among vertebrate LepRs (fig. 6). Also necessary are amino acids nineteen–24 (CSWAQG), which are completely conserved amid tetrapods and largely conserved amid fishes; and amino acids 31–48. At that place is flexibility in the sequence requirements for amino acids 37–48, simply the sequence of amino acids 31–36 plays a disquisitional office in Jak2 activation [107]. The latter sequence shows some degree of conservation among vertebrates, while the one-time sequence is only conserved within tetrapods (fig. 6a, c). Bahrenberg et al. [108] showed that the two hydrobic residues, L and F, located at positions 36 and 37 were indispensable for receptor signaling. These two residues are completely conserved among tetrapods and largely conserved in fishes (indicated by stars above the sequences in fig. half dozen).

Fig. v

Intracellular signaling pathways engaged by the leptin receptor (mouse LepRb). The LepRb forms a homodimer in the membrane. Leptin binds to the extracellular domain leading to a conformational change in the receptor, activating Janus kinase 2 (Jak2) which so phosphorylates (designated by 'p') 3 evolutionarily conserved tyrosine residues within the cytoplasmic domain of LepRb. The Y⁹⁸⁵ is required for activation of the SH2-containing tyrosine phosphatase-2 (SHP2)/extracellular signal-regulated kinase (ERK) pour (activation of ERK via growth factor receptor binding protein 2 – GRB2) which leads to the phosphorylation of ribosomal poly peptide S6 and increased translation. Y¹⁰⁷⁷ is required for signaling by indicate transducer and activator of transcription 5 (STAT5), while Y¹¹³⁸ mediates STAT3 signaling. STAT3 transactivates the suppressor of cytokine signaling three (SOCS3) factor, and SOCS3 mediates negative feedback on LepRb signaling via Y⁹⁸⁵. The LepRb besides activates the phosphatidylinositol 3 kinase (PI3K) and mammalian target of rapamycin 1 (mTORC1) pathways, but the mechanism is not understood. The diagram is based on effigy 1 of Villanueva and Myers [104].

Fig. 6

Conserved structural features of the cytoplasmic domains of vertebrate leptin receptors. The alignment colour scheme is every bit described in the fable of effigy ii. a Alignment of tetrapod LepR cytoplasmic domains. Shown are frog [Silurana (Xenopus) tropicalis; NP001037866] lizard (Anolis carolinensis; online suppl. table two), chicken (Gallus domesticus; NM204323), mouse (Mus musculus; AAC52705) and human (Homo sapiens; AAB09673). Box homology motifs 1, 2 and 3 are regions conserved among course I helical cytokine receptors [102]. Sequences within the Box 1 homology motif are important for Jak2 recruitment and activation [107,108]. The existence, position and part of the putative Box 2 homology motif in vertebrate LepRs is uncertain ( [107,108]; the 2 predicted Box 2 homology motifs are shown with question marks). A region immediately North-terminal to the 2nd Box 2 homology motif shows evolutionary conservation and is indicated by the dotted green box. Blue bars beneath the sequence alignments stand for to amino acids 13–24 (long bar) and 31–36 (brusk bar) in the mouse LepRb (numbering from the get-go of the cytoplasmic domain) that have been shown to be disquisitional for Jak2 binding and activation [107]. The red bar corresponds to amino acids 37–48 which have also been shown to function in Jak2 activation but whose sequence requirements are more flexible than amino acids 31–36 [107]. Stars above the sequences indicate the two hydrophobic residues, leucine and phenylalanine, located at positions 36 and 37 shown by Bahrenberg et al. [108] to be indispensable for receptor signaling. The Box 3 homology motif is critical for recruitment and activation of STAT3 [109,111]. The asterisks designate conserved cysteine residues. Arrows testify conserved tyrosine residues in the mouse LepRb necessary for signaling via SH2-containing tyrosine phosphatase-ii (Y⁹⁸⁵ mouse; Y⁹⁸⁶ human; Y⁹⁷⁶ chicken; Y⁹⁷³ frog), STAT5 (Y¹⁰⁷⁷ mouse; Y¹⁰⁷⁹ human: Y¹⁰⁷¹ chicken; Y¹⁰⁶⁶ frog) and STAT3 (Y¹¹³⁸ mouse; Y¹¹⁴¹ human; Y¹¹²⁹ chicken; Y¹¹²⁷ frog). The comparable positions in the lizard LepR could not be fixed because the precise N-terminus of the protein has non yet been determined. b Alignment of iii fish LepR cytoplasmic domains: salmon (Salmo salar; AB489201), zebrafish (Danio rerio; DQ007541) and pufferfish (Takifugu rubripes; AB385663). Shown are the predicted Box i, two and 3 homology domains. The dotted greenish box corresponds to the conserved region in tetrapod LepRs that is N-terminal to the second putative Box two motif. The three conserved tyrosine residues are indicated by the arrows (with the corresponding amino acid position for the mouse LepRb). c Alignment of the LepR cytoplasmic domains of a representative teleost fish (zebrafish – Danio rerio; DQ007541), nonamniote tetrapod [amphibian – Silurana (Xenopus) tropicalis] and amniote tetrapod (homo – Homo sapiens). Shown are the predicted Box 1, 2 and three homology domains. The dotted light-green box corresponds to the conserved region in tetrapod LepRs that is N-terminal to the 2d putative Box 2 motif. The 3 conserved tyrosine residues are indicated by the arrows (with the corresponding amino acrid position for the mouse LepRb).

Two putative Box two homology motifs were proposed for the LepRb (intracellular amino acids 49–60 and 202–213 [107,109,110], shown in effigy 6a with question marks) but were constitute not to exist required for Jak2 signaling [107,108]. Nonetheless, the relatively high evolutionary conservation of residues inside the first putative Box 2 motif (a.a. 49–60) suggests that it has some, as nonetheless undiscovered role in LepR signaling. The second putative Box 2 motif (a.a. 202–213) shows very niggling sequence conservation across taxa. The Box three homology motif is critical for recruitment and activation of STAT3 [109,111].

Hormone binding induces Jak2 autophosphorylation, which increases the Vmax of the kinase, and Jak2 and so phosphorylates three tyrosine residues located in the cytoplasmic domain of LepRb [111] (fig. 5 and half dozen). These three tyrosine residues are conserved amidst all vertebrates that have been studied. Phosphorylation of Y⁹⁸⁵ in mouse LepRb leads to recruitment of SH2-containing tyrosine phosphatase-ii (SHP2; [112,113] which then promotes activation of the extracellular signal-regulated kinase (ERK) pour [104]. Leptin activation of the ERK cascade has been linked to phosphorylation of ribosomal protein S6 and cap-dependent protein translation [104]. The Y⁹⁸⁵ site besides mediates feedback inhibition of the LepRb by suppressor of cytokine signaling 3 (SOCS3) [114]. The residues immediately following Y⁹⁸⁵ are completely conserved among tetrapods (consensus sequence YAT; fig. 6a) but not among fishes (fig. 6b).

Phosphorylation of Y¹⁰⁷⁷ leads to recruitment and phosphorylation of STAT5 [115] (fig. v). Mutation of Y¹⁰⁷⁷ leads to obesity in mice, but the molecular physiological pathways regulated by STAT5 are unclear [104]. Phosphorylation of Y¹¹³⁸ leads to recruitment and phosphorylation of STAT3 [104]. The STAT3 pathway has been shown to be disquisitional for mediating leptin deportment on food intake, glucose metabolism, and weight gain, but does non influence fertility [116]. The Box 3 sequence surrounding Y¹¹³⁸ that forms the binding site for STAT3 is evolutionarily conserved from fishes to mammals (fig. 6c). The consensus STAT3 binding sequence is considered to exist YXXQ; yet, the complete conservation of the P in position 3, and the post-obit two residues FQ (FR in fishes) suggests that the critical residues in the LepR are YXPQFQ/R. This site has been functionally conserved through tetrapod development as evidenced by the finding that the frog LepR activated STAT3 signaling when tested in transient transfection assay [30], leptin injection increased pSTAT3 immunoreactivity in frog brain [C. Hu, C. Pelletier and R.J. Denver, unpublished], and mutation of Y¹¹²⁷ of the frog LepR (homologous to Y¹¹³⁸ in the mouse LepR) abrogated STAT3 signaling [A. Dziuba and R.J. Denver, unpublished]. Activation of STAT3 leads to the upregulation of SOCS3, which binds to Y⁹⁸⁵ on LepRb and inhibits signaling [104]; fig. 6). The phosphoinositol 3 kinase and mammalian target of rapamycin pathways are also engaged by LepRb signaling but the mechanisms for their activation are poorly understood [104].

Tissue Sites of Leptin Receptor Expression

In mammals the LepRb is highly expressed in the hypothalamus and at lower levels in several other tissues including liver [91], kidney, lung [117], stomach [118], pancreatic β cells [119], and immune cells [120]. Leptin'south role in energy residuum/torso weight control is mediated past LepRb expressed in the encephalon [104,121].

In the few nonmammalian species that have been studied, LepR mRNA is every bit highly expressed in the encephalon as information technology is in mammals [30,63,122]. Liu et al. [122] reported that LepR mRNA expression was restricted to the adult zebrafish hindbrain and hypothalamus. Using increased pSTAT3 immunoreactivity following leptin injection nosotros mapped biologically agile LepR in frog to the anterior preoptic area (location of neurosecretory neurons in the frog brain), ventral hypothalamus and anterior pituitary gland [C. Hu and R.J. Denver [,]unpublished]. Like to mammals, LepR mRNA is plant in many unlike tissues in ectotherms [30,63,122] supporting that the hormone has the potential to have diverse influences on development and physiology. In the frog, LepR mRNA is highly expressed in brain, but of all tissues analyzed, LepR mRNA was highest in the pituitary gland [xxx], and leptin increased pSTAT3 immunoreactivity in the frog pituitary [C. Hu and R.J. Denver, unpublished]. These findings advise that leptin could play a role in pituitary evolution and/or function [97].

Evolutionary Conservation of Leptin Actions on Food Intake and Metabolism

The major site of leptin action in mammals is the brain where it acts to inhibit appetite and increase energy expenditure [123,124]. Leptin influences free energy residue through its principal actions on hypothalamic feeding and autonomic control centers, and secondarily through its influence on hypothalamo-pituitary-adrenal (HPA) and hypothalamo-pituitary-thyroid (HPT) axes [46,125,126].

Before Lep genes were isolated from nonmammalian species, injections of recombinant mouse (or 'craven') leptin into chickens was constitute to reduce food intake in one study [127] but not another [128]. In a lizard, injections of mouse leptin reduced nutrient intake and increased metabolic rate [77]. Mouse leptin inhibited food intake in the goldfish [129] but not in other fishes (e.g. coho salmon, catfish, and light-green sunfish; [27]). Although these findings pointed to a similar role for leptin in the regulation of nutrient intake and free energy metabolism in nonmammals equally in mammals, the use of heterologous hormone and the conflicting results obtained in dissimilar species prohibited definitive conclusions.

Contempo comparative studies in nonmammalian species using homologous leptin preparations support an aboriginal role for leptin in regulating food intake and metabolism [30,31,32,130]. The get-go demonstration of an anorexigenic issue of a homologous leptin in a nonmammalian species was shown in the frog, Xenopus laevis [thirty]. Intracerebroventricular injections of recombinant frog leptin (rxLeptin) in juvenile frogs strongly inhibited nutrient intake, and this action developed in the tadpole during prometamorphosis. As in mammals, chronic assistants of recombinant frog leptin reduced trunk weight of prometamorphic tadpoles [30]. Also, frog leptin injections acquired food-deprived tadpoles to lose more than weight than vehicle-injected controls, thus showing that leptin increases energy expenditure in prometamorphic tadpoles as it does in mammals. Murashita and colleagues [31,130] showed that injections of recombinant rainbow trout leptin into trout decreased growth, inhibited feeding, decreased hypothalamic neuropeptide Y mRNA, and increased hypothalamic POMC mRNA. Li et al. [32] reported an anorexigenic issue of injected recombinant grass carp leptin on food intake in the carp.

Nutritional Regulation of Leptin and Leptin's Office in Body Weight Regulation

In mammals, leptin is secreted in proportion to fatty stores and thus signals to the encephalon long-term energy balance [131,132]. Fasting decreases plasma leptin concentration, while refeeding reverses this reject. Piddling information is available in nonmammalian species relating nutrition to leptin production. In bother, hepatic Lep mRNA levels were increased subsequently feeding but did not alter during long-term fasting [57]. The only nonmammalian species for which a leptin radioimmunoassay has been developed is the rainbow trout [133]. Paradoxically, plasma leptin concentration was elevated during fasting in rainbow trout [133]. This led the authors to conclude that the regulation of circulating leptin concentration in fish differs from mammals, and that leptin may not function as an adiposity signal in fish (at least not in salmonids). However, the fact that leptin injections inhibit appetite in nonmammals suggests that information technology can signal to the brain information near energy balance, and and then more work needs to be done to test the hypothesis that leptin functions as an adipostat in nonmammalian species.

Summary and Directions for Hereafter Research

Recent molecular cloning and functional studies have increased our agreement of the diversity of functions and evolutionary history of the hormone leptin. Despite depression primary amino acid sequence conservation, leptins from diverse species are predicted to form like tertiary structures and demark to the LepR, which leads to activation of mutual intracellular signaling pathways via highly conserved structural motifs located inside the LepR cytoplasmic domain.

A major role for leptin in mammals is as an adiposity point, acting on the hypothalamus to suppress food intake and increase metabolic energy expenditure. Leptin's actions on the brain are mediated by the long form leptin receptor (LepRb), which leads to the activation of central melanocortin pathways that inhibit feeding. Similar actions of leptin on feeding have been discovered in nonmammalian species, although the hypothesis that leptin functions as an adiposity signal in nonmammals remains to be tested.

In mammals, leptin is an important indicator of body condition/nutritional state, signaling bachelor energy for evolution, growth, and metabolism. Insufficient energy stores delay creature growth and development, and leptin'due south role equally an adipostat suggests that information technology tin can influence the timing of energy-requiring developmental processes such equally reproductive maturation (puberty in mammals) [96,134,135]. Females must achieve a minimum torso size and body status (i.e. fat stores) to initiate puberty. Leptin may play similar roles in nonmammalian species, signaling appropriate timing for developmental processes such as metamorphosis, a critical life history transition, and the onset of reproductive maturity, an energetically expensive process.

Findings of links betwixt birth weight and adult onset metabolic disorders have focused on the relationships among leptin, growth and development during embryonic and fetal stages [136,137,138,139,140]. Circulating leptin is elevated in the human fetus during late gestation and correlates with fat mass and birth weight [141,142]. Earlier the formation of adipose tissue, leptin and LepR are expressed in liver, heart, hair follicles, and primordial bone of fetal mouse [94,143], and leptin is found in the circulation of fetal sheep [144]. Plasma leptin exhibits a surge during early postnatal development in rodents, and recent findings advise that leptin plays an important role in controlling neurogenesis, and the maturation of feeding circuits in the hypothalamus [124]. Lep mRNA is expressed in frog oocytes and embryos before feeding stages and before adipose tissue formation [30]. LepR mRNA is expressed in tadpole hind limb, and injections of recombinant frog leptin accelerated hind limb development [xxx]. Thus, in addition to its integral role as a regulator of appetite and energy balance in juveniles and adults, these findings highlight the potential for important roles for leptin during early development.

Acknowledgements

The preparation of this paper was supported past NSF grant IOS 0641587 to RJD. We are grateful to 4 reviewers whose helpful comments improved the paper.

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