Leptin and its receptors: regulators of whole-body energy homeostasis

Leptin and its receptors: regulators of whole-body energy homeostasis

DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 15(6):457– 475, 1998 LEPTIN AND ITS RECEPTORS: REGULATORS OF WHOLE-BODY ENERGY HOMEOSTASIS K.L. Houseknecht1 and C...

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DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 15(6):457– 475, 1998

LEPTIN AND ITS RECEPTORS: REGULATORS OF WHOLE-BODY ENERGY HOMEOSTASIS K.L. Houseknecht1 and C.P. Portocarrero Laboratory of Endocrinology and Metabolism, Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-1151 Received May 7, 1998 Accepted July 12, 1998

Leptin is the adipocyte-specific product of the ob gene. Expression of leptin in fully fed animals reflects adipocyte size and body-fat mass. Leptin signals the status of body energy stores to the brain, where signals emanate to regulate food intake and whole-body energy expenditure. The leptin gene was identified in the leptin-deficient, obese ob/ob mouse by positional cloning techniques. Recently, leptin has been cloned in domestic species including pigs, cattle, and chickens. The leptin receptor has at least five splice variants; the long form of the receptor is primarily expressed in the hypothalamus and is thought to be the predominant signaling isoform. Leptin receptors are members of the cytokine family of receptors and signal via janus-activated kinases (JAK)/signal transducers and activators of transcription (STAT) and mitogen-activated protein kinase (MAPK) pathways. Mutations in the leptin or leptin receptor genes results in morbid obesity, infertility, and insulin resistance in rodents and humans. Leptin regulates food intake and energy expenditure via central and peripheral mechanisms. Leptin receptors are expressed in most tissues, and in vitro evidence suggests that leptin may have direct effects on some tissues such as adipose tissue, the adrenal cortex, and the pancreatic b-cell. Leptin is thought to influence whole-body glucose homeostasis and insulin action. Studies are underway to determine the role that leptin plays in the biology of domestic animals. © Elsevier Science Inc. 1998

INTRODUCTION Leptin is an adipocyte-specific protein that functions as an “adipostat” to sense and regulate body energy stores in rodents and humans. The discovery of leptin has helped to define a newly emerging endocrine role for the fat cell and has increased our understanding of how food intake and energy metabolism are regulated. Coordination of whole-body energy balance involves complex regulation of energy intake and expenditure in response to acute (homeostatic) signals (e.g., insulin, catecholamines) and longer term, chronic (homeorhetic) signals. The chronic signals reflect energy demands because of changes in physiological state (e.g., pregnancy), nutritional status (e.g., starvation), or in response to disease (e.g., inflammation, cachexia). Leptin seems to fill the niche as a homeorhetic regulator of energy homeostasis. For many years scientists have been seeking fat-derived hormone/metabolite messengers that signal the status of body energy reserves to the brain and other tissues so that appropriate changes in food intake, energy expenditure, and nutrient partitioning could occur to maintain whole-body energy balance. Such adipocyte-derived mediator(s) would serve as an “adipostat.” Kennedy (1) first proposed this lipostatic hypothesis of bodyweight regulation. Evidence to support this notion was provided by Hervey (2) in 1958 when he conducted now classic parabiosis studies in which the circulatory systems of obese rats (rendered obese by lesion of the ventromedial hypothalamus) were surgically © Elsevier Science Inc. 1998 655 Avenue of the Americas, New York, NY 10010

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Figure 1. The mouse leptin gene. The mouse leptin gene consists of three exons (shaded boxes). The promoter contains transcription response elements including a TATA box, a CAAT/enhancer binding protein (C/EBP) element, a leptin promoter specific factor (LP1), an Sp1 site, and a DR-1 site. All positions indicated in parentheses are relative to transcription start site (arrow). Figure is not drawn to scale. (aa, amino acid).

joined to those of lean rats. The lean animal starved to death, presumably because of exposure to a factor over-produced in the obese (VMH lesioned) animal. Apparently, the VMH lesion rendered the obese animal resistant to the circulating factor. Additional parabiosis studies using obese rodents including ob/ob mice (3), db/db mice (4) and fa/fa rats (5) supported Hervey’s findings. Specifically, obesity was partially cured when ob/ob mice were parabiosed to lean mice (3), suggesting that ob/ob mice lack the circulating adipocyte “factor.” In the case of db/db mice (4) or fa/fa rats (5), parabiosis to lean controls culminated in death by starvation for the lean animals, reminiscent of the experiments with VMH lesioned animals (above). Thus, it was proposed that the VMH lesioned animals and the db/db and fa/fa rodents have a defect in the receptor for the adipostatic factor. In the years after these experiments, biochemical approaches failed to identify the adipostatic factor and its receptor(s). Finally, in 1994, Friedman’s group at Rockefeller University cloned the adipostatic factor we now know as leptin (6). Since that time, numerous studies have been conducted that indicate a pivotal role for leptin in regulating not only food intake, but also energy expenditure. In fact, it seems that leptin may affect essentially every tissue and organ system in the complex process of energy balance regulation. Additionally, evidence suggests that leptin’s regulatory role in wholebody energy homeostasis is plastic, in that leptin responds to acute signals such as insulin and b-adrenergics and is modulated chronically by food deprivation, disease, and changes in physiological state. Leptin: The Gene. Cloning. The morbidly obese ob/ob mouse was first described in 1950 (7). The phenotype of this strain is attributable to a single gene mutation resulting in obesity, hyperphagia, hypothermia, extreme insulin resistance, infertility, and myriad endocrine abnormalities. Zhang et al. (6) identified the ob gene using positional cloning strategies. The ob gene consists of three exons with the two coding regions separated by two introns (Figure 1). The ob gene encodes a 4.5-kb mRNA product with a 167-amino acid open reading frame and a 21-amino acid signal sequence, indicative of a secretory protein (6). Later studies confirmed that leptin is secreted from adipocytes into the bloodstream. The ob mutation described for the original coisogenic C57BL/6J ob/ob strain was mapped to mouse Chromosome 6 (6). In the C57BL/6J ob/ob strain, a non-sense C 3 T mutation changes an arginine at Position 105 to a stop codon (6). This mutation results in a 20-fold increase in abnormal ob mRNA (6). Mutation of the ob gene in the coisogenic SM/Ckc-1Dacob2J/ob2J mouse strain results in the absence of leptin mRNA production, presumably because of a mutation in the ob promoter (6). A mutation in the human ob gene leading to leptin deficiency reminiscent of ob/ob mice was reported in two children with the same consanguineous pedigree (8). The homozygous frameshift mutation is the result of the deletion of a single guanine nucleotide in

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codon 133. These children produce very little leptin, present early onset morbid obesity, have seemingly uncontrolled hyperphagia, but have normal body temperatures and normal plasma cortisol and glucose concentrations (8). Additionally, the older child presents hyperinsulinemia consistent with insulin resistance observed in ob/ob mice; this may be influenced by age. Additional mutations in the human leptin gene result in less severe hypoleptinemia than reported (8). Genetic screening of morbidly obese patients revealed a polymorphism in Exon 1 of the ob gene (A 3 G, base 19), which resulted in lower BMI-corrected leptin concentrations compared to obese patients with no mutation (9). The ob gene is highly conserved among vertebrates. Human leptin shares 84% sequence identity to mouse leptin (6). Recently, the ob gene has been cloned in several domestic animal species. The porcine ob gene was cloned by Bidwell et al. (10); pleptin shares 95%, 92%, and 89% sequence homology to bovine, human, and murine leptin, respectively. Partial cloning of the bovine leptin gene by Ji et al. (11) revealed approximately 87% sequence homology to mouse and human leptin. Cloning and tissue distribution of the ob gene in chickens revealed that leptin expression is not restricted to adipose tissue in this species, but is expressed in liver as well (12). Chicken leptin is 97, 96, and 83% homologous to mouse, rat and human leptin, respectively (12). Promoter. Leptin gene expression is restricted to adipose tissue and placenta in mammals and is regulated by adipose tissue mass and hormones such as insulin and glucocorticoids [see reviews (13,14)]. Specific transcription factor binding motifs in the leptin promoter may explain endocrine regulation of leptin gene expression. To date, several important regulatory domains have been identified (see Figure 1). These include binding domains for CAAT/enhancer binding proteins (C/EBP; 15–17), SP-1 (17), glucocorticoids [GRE, cAMP, CREB (18)] and peroxisome proliferator activated receptor g (PPARg; 19). C/EBPa is expressed in multiple cell types, functions as a transcriptional activator of many adipocyte genes, (20,21), and plays a role in terminal adipocyte differentiation (22,23). Functional binding sites for C/EBP are located in the region -58 to -42 relative to the transcriptional start site (24 –26). The C/EBPa binding site, which lies 15 bp upstream from the TATA box in the mouse leptin promoter, is required for maximal promoter activity (15–17). Mutation analysis of the leptin promoter revealed a novel transcription site (LP1) in addition to the C/EBP, SP1 and TATA regulatory motifs (17). These regulatory motifs are important for leptin gene expression because, in each case, point mutations resulted in two- to threefold reductions in leptin promoter activity (17). Additionally, when multiple regulatory motifs were mutated there was an additive effect on inhibiting leptin promoter activity, indicating that each promoter regulatory site contributes independently to leptin gene transcription (17). Transcriptional regulation of the leptin gene also seems to be controlled by PPARg. PPARg1 and 2 are members of the family of orphan nuclear receptors that function as trans-activators of fat-specific genes such as aP2, and thus are dominant activators of fat cell differentiation (27–29). Thiazolidinediones (TZD), pharmacological ligands for the PPARg (30,31), down-regulate leptin mRNA abundance in adipocytes. These data are consistent with a role of PPARg in regulating the leptin promoter (32–34). A canonical DR-1 PPARg binding site is located between residues -3951 and -3939 of the mouse 59-flanking sequence of the leptin gene (19). The mechanism by which PPARg ligands down-regulate leptin gene expression seems to be via inhibition of C/EBP a-mediated transactivation (19). However, not all investigators have been able to show PPARg regulation of the leptin promoter (17). Leptin expression (mRNA and protein) in humans, rodents, and pigs (fed state) is highly correlated with fat mass, BMI, and adipocyte size (10,13,14,). Transfection of

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leptin promoter reporter constructs into adipocytes from obese fa/fa rats resulted in increased reporter activity, consistent with the notion that leptin expression is regulated by adipocyte size (17). Expression of other adipocyte genes (e.g., GLUT4 and GAPDH) is also correlated positively to adipocyte size (35,36). The mechanisms underlying cell size-dependent regulation have not been fully elucidated. Leptin expression is regulated by hormones such as insulin and glucocorticoids [see reviews (13,14,37)]. The transcription factor, ADD1/SREBP1 provides a key link between insulin and changing nutritional status to the expression of genes important in the regulation of energy homeostasis (38). ADD1/SREBP1 strongly transactivates the leptin promoter (38); however, the response element for ADD1/SREBP1 has yet to be identified in the leptin promoter. Hormones such as insulin may also regulate leptin expression via regulation of expression and phosphorylation status of the phosphoprotein transcription factors C/EBPa and SP1 (39 – 42). The factor that binds LP1 and activates the leptin promoter has yet to be discovered. However, it seems to be present in preadipocytes and adipocytes but not other cell types (17). Thus, the LP1 factor may be important for fat-specific expression. It remains to be determined if this factor is regulated by hormonal stimulation of transcription and/or phosphorylation status. Leptin concentrations in body fluids and tissues undergo dynamic changes during pregnancy and lactation. Leptin is found in human milk, is higher in whole compared to skim milk, and correlates with maternal adiposity and plasma leptin concentrations (43). A recent report (44) suggests that milk-borne leptin is produced by mammary epithelia and is associated with milk-fat droplets. It remains to be determined what proportion of milk leptin is derived from maternal circulation or made in the breast. Leptin is expressed in the placenta (18,45,46), and serum leptin concentrations increase during pregnancy in women and rodents (45–50). Human placental leptin mRNA is expressed at levels 100-fold lower than found in adipose tissue (51). The leptin mRNA transcript is the same size as that expressed in adipose tissue and the same promoter is used for placental and adipose tissue leptin expression (51). Studies that examined the regulation of leptin expression in placenta reported that when leptin promoter-luciferase constructs containing 1775–2992 bp of 59 flanking region of the leptin promoter were transfected into human trophoblastic cell lines, there was significant promoter activity (51,52). Furthermore, mutations between -1885 and -1830 significantly reduced promoter activity (52). When this region was mutated and then transfected into rat adipocytes, leptin promoter activity was unaltered, indicating trophoblast-specific regulatory elements in this region of 59flanking sequence (52). Oksana et al. (51) identified the 100 base-pair region from -1847 to -1946 as the location of the placental leptin enhancer. These authors identified two novel placenta-selective binding sites in this region denoted PLE1 and PLE3, and found that the placental enhancer and transcription factor binding to the PLE3 site are placentalspecific (51). Leptin structure. Leptin has been classified as a cytokine based upon the structural similarity between the leptin receptor and gp130, a member of the interleukin-6 family of receptors (53). Threading analysis of leptin sequence indicated a cytokine folding pattern (54). Nuclear magnetic resonance (NMR) methodology revealed that leptin is a four-helix bundle cytokine (55). Crystal structure analysis of a mutant form of the human leptin protein (E-100) confirmed the four-helix bundle structure for leptin, and provided evidence that the structure is similar to the long-chain helical cytokine family (56). Leptin contains four antiparallel a helices connected by two long crossover links and one short loop arranged in a left-handed twisted helical bundle (56). Additionally, NMR (55) and crystal structure (56) analyses revealed that leptin contains a single disulfide linkage

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connecting the CD loop to the carboxyl terminus. This disulfide linkage is crucial for leptin function, since disruption of the cysteine leads to loss of bioactivity (56,57). Leptin: Its Receptors. Cloning and structure. The leptin receptor was first identified by expression cloning techniques (53). Tagged leptin constructs were used to identify tissues with high levels of leptin binding. Leptin-specific binding was abundant in mouse choroid plexus. Subsequently, a choroid plexus cDNA library was prepared and screened; proteins were identified that bound leptin with high affinity (Kd 5 0.7 nM) (53). The human homolog of the mouse leptin receptor was cloned using a human infant total brain library (53). A major difference between the human and mouse OB-R, is the longer intracellular domain expressed in the human receptor, which most closely resembles gp130 (53). The extracellular domain of OBR consists of 816 amino acids (53) containing two cytokine binding domains (amino acids 251–325 and 551– 627), each containing a single copy of the characteristic Trp-Ser-X-Trp-Ser motif, and a fibronectin type III domain (58). The duplicated ligand binding domains are found in 8 exons (59). Deletion/substitution mutation experiments determined that the first potential leptin binding domain is unnecessary for leptin binding and receptor activation (60). However, modification of the second domain renders the receptor inactive (60), indicating that the leptin binding domain is localized in the second cytokine binding region. These structural motifs are distinct for the leptin receptor, because most members of the cytokine family of receptors contain only one CK-F3 domain (60). The extracellular domain is followed by a 23-amino acid transmembrane domain [coding Exon 16; (59)] and intracellular domain which varies in length from 30 to 303 amino acids, depending on alternative splicing (see below). The first 29 amino acids of the cytoplasmic domain are identical in OBR-l and OBR-s isoforms (61). Alternative splicing. At least five different isoforms of the leptin receptor exist because of alternative splicing of a single leptin receptor gene (62,63). The extracellular and transmembrane domains are identical between the short and long isoforms; differences are due entirely to changes in the length of the cytoplasmic domain (62). The long form, denoted OB-Rl, is predicted to have 302 cytoplasmic residues compared with the short isoforms (OB-Rs) whose cytoplasmic residues range from 32– 40 amino acids in length (62). Additionally, a soluble form of the leptin receptor (OB-Re) is predicted to contain no intracellular motifs or transmembrane residues, thus consisting entirely of the extracellular domain of the receptor. The OB-Re transcript includes 14 coding exons and an alternatively spliced 39 terminal exon (59). Isoforms of the leptin receptor have been identified in multiple tissues. The long form of the receptor, OB-Rl, is most highly abundant in the hypothalamus, especially in the ventromedial hypothalamus, a key regulatory center for appetite control (53). The high abundance of OB-Rl in the hypothalamus coincides with the multiplicity of leptin effects which are mediated centrally (see discussion). OB-Rl is also expressed at lower levels in multiple tissues including: lung, kidney, liver, adipose tissue, and pancreatic b-cells (64,65). The short isoforms of the leptin receptor can be found in almost all tissues tested to date. This may explain the apparent direct effects of leptin on target tissues such as adipocytes, liver, pancreas and skeletal muscle in vitro (66 – 68). It is unclear what the relative importance of peripheral leptin receptors is to the overall actions of leptin on the regulation of whole-body energy homeostasis. Leptin receptor mutations. Multiple obese rodent models have mutations in the leptin receptor gene, highlighting the importance of leptin and its receptors in the regulation of whole-body energy homeostasis. The leptin receptor gene maps to Chromosome 4 of the mouse and Chromosome 5 of the rat in regions that contain the db/db and Zucker fa/fa

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mutations (69). The db mutation consists of a point mutation (G 3 T transversion) that results in a novel splice donor site in the extreme C-terminal portion of the 39 untranslated region of the penultimate exon (62,63). This causes a frameshift that results in inappropriate splicing so that OB-Rl has a truncated intracellular domain similar to the predominant OB-Rs isoform. The fa mutation is a single amino acid mutation in Codon 269, which lies within the first cytokine receptor motif of the extracellular domain of the receptor (70 –71). The fa mutation has no effect on mRNA abundance, or on affinity for leptin; however, abundance of OBR at the cell surface is greatly reduced (71). The obese Koletsky rat also has a mutation in the leptin receptor gene (71). Codon 763 in the extracellular domain of the receptor is mutated to a stop codon (Tyr 3 Stop) that results in virtual absence of LR mRNA (71). The human leptin receptor gene shares 78% homology to the mouse gene at the amino acid level (53) and maps to chromosome 1p31 (53). Polymorphisms in the leptin receptor gene have been reported in both obese and non-obese humans (72–74). Recently, a homozygous mutation in human OB-R has been reported (75). This mutation results in a truncated receptor that lacks the transmembrane and intracellular domains. Patients who are homozygous for this mutation are obese, do not achieve puberty and have abnormal growth hormone and thyroid stimulating hormone secretion (75). Furthermore, these patients have normal glycemia and normal glucose tolerance despite their obesity, and, unlike rodents with leptin or leptin receptor mutations, exhibit no defect in the hypothalamic-pituitary-adrenal-axis (75). Leptin receptor signaling. Recent studies have shed light on the mechanisms by which leptin receptors transduce their intracellular signals. Initially, it was believed that only the long form of the leptin receptor was functional; it was thought that the short leptin receptor isoforms were incapable of signal transduction, or at best had greatly reduced capacity to signal (62,63,65,76). Figure 2 illustrates what is currently known about leptin receptor signal transduction. Upon leptin binding to its receptor, receptor dimerization occurs; this seems to be required for signaling activity (77–78). Leptin receptors undergo homodimerization, a characteristic shared with other members of growth hormone subfamily of receptors (77–78). However, unlike many cytokine receptors, OB-R does not form heterodimeric complexes with structurally related receptors. Because OB-R shares significant homology to cytokine receptors, it is not surprising that the long form of the receptor signals via JAK/STAT activation (79). Leptin infusion of normal but not db/db mice resulted in hypothalamic STAT activation, presumably via OB-Rl (80). Sequence analysis of the intracellular domain of OB-Rl identified a JAK binding domain (amino acids 869 – 876) and a STAT3 binding domain (amino acids 1138 –1141; (53). In cultured cells transfected with OBR expression vectors, OB-Rl can activate JAK and STAT proteins (79,81,82). Specifically, OB-Rl can activate JAK2, STAT3, STAT5, and STAT6 but not STAT1, STAT2, and STAT4 in transient transfection studies (65). In contrast, the short isoforms of the leptin receptor are unable to activate STAT proteins (65). Signal transduction by members of the class I cytokine receptor family is not limited to the JAK-STAT system. Some of these receptors are linked to mitogen-activated protein kinase (MAPK) or phosphotidyl inositol-3 (PI-3) kinase pathways (83) In C2C12 cells, leptin stimulates glucose transport (84) and activates PI-3 kinase via JAK2 and IRS-2 dependent pathways (85). Recently an elegant study by Bjorbaek et al. (82) shed new light on the differential signaling pathways for the short and long isoforms. Leptin-dependent tyrosine phosphorylation of its receptors (both long and short isoforms) occurs in cells co-transfected with JAK2 (82). When OB-Rl or OB-Rs were transfected with JAK2 and

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Figure 2. Schematic illustration of leptin receptor signaling pathways. After leptin binding and receptor homodimerization, the long form of the leptin receptor (OB-Rl) activates janus-activated kinases (JAK)/STAT (signal transducers and activators of transcription) pathways leading to the activation of c-fos. OB-Rl activation may also phosphorylate JAK leading to the activation of insulin receptor substrate-1 (IRS-1) and mitogenactivated protein kinase (MAPK). Upon leptin binding, the short form of the leptin receptor (OB-Rs) phosphorylates IRS-1 and consequently activates MAPK. The activation of MAPK leads to the activation of pp90 S6-Kinase S6-K). (1, activation; P, phosphorylation).

insulin receptor substrate (IRS)-1, leptin binding induced tyrosine phosphorylation of IRS-1 (82). Studies in transfected cells revealed leptin-stimulated activation of JAK/STAT pathways ultimately leading to the activation of c-fos (82). In contrast, signaling by the short isoforms leads to JAK activation but not STAT activation (82). Additionally, the short isoforms were able to signal MAPK activation. Thus, the data indicate that the OB-Rs can perform signaling functions, however; the physiological relevance of leptin signaling via OB-Rs has yet to be determined. Physiological regulation. To date, very little data exists concerning leptin receptor promoter function/regulation. However, it has been reported that the OB-R promoter can drive expression of a different protein by “leakage” during transcription (86). Sensitivity to leptin is of importance in the regulation of leptin action with obesity and during fasting, anorexia and disease. Central sensitivity to leptin could be impacted by leptin transport across the blood– brain barrier, by alteration in leptin receptor signaling and/or changes in the expression of OB-RL and/or OB-Rs isoforms. Expression of OB-Rl in the arcuate nucleus of ob/ob mice is attenuated with systemic leptin treatment (87). Expression of OB-Rl is also affected by fasting; in neuropeptide Y (NPY) knockout and normal mice that were fasted for 48 hr, there was a significant increase in OB-Rl mRNA abundance [400% and 247%, respectively; (87)]. Additionally, moderate undernutrition reduced leptin receptor abundance in ovine hypothalamus (88). Thus, increased expression of OB-Rl in the hypothalamus may result in increased leptin sensitivity. The level of OB-R expression in adipocytes may be important in the regulation of leptin gene expression (89). It was recently shown that leptin expression in adipocytes from fa/fa rats could be

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predicted by the level of OB-R expression, suggesting that leptin down-regulates its own expression (89). Leptin in circulation: Role of binding proteins. Data from several groups indicate that leptin circulates bound to proteins in serum of rodents and humans (90,91). Furthermore, leptin binding to serum proteins appears to be saturated with obesity (90,91) and altered with pregnancy (92). Although specific leptin binding proteins have not been cloned, evidence suggests that a soluble form of the leptin receptor may be a serum leptin binding protein (91). Sequence analysis for the leptin receptor presented the possibility of a soluble leptin receptor due to alternative splicing (62); many members of the cytokine family circulate bound to soluble receptors in serum (93,94). Polymerase chain reaction evidence (62,64) indicated that OB-Re is expressed in multiple tissues including adipose tissue, hypothalamus, thymus, liver heart, and testes. Additionally, expression of the soluble OB-R in the placenta, and its appearance in serum is increased during mid to late pregnancy in the mouse (49,92). Although it seems that a soluble leptin receptor exists in rodents, it is not yet clear that the soluble receptor is expressed in humans. Expression of the cDNA for the extracellular domain of human OB-R in COS cells revealed that the soluble leptin receptor secreted into cell culture media binds leptin as a dimer with high affinity (95). However, Sinha et al. (91) reported that less than 10% of circulating leptin is bound to the soluble receptor in humans. Thus, the physiological relevance of a soluble leptin receptor in humans remains unclear. Regulation of Whole-Body Energy Homeostasis. The lipostatic theory of body weight regulation proposes complex mechanisms by which body energy reserves are “sensed” and peripheral signals are transmitted to the brain (and perhaps peripheral tissues), resulting in altered energy expenditure, nutrient partitioning, and energy intake. Rodents and humans that have mutations in the leptin or leptin receptor genes give credence to the notion that leptin and its receptors play a profound, pivotal role in the regulation of whole-body energy homeostasis. Leptin (ob/ob mice) and leptin receptor mutants (db/db mice, fa/fa rats) have lower core body temperatures, are more susceptible to cold stress soon after birth, and are hyperphagic and obese by the time of weaning (96). Children with mutations in the leptin gene (8) are likewise hyperphagic and obese, however lack the hypothermia, stunted growth, and reduced lean body mass observed in ob/ob mice. Although such mutations in humans are extremely rare, extreme obesity in human subjects has been linked to genetic markers proximal to the leptin gene (97,98). Furthermore, it was reported recently that a homozygous mutation in the human leptin receptor caused morbid obesity and infertility in a group of siblings (75). Central versus peripheral effects. The hypothalamus is the major regulatory center for food intake and body-weight maintenance. Experimentally induced hypothalamic lesions in rodents dramatically illustrate the profound role the hypothalamus plays in energy homeostasis regulation. Specifically, lesions in the ventromedial hypothalamus result in hyperphagic obesity, whereas lesions in the lateral hypothalamus result in weight loss and reduced food intake (99 –101). The majority of data suggest that leptin regulation of whole-body energy homeostasis is mediated centrally, presumably at the level of the hypothalamus where the long form of the leptin receptor predominates. Leptin regulates the synthesis and secretion of myriad neurotransmitters involved in food-intake regulation, as well as the secretion of growth hormone from the pituitary (102,103). Growth hormone has nutrient partitioning effects and may mediate leptin effects on peripheral tissues. However, data are accumulating that leptin may have direct, peripheral effects as well. Leptin receptors are expressed throughout the body; although in most tissues the short isoforms predominate. Recently, multiple in vitro studies using cell lines and in some cases, primary cell culture, have revealed

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direct effects of leptin on adipocytes, skeletal muscle, ovary, adrenal cortex, and pancreatic b-cells [see reviews (14,104)]. Much more data are needed, however, to determine the relative importance of peripheral versus central leptin effects and the physiological relevance of the short isoforms of the leptin receptor. The latter point is highlighted by the fact that the short isoforms of the leptin receptor exist in peripheral tissues of db/db mice, yet the obese, diabetic phenotype remains (62,63). Leptin affects energy intake. The most well-known function of leptin is to regulate food intake. Uncontrolled hyperphagia is a symptom of leptin deficiency or leptin signaling deficiency in rodents and humans. Leptin regulation of food intake is mediated at the level of the hypothalamus. NPY has been implicated in mediating many leptin-induced effects on food intake and thermogenesis (105). NPY potently stimulates food intake, inhibits thermogenesis, and increases plasma insulin and glucocorticoid concentrations (106,107). NYP expression in the mediobasal hypothalamus is increased in many obese rodent models (including ob/ob) and with fasting (108,109). Leptin acts centrally to inhibit the effects of NPY by inhibiting its synthesis in the arcuate nucleus (105,110). Leptin administration to hyperphagic ob/ob mice results in a rapid reduction in NPY mRNA abundance, protein secretion, and reduced food intake before any change in body weight (111,112). More direct evidence for leptin regulation of NPY expression is that leptin treatment directly suppressed NPY release from perfused rat hypothalami isolated from normal animals (111). Despite this compelling evidence, it is now clear that NPY is not the only neuroendocrine target of leptin. Elegant studies in which NPY was knocked out in ob/ob (105) illustrated that the absence of NPY attenuated, but did not completely normalize, all aspects of the obesity phenotype. Furthermore, wild-type mice that lack NPY control their food intake and body weight normally, and have normal food intake response to leptin (113). Other potential mediators of leptin action in the mediobasal hypothalamus include proopiomelanocortin (POMC), melanocortin stimulating hormone (a-MSH) and agoutirelated peptide (AGRP). Studies using the obese, leptin-resistant agouti (Ay/a) mouse helped to elucidate the roles these neuropeptides play in the regulation of feeding behavior. The agouti mutation results in high ectopic expression of agouti, which is an antagonist of MC4 and other members of the melanocortin receptor family (114). The MC4 melanocortin receptor has been shown to play a pivotal role in energy homeostasis regulation; deletion of the MC4 gene results in obesity reminiscent of that observed in the agouti mouse (115). a-MSH (derived from POMC precursors) acts as an agonist for MC4; MSH activation of MC4 culminates with reduced food intake (116). POMC neurons in the hypothalamus express OB-Rl, and leptin stimulates POMC expression (117). In contrast, AGRP (expressed in the arcuate nucleus) antagonizes the MC4 receptor (118). AGRP is overexpressed in the hypothalamus of ob/ob mice (118), and overexpression of AGRP results in obesity (118). The role of the MC4 receptor in the regulation of leptin’s satiety effects is further supported by the observation that pretreatment with MC4 receptor antagonists blocks the satiety effect of leptin treatment (119). Melanin concentrating hormone (MCH), like a-MSH, is involved in regulation of skin color and seems to be a functional antagonist to a-MSH (120). MCH mRNA is overexpressed in ob/ob mice (121) and with fasting (121) and ICV injection of MCH stimulates feeding behavior (121). Additionally, intracerebro ventricular (i.c.v.) leptin administration reduced hypothalamic MCH and galanin (GAL) mRNA expression while increasing neurotensin (NT) expression (122). These leptin-induced changes in gene expression are independent of reduced food intake since expression of MCH, GAL, and NT was unchanged in pair-fed controls (122). Recently, two new lateral hypothalamic neuropep-

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tides, orexins A and B have been identified as hyperphagic agents (123). It is not yet known if leptin regulates the expression or function of the orexin peptides. Much of what is known concerning the effects of leptin on food-intake regulation has been elucidated using the leptin-deficient ob/ob mouse. However, leptin deficiency, per se, is not the major cause of hyperphagia and obesity in most obese rodents and humans. Leptin expression and secretion are positively correlated to adipose tissue mass and fatcell size in fully fed animals and humans (13,14). Thus, hyperleptinemia, not leptin deficiency is the hallmark of obesity. This observation has led to the hypothesis that obesity is associated with leptin resistance more reminiscent of the db/db mutation (13,14). The cause of this so-called leptin resistance has yet to be fully elucidated. Leptin resistance could result from leptin receptor mutation leading to an absence of or inappropriate leptin signaling. The recent discovery of such a mutation in humans (75) reinforces this notion. Leptin resistance may also be the result of a transport defect at the level of the blood– brain barrier. Leptin is transported across the blood– brain barrier via a saturable system (124 –126), and obese subjects with very high plasma leptin levels have been shown to have CSF levels more similar to normal-weight subjects (125), suggesting that the hypothalamic leptin receptors never “see” the additional leptin. This may be an important mechanism for anorexics, because CSF leptin concentrations are normalized before normalization of plasma concentrations and body weight (127). Thus, CSF leptin concentrations my “sabotage” efforts to normalize body weight in these patients. Additionally, leptin circulates bound to proteins in serum (90 –91), and this may affect leptin delivery to target tissues and/or half-life in serum. Most circulating leptin in bound in lean animals and humans; it is unknown if the binding status is important for transport into the brain. Recently, Bjorbaek et al. (128) have provided exciting new molecular evidence for mechanism(s) underlying leptin resistance. These authors report that leptin induces the expression of a member of the suppressors-of-cytokine-signaling (SOCS) family, SOCS-3. Peripheral leptin treatment of ob/ob (but not db/db) mice resulted in a significant increase in SOCS-3 (but not other SOCS isoforms) mRNA in leptin-responsive hypothalamic neurons (128). Additionally, SOCS-3 mRNA was increased in the arcuate and dorsomedial hypothalamus of the leptin-resistant, obese agouti mouse (Ay/a). Bjorbaek et al. (128) also directly tested the specific effect of SOCS-3 expression on leptin signaling by transiently transfecting various members of the SOCS family into mammalian cell lines. They found that SOCS-3 but not SOCS-2 or CIS (cytokine-inducible sequence) attenuated leptin-induced signaling events (128). Overexpression of SOCS-3 in the hypothalamus of obese subjects may be a mechanism underlying leptin resistance, and SOCS-3 may be an important target for anti-obesity drug therapy. Leptin affects energy expenditure. Leptin has been implicated in the regulation of whole-body energy expenditure based on observations from in vivo and in vitro experimentation. Leptin administration to ob/ob mice increases basal metabolic rate and thermogenesis, which, coupled with reduced food intake, results in loss of body weight (129). Leptin’s effects on weight loss are not limited to changes in food intake as evidenced by studies in which normal rats were administered a leptin-adenovirus construct. In these animals, leptin treatment abolished adipose tissue depots, and the loss of body fat was greater than observed in pair-fed controls (130). In a separate study, central leptin administration caused deletion of adipocyte stores by stimulating apoptosis (131). Additionally, in healthy lean human subjects, increased energy expenditure during hyperinsulinemic euglycemic clamp was positively correlated with plasma leptin concentrations (132). It remains to be determined if leptin effects on whole-body energy expenditure are mediated centrally, peripherally, or by both systems.

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The thermogenic effects of leptin may be mediated via the sympathetic nervous system. Leptin increases norepinephrine turnover in brown adipose tissue (133) and increased sympathetic nervous activity to brown fat, hindlimb, adrenal gland and kidney of lean Sprague–Dawley rats (134). The latter effects were not observed in obese Zucker rats that are known to have a mutation in the OB-R gene (134). Leptin also increases lipolysis in white adipose tissue, a process that is activated by sympathetic stimulation (135,136). A potential mechanism for leptin-induced thermogenesis could be the down-regulation of NPY expression and secretion. NPY regulates sympathetic stimulation of brown adipose tissue, uncoupling protein 1 (UCP1) expression, and UCP1 activation in rodents, culminating in reduced energy expenditure (106). In contrast, leptin increases the expression of UCP1 in brown adipose tissue (137), perhaps via inhibition of NPY. Leptin also increases the expression of the newly discovered UCP2 and UCP3 in peripheral tissues (137–139). Whereas UCP 1 is only expressed in brown adipose tissue, UCP2 is ubiquitously expressed (140,141) and UCP 3 is highly expressed in skeletal muscle and in very low levels in heart and brain (142–144). Most of the variation in basal metabolic rate between individuals can be explained by differences in energy expenditure in skeletal muscle (145). Thus, UCP3 may be a major determinant of whole-body energy expenditure. Furthermore, leptin increases fatty acid oxidation in skeletal muscle (68), which may be linked to increased expression and/or activation of UCP3. Metabolic rate and therefore energy expenditure are regulated by other endocrine factors including thyroid hormone. Evidence is mounting for a connection between leptin and thyroid hormone; leptin administration prevented the fasting-induced reduction in TRH mRNA in the medial parvocellular neurons (146) and leptin increases circulating T3 and T4 concentrations (147,148). Furthermore, thyroid hormone, as well as leptin and b-adrenergic agonists, stimulates the expression of UCP3 in skeletal muscle (149). Thus, each of these components may work synergistically to increase energy expenditure in leptin-treated animals. Leptin affects glucose metabolism and insulin action. An important regulator of energy homeostasis is insulin. Insulin stimulates glucose and amino acid uptake by tissues and tissue anabolism. It is not surprising that a link between leptin and insulin exists in the chronic regulation of energy homeostasis. First, insulin stimulates leptin gene expression in adipose tissue and plasma leptin concentrations [see reviews (13,14,104)]. The ability of insulin to regulate leptin expression seems to be chronic, requiring in most cases 3– 4 hr for stimulation (150 –153). Unlike insulin, plasma leptin concentrations are not tightly linked to meal feeding but can be elevated with chronic hyperinsulinemia both in vivo and in vitro (1540 –154). However, to date there is no link between leptin and Type 2 diabetes. A classic characteristic of a chronic, homeorhetic regulator of metabolism is its ability to alter tissue response to acute homeostatic signals such as insulin. This seems to be the case for leptin; both in vitro and in vivo evidence indicate that leptin regulates glucose metabolism and insulin action. Skeletal muscle, adipose tissue, and liver are the key insulin-responsive organs involved in glucose homeostasis. Leptin receptors are expressed in each of these tissues although the short isoform is predominant (64). However, in the case of adipose tissue and liver, there is low-level expression of OB-Rl (155). Thus, it is possible that leptin could directly regulate insulin action and glucose metabolism in these tissues. Leptin treatment (physiological concentration) attenuated the ability of insulin to stimulate lipogenesis, glycogen synthesis, amino acid uptake, and antilipolysis in isolated rat adipocytes (67), suggesting that leptin could be involved in the peripheral insulin resistance associated with obesity. Leptin stimulated glucose uptake into C2C12 muscle cells (84) but not L6 muscle cells (156), isolated rat and mouse skeletal muscle (68,157),

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Figure 3. Schematic illustration of leptin coordination of energy homeostasis: central and peripheral mechanisms. Leptin is secreted by adipocytes and acts in an autocrine fashion to increase adipocyte expression of uncoupling protein 2 (UCP2) and attenuate insulin action. Leptin acts on the hypothalamus to control food intake, thermogenesis, and insulin action by regulating expression and secretion of multiple neurotransmitters including neuropeptide Y (NPY), galanin (GAL), and melanin-concentrating hormone (MCH). Leptin also upregulates neurotensin (NT) and proopiomelanocortin (POMC) and increases sympathetic tone. Leptin increases fatty acid oxidation, and upregulates uncoupling proteins 2 and 3 (UCP2 and UCP3) in skeletal muscle. The synthesis and secretion of cortisol is inhibited by leptin treatment. Leptin inhibits insulin secretion, upregulates UCP 2, and increases FA oxidation in the pancreas. In liver, the role of leptin in insulin action is equivocal. In brown adipose tissue (BAT) leptin upregulates uncoupling protein 1 (UCP1), which leads to increased thermogenesis. (1, increase; 2, decrease; ?, unknown/unclear).

or isolated rat and mouse adipocytes (156,157). Leptin increased glucose utilization of brown adipose tissue (135), lipolysis in white adipose tissue (135,136) and expression of malic enzyme and lipoprotein lipase in brown adipocytes in culture (135). Additionally, treatment of preadipocytes with leptin reduced expression of acetyl coA carboxylase and de novo lipid synthesis (158). Cohen et al. (66) reported that leptin treatment of HepG2 cells attenuated some insulin signaling events, but others have been unable to repeat these findings (155). One possible explanation for these discrepancies could be attributable to different levels/isoform distribution of leptin receptor expression in cultured versus primary cells and differing cell culture conditions. In vivo evidence suggests that leptin may regulate (at least acutely) whole-body glucose metabolism. Siegrist-Kaiser et al. (135) reported increased glucose utilization in brown adipose tissue following i.v. leptin administration. Kamohara et al. (147) reported that leptin acutely regulates whole-body glucose metabolism independent of changes in plasma insulin concentrations. Leptin administration (i.v. or low-dose i.c.v. infusion) to lean mice for 5 hr increased glucose uptake into skeletal muscle and brown adipose tissue, increased whole-body glucose turnover, and increased glucose oxidation (147). Furthermore, the leptin-induced increase in glucose uptake into soleus muscle was partially attenuated by denervation (147). Taken together, these data indicate that the effects of acute leptin administration on glucose metabolism are mediated centrally, and effects in

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skeletal muscle are dependent, at least in part, on neuronal signals. These effects could be mediated by increased whole-body insulin sensitivity or by insulin independent pathways. Leptin affects insulin secretion. Leptin has also been implicated in the regulation of insulin secretion from pancreatic beta cells. Leptin receptors are found on beta-cells (159,160), and leptin effects on insulin secretion seem to be mediated via alteration in ionchannel function (160,161). Deficiency in leptin action attributable to leptin receptor mutation in Zucker diabetic fatty rats results in lipid accumulation and b-cell death via lipotoxemia (162–164). This phenomenon is reversed with expression of leptin receptors in pancreatic islets (165) or with caloric restriction (166). SUMMARY Data are rapidly accumulating to support the notion that leptin plays a pivotal, integrative role in the regulation of whole-body energy homeostasis. Figure 3 summarizes the myriad effects of leptin on the hypothalamus and peripheral tissues. Although it remains to be determined if effects on peripheral tissues are mediated directly by leptin or centrally via a leptin-induced mediator, it is clear that leptin is acting to orchestrate the complex array of signals which regulate food intake, energy expenditure and nutrient partitioning. Leptin has been cloned in several domestic animal species, recombinant speciesspecific leptin is being generated, and the race is on to determine the role(s) leptin is playing in the regulation of animal growth, reproduction, health, and well-being. It is likely, as in rodents and humans, that leptin will prove to play an important role in the physiology of energy metabolism in domestic animals. ACKNOWLEDGMENTS/FOOTNOTES Authors thank B. Barlow for assisting with the artwork included in this paper. This work was supported by Purdue University Agricultural Research programs and United States Department of Agriculture Grant No. 97–35206-5093 (K.L.H.). 1 Address all correspondence to: Dr. Karen L. Houseknecht, Endocrinology and Metabolism, Department of Animal Sciences, 1151 Lilly Hall, Purdue University, West Lafayette, IN 47907-1151, USA.

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