Leptin
3. LEPTIN RECEPTORS AND SIGNAL TRANSDUCTION IN THE BRAIN
Soon after it was shown that low doses of leptin, when administered directly into the brain, decreased food intake and body weight (14), leptin receptors were discovered through expression cloning in mouse brain and by positional cloning of the db locus (7,8).
The leptin receptor (LEPR) belongs to the cytokine receptor class I superfamily (38). Five alternatively spliced isoforms, a, b, c, d, e, differing in the lengths of their carboxy termini, have been identified (refs. 10,38;Fig. 1). The short leptin receptor isoform, LEPRa, is expressed in several peripheral tissues, choroid plexus, and brain microvessels, and thought to be involved in the transport of leptin across the BBB or efflux from the brain (48–50). The long leptin receptor (LEPRb) that has intracellular domains necessary for signaling via the JAK–STAT pathway is highly expressed in the hypothalamus—e.g., arcuate (Arc), dorsomedial (DMN), ventromedial (VMN), and ventral premamillary nuclei (PMN). Moderate LEPRb expression is found in the periventricular and lateral hypothalamic areas, and nucleus solitarius and various brainstem nuclei (51). The PVN, which integrates energy balance, neuroendocrine function, and glucose homeostasis, has a very low level of LEPRb (51). LEPRb has been colocalized with neuropeptides involved in energy homeostasis (52). Neuropeptide Y (NPY) and agouti-related protein (AGRP), which stimulate feeding, are present in the same neurons in the medial Arc. An increase in leptin directly suppresses NPY and AGRP. Leptin increases the levels of anorectic pep- tides, F-melanin-stimulating hormone (F-MSH) derived from proopiomelanocortin (POMC), and cocaine and amphetamine-regulated transcript (CART), in the lateral Arc (53). Second-order neurons that synthesize CRH, TRH, and oxytocin in the PVN are con- trolled indirectly by leptin targets in the Arc, and mediate the inhibitory effects of leptin on food intake, stimulation of thermogenesis, and neuroendocrine secretion (52). Other orexigenic peptides, such as melanin-concentrating hormone (MCH) and orexins, expressed in the lateral hypothalamus, are inhibited indirectly by leptin (51). Outside the hypothalamus, LEPRb mRNA has also been found in the thalamus and cerebellum (52).
Binding of leptin to LEPRb in hypothalamic and brainstem neurons results in rapid activation of intracellular JAK-2, leading to tyrosine phosphorylation of LEPRb on amino acid residues 985 and 1138, which provide binding motifs for src homology 2 (SH2)-domain containing proteins—i.e., STAT-3 and SH-2-domain-phosphotyrosine phosphatase (SHP-2) (ref. 54; Fig. 2). STAT-3 binds to Y1138, becomes tyrosine-phos- phorylated by JAK-2, then dissociates and forms dimers in the cytoplasm, which are translocated to the nucleus to regulate gene transcription (Fig. 2). The importance of Y1138 has been demonstrated in mice by replacing this amino acid with serine (55).
The Y1138S (LeprS1138) mutation disrupted STAT-3 activation, resulting in hyperpha- gia, impairment of thermoregulation, and obesity (55). However, in contrast to Leprdb/db mice, Y1138S mutation did not affect sexual maturation and growth, and glucose levels were lower, pointing to a specific physiological role of this domain (55).
Leptin regulates insulin receptor substrate 1 (IRS-1) and IRS-2, mitogen-activated pro- tein kinase, extracellular-regulated kinase, Akt, and phosphatidylinositol-3 (PI3)-kinase Fig. 1. Leptin receptors. Five leptin receptor isoforms result from alternate splicing of lepr mRNA transcript. Receptors LEPRa–LEPRd contain identical extracellular ligand binding and cytoplasmic signaling domains. Despite having the same extracellular N-terminal, each isoform has a different C-terminal. However, unlike the other leptin isoforms, LEPRe has neither a transmembrane nor intra- cellular domains. LEPRa is the principal “short leptin receptor” and lacks the cytoplasmic domain necessary for signaling through the JAK–STAT pathway. LEPRb, the “long leptin receptor,” is prima- rily responsible for the leptin-mediated effects on energy homeostasis and endocrine function through activation of the JAK–STAT pathway. Terminal amino acid residues for each isoform are represented by the alphabetic code.
through LEPRb, raising the possibility of crosstalk between leptin and insulin (56). Leptin enhances IRS-2-mediated activation of PI3-kinase in the hypothalamus, concomitant with its ability to inhibit food intake (56,57). In contrast, blockade of PI3-kinase activity pre- vents the anorectic action of leptin (56).
Bjørbaek et al. (58,59) first proposed a role of SOCS-3 in leptin signaling, based on the observation that leptin rapidly increased the levels of this cytokine mediator in hypo- thalami of Lepob/obbut not Leprdb/dbmice. In situ hybridization for SOCS-3 mRNA in Fig. 2. Leptin signal transduction. Leptin binding to the hypothalamic LEPRb results in activation and autophosphorylation of JAK-2. Subsequently STAT-3 is phosphorylated then translocated to the nucleus, where it acts together with a variety of transcription factors to regulate the expression of neuropeptides and other genes. In congenital or acquired leptin deficiency or leptin receptor defect, failure to activate LEPRb results in increased orexigenic neuropeptide expression (NPY and AGRP), and decreased anorexigenic neuropeptide expression (POMC), and uncoupling protein (UCP)-1 expression in brown adipose tissue (BAT). The net effect is hyperphagia and a decrease in metabolic rate, resulting in severe obesity, markedly elevated insulin levels and steatosis. Another manifestation of leptin deficiency is hypothalamic hypogonadism. The fall in leptin during fasting triggers hyperpha- gia, metabolic and neuroendocrine responses similar to congenital leptin deficiency. An increase in lep- tin within the physiological range inhibits feeding and increases the metabolic rate, resulting in weight loss. In contrast, polygenic (diet-induced) obesity results in elevated leptin levels, which promote phos- phorylation of Tyr1138 on the intracellular domain of LEPRb, resulting in inhibition of leptin signal- ing by induction of SOCS-3 and inhibition of JAK–STAT and SHP-2 pathways. These mechanisms result in “leptin resistance” that blunts the ability of leptin to suppress feeding and increase metabolic rate, resulting in obesity. The degree of obesity, steatosis, insulin resistance, and hyperlipidemia in diet- induced obesity is less severe than that seen in congenital leptin deficiency or lipodystrophy.
the rat and mouse brains showed that leptin-induced SOCS-3 expression colocalized with LEPRb and neuropeptides in the hypothalamus (58). Furthermore, expression of SOCS-3 prevented the tyrosine phosphorylation of LEPRb and downstream signaling (59). STAT-3 DNA binding elements are present in the socs-3 promoter, and because the lack of the STAT-3 binding site on LEPRb prevents induction of SOCS-3 mRNA by leptin, this indicates that leptin stimulates socs-3 transcription via the STAT-3 pathway (59). The crucial role of SOCS-3 as a negative regulator of leptin signaling was demon- strated in two studies (60,61). SOCS-3 haploinsufficiency increased leptin sensitivity and prevented diet-induced obesity in mice (60). More specifically, neuron-specific ablation of SOCS-3 enhanced leptin sensitivity, resulting in activation of STAT3, increase in hypothalamic POMC expression, reduction in food intake, and resistance to obesity, hyperlipidemia, and diabetes (61).
Protein tyrosine phosphatase 1B (PTP1B), an insulin receptor phosphatase that inhibits insulin signaling, was implicated in leptin action, based on the finding that mice lacking PTP1B were less hyperphagic and resistant to obesity despite having low serum leptin levels (62–64). In vitro studies revealed that PTP1B directly inhibited JAK2 kinase, and leptin-induced tyrosine phosphorylation of JAK-2 and STAT-3 was attenu- ated in cells overexpressing PTP1B (63). PTP1B mRNA is colocalized with STAT-3 and neuropeptides in the Arc and various hypothalamic nuclei (64). Importantly, STAT-3 phosphorylation in the hypothalamus is enhanced following leptin treatment in mice lacking PTP1B (PTP1B–/–), suggesting that PTP1B inhibits the signal transduction of leptin in the brain (63,64). This idea was tested by comparing the effects of leptin treat- ment on energy balance in wild-type (WT) and PTP1B –/– mice.As predicted, PTP1B –/–
and heterozygotes PTP1B+/– mice exhibited greater leptin sensitivity than WT (63).