Activation of downstream signals by the long form of the leptin receptor

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.     

Disruption of leptin receptor-STAT3 signaling enhances leukotriene production and pulmonary host defense against pneumococcal pneumonia

The adipocyte-derived hormone leptin regulates energy homeostasis and the innate immune response. We previously reported that leptin plays a protective role in bacterial pneumonia, but the mechanisms by which leptin regulates host defense remain poorly understood. Leptin binding to its receptor, LepRb, activates multiple intracellular signaling pathways, including ERK1/2, STAT5, and STAT3. In this study, we compared the responses of wild-type and s/s mice, which possess a mutant LepRb that prevents leptin-induced STAT3 activation, to determine the role of this signaling pathway in pneumococcal pneumonia. Compared with wild-type animals, s/s mice exhibited greater survival and enhanced pulmonary bacterial clearance after an intratracheal challenge with Streptococcus pneumoniae. We also observed enhanced phagocytosis and killing of S. pneumoniae in vitro in alveolar macrophages (AMs) obtained from s/s mice. Notably, the improved host defense and AM antibacterial effector functions in s/s mice were associated with increased cysteinyl-leukotriene production in vivo and in AMs in vitro. Augmentation of phagocytosis in AMs from s/s mice could be blocked using a pharmacologic cysteinyl-leukotriene receptor antagonist. Phosphorylation of ERK1/2 and cytosolic phospholipase A(2) α, known to enhance the release of arachidonic acid for subsequent conversion to leukotrienes, was also increased in AMs from s/s mice stimulated with S. pneumoniae in vitro. These data indicate that ablation of LepRb-mediated STAT3 signaling and the associated augmentation of ERK1/2, cytosolic phospholipase A(2) α, and cysteinyl-leukotriene synthesis confers resistance to s/s mice during pneumococcal pneumonia. These data provide novel insights into the intracellular signaling events by which leptin contributes to host defense against bacterial pneumonia.     

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb ^+/+ mice and in Leprb ^db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.     

Leptin Acts via Leptin Receptor-Expressing Lateral Hypothalamic Neurons to Modulate the Mesolimbic Dopamine System and Suppress Feeding

The lateral hypothalamic area (LHA) acts in concert with the ventral tegmental area (VTA) and other components of the mesolimbic dopamine (DA) system to control motivation, including the incentive to feed. The anorexigenic hormone leptin modulates the mesolimbic DA system, although the mechanisms underlying this control have remained incompletely understood. We show that leptin directly regulates a population of leptin receptor (LepRb)-expressing inhibitory neurons in the LHA and that leptin action via these LHA LepRb neurons decreases feeding and body weight. Furthermore, these LHA LepRb neurons innervate the VTA, and leptin action on these neurons restores VTA expression of the rate-limiting enzyme in DA production along with mesolimbic DA content in leptin-deficient animals. Thus, these findings reveal that LHA LepRb neurons link anorexic leptin action to the mesolimbic DA system.     

Insulin receptor substrate 4 couples the leptin receptor to multiple signaling pathways

Leptin is an adipokine that regulates food intake and energy expenditure by activating its hypothalamic leptin receptor (LR). Members of the insulin receptor substrate (IRS) family serve as adaptor proteins in the signaling pathways of several cytokines and hormones and a role for IRS2 in central leptin physiology is well established. Using mammalian protein-protein interaction trap (MAPPIT), a cytokine receptor-based two-hybrid method, in the N38 hypothalamic cell line, we here demonstrate that also IRS4 interacts with the LR. This recruitment is leptin dependent and requires phosphorylation of the Y1077 motif of the LR. Domain mapping of IRS4 revealed the critical role of the pleckstrin homology domain for full interaction. In line with its function as an adaptor protein, IRS4 interacted with the regulatory p85 subunit of the phosphatidylinositol 3-kinase, phospholipase Cgamma, and the suppressor of cytokine signaling (SOCS) family members SOCS2, SOCS6, and SOCS7 and thus can modulate LR signaling.     

The Influence of LepR Tyrosine Site Mutations on Mouse Ovary Development and Related Gene Expression Changes

Leptin exerts many biological functions, such as in metabolism and reproduction, through binding to and activating the leptin receptor, LepRb, which is expressed in many regions of the brain. To better understand the roles of LepR downstream signaling pathways, Y123F mice, which expressed mutant leptin receptors with phenylalanine (F) substituted for three tyrosines (Y) (Tyr985, Tyr1077 and Tyr1138), were generated. The body weight and abdominal fat deposits of Y123F homozygous mice (HOM) were higher than those of wild-type mice (WT). HOM ovaries were atrophic and the follicles developed abnormally; however, the HOM ovaries did not exhibit polycystic phenotypes. Moreover, Y123F HOM adults had no estrous cycle and the blood estrogen concentration remained stable at a low level below detection limit of 5 pg/ml. LepR expression in HOM ovaries was higher than in WT ovaries. Using cDNA Microarrays, the mRNA expressions of 41 genes were increased, and 100 were decreased in HOM vs. WT ovaries, and many signaling pathways were evaluated to be involved significantly. The expressions of 19 genes were validated by real-time quantitative PCR, most of which were consistent with the microarray results. Thus, Y123F HOM mice were suggested as a new animal model of PCOS for research that mainly emphasizes metabolic disorders and anovulation, but not the polycystic phenotype. Meanwhile, using the model, we found that JAK-STAT and hormone biosynthesis pathways were involved in the follicular development and ovulation disorders caused by LepR deficiency in ovaries, although we could not exclude indirect actions from the brain.     

Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis within the individual cell. Recent reports have suggested that leptin, an adipocyte-secreted hormone, phosphorylates AMPK in skeletal muscle directly. However, little is known about the interaction between leptin signaling and AMPK activation. Here, we report that the leptin-induced phosphorylation of AMPK was detected in Huh7 cells expressing long form leptin receptor (OBRb) as well as short form leptin receptor (OBRa). In addition, we demonstrate that AMPK activation does not require the phosphorylation of either Tyr-985 or Tyr-1138 within the OBRb and may occur via a STAT3-independent signaling pathway. We also show that Huh7 cells expressing OBRb and SOCS3 (inhibitor of JAK2) resulted in a marked reduction of AMPK activation in response to leptin. These findings suggest that the activation of JAK2, but not STAT3, may play a critical role in leptin-induced AMPK activation in Huh7 cells.     

Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice

Leptin regulates energy balance and body weight by activating its receptor LEPRb and multiple downstream signaling pathways, including the STAT3 and the IRS2/PI 3-kinase pathways, in the hypothalamus. Leptin stimulates activation of LEPRb-associated JAK2, which initiates cell signaling. Here we identified SH2-B, a JAK2-interacting protein, as a key regulator of leptin sensitivity, energy balance, and body weight. SH2-B homozygous null mice were severely hyperphagic and obese and developed a metabolic syndrome characterized by hyperleptinemia, hyperinsulinemia, hyperlipidemia, hepatic steatosis, and hyperglycemia. The expression of hypothalamic orexigenic NPY and AgRP was increased in SH2-B(-/-) mice. Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hypothalamic STAT3 and IRS2 were significantly impaired in SH2-B(-/-) mice. Moreover, overexpression of SH2-B counteracted PTP1B-mediated inhibition of leptin signaling in cultured cells. Our data suggest that SH2-B is an endogenous enhancer of leptin sensitivity and required for maintaining normal energy metabolism and body weight in mice.     

Neotomodon alstoni mice present sex differences between lean and obese in daily hypothalamic leptin signaling

This article compared the effects of spontaneous obesity on the daily profile in the relative amount of the leptin receptor (LepRb), and its output. That is the precursor Pro-opiomelanocortin (POMC) over a 24-hour period and compared with differences in locomotion and food intake in periods of artificial light. Differences between lean and obese mice were examined, as were sex differences. Body weight, food intake and locomotor activity were monitored in freely moving lean and obese mice. Hypothalamic tissue was collected at 5 h, 10 h, 15 h, 19 h and 24 h. Samples were analyzed by western blotting to determine the relative presence of protein for LepRb, STAT3 phosphorylation (by pSTAT3/STAT3 ratio) and POMC. Obese mice were 60% less active in locomotion than lean mice during the night. While both locomotor activity and food intake were noticeably greater during the day in obese mice than in lean mice, the hypothalamus in obese mice showed a lower relative abundance of POMC and reduced pSTAT3/STAT3 ratio and leptin receptors. Behavioral and biochemical differences were more evident in obese females than in obese males. These results indicate that obesity in N. alstoni affects hypothalamic leptin signaling according to sex.     

Leptin action via neurotensin neurons controls orexin, the mesolimbic dopamine system and energy balance

Leptin acts on leptin receptor (LepRb)-expressing neurons throughout the brain, but the roles for many populations of LepRb neurons in modulating energy balance and behavior remain unclear. We found that the majority of LepRb neurons in the lateral hypothalamic area (LHA) contain neurotensin (Nts). To investigate the physiologic role for leptin action via these LepRb(Nts) neurons, we generated mice null for LepRb specifically in Nts neurons (Nts-LepRbKO mice). Nts-LepRbKO mice demonstrate early-onset obesity, modestly increased feeding, and decreased locomotor activity. Furthermore, consistent with the connection of LepRb(Nts) neurons with local orexin (OX) neurons and the ventral tegmental area (VTA), Nts-LepRbKO mice exhibit altered regulation of OX neurons and the mesolimbic DA system. Thus, LHA LepRb(Nts) neurons mediate physiologic leptin action on OX neurons and the mesolimbic DA system, and contribute importantly to the control of energy balance.     

Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways

Leptin controls body weight by activating the long form of the leptin receptor (LEPRb). Janus kinase 2 (JAK2) is associated with LEPRb and autophosphorylates in response to leptin. JAK2 also phosphorylates LEPRb, STAT3, and multiple other downstream molecules. Surprisingly, here we show that JAK2 is not required for leptin stimulation of STAT3 phosphorylation. Leptin time- and dose-dependently stimulated tyrosine phosphorylation of STAT3 in both human and mouse JAK2-null cells. Leptin also increased the viability of JAK2-null cells. Overexpression of c-Src or Fyn, two Src family members, promoted STAT3 phosphorylation, whereas inhibition of the endogenous Src family members by either pharmacological inhibitors or dominant negative Src(K298M) decreased the ability of leptin to stimulate the phosphorylation of STAT3 and ERK1/2. Leptin also stimulated tyrosine phosphorylation of kinase-inactive JAK2(K882E) in JAK2-null cells. Overexpression of JAK2(K882E) enhanced the ability of leptin to stimulate STAT3 phosphorylation in JAK2-null cells. Tyr1138 in LEPRb was required for leptin-stimulated phosphorylation of STAT3 but not JAK2(K882E). These data suggest that leptin stimulates non-JAK2 tyrosine kinase(s), including the Src family members, which phosphorylate JAK2, STAT3, and other molecules downstream of LEPRb. JAK2 mediates leptin signaling by both phosphorylating its substrates and forming a signaling complex as a scaffolding/adaptor protein. The non-JAK2 kinase(s) and JAK2 may act coordinately and synergistically to mediate leptin response.     

Analysis of Tyr to Phe and fa/fa leptin receptor mutations in the PC12 cell line

Weight regulation through body-fat content and energy homeostasis, is regulated mainly through the actions of leptin. Herein, we analyse the effect of mutations in the mouse leptin receptor using the PC12 pheochromocytoma cell line as a model system. Both the induction of pancreatitis associated protein 1 and metallothionein-II, two leptin regulated genes in PC12, was evaluated. Tyr to Phe mutations in the cytoplasmic tail of the mouse leptin receptor confirmed the critical role of Tyr1138 (a YxxQ motif) and STAT-3 activation for induction of leptin-induced genes in PC12. In addition, the Tyr985Phe mutation showed enhanced responsiveness to leptin, which was even more pronounced in combination with Tyr1077Phe. The short isoform of the leptin receptor showed complete loss of stimulation of both genes. In contrast, a leptin receptor devoid of all Tyr residues in its cytoplasmic tail was still capable of a limited induction of the PAP 1 gene. A mutant mouse leptin receptor containing the fa/fa mutation showed constitutive signalling and impaired responsiveness to leptin. Treatment with the adenylate cyclase activator forskolin alone, in the absence of leptin was sufficient to obtain full induction of both genes.