The adipose-derived hormone, leptin, acts via its receptor (LRb) to convey the status of body energy stores to the brain, decreasing feeding and potentiating neuroendocrine energy expenditure. The failure of high levels of leptin in most obese individuals to promote weight loss defines a state of diminished responsiveness to increased leptin, termed leptin resistance. Leptin stimulates the phosphorylation of several tyrosine residues on LRb to mediate leptin action. We homologously replaced LRb in mice with a receptor with a mutation in one of these sites (Tyr985) in order to examine its role in leptin action and signal attenuation in vivo. Mice homozygous for this mutation are neuroendocrinologically normal, but females demonstrate decreased feeding, decreased expression of orexigenic neuropeptides, protection from high-fat diet-induced obesity, and increased leptin sensitivity in a sex-biased manner. Thus, leptin activates autoinhibitory signals via LRb Tyr985 to attenuate the anti-adiposity effects of leptin, especially in females, potentially contributing to leptin insensitivity in obesity. IntroductionThe prevalence of obesity continues to increase at alarming rates throughout the world, fostering the rise in obesity-related comorbidities, such as diabetes and cardiovascular disease. While body energy homeostasis is closely regulated, only recently have we begun to understand the physiologic mechanisms that regulate feeding and body weight to effect this balance. One important effector of body energy homeostasis is leptin, which is produced by adipocytes as a signal of the repletion of body energy stores. Leptin acts in the CNS to promote satiety and enable neuroendocrine energy expenditure (1-7). The lack of leptin action due to mutations in leptin (e.g., ob/ob mice) or in the active (b) form of the leptin receptor (LRb; e.g., db/db mice) or as a consequence of lowered fat stores results in increased appetite and an energy-sparing neuroendocrine starvation response that includes infertility and growth retardation (3,8). In ob/ob and db/db animals, hyperphagia paired with decreased energy expenditure results in morbid obesity and a propensity to develop type 2 diabetes. Conversely, in normal leptinsensitive animals, high leptin levels tend to reduce appetite and permit neuroendocrine energy expenditure, and leptin administration decreases feeding and body weight while preserving metabolic energy utilization. The failure of elevated leptin levels to mediate weight loss in common forms of human obesity suggests the attenuation of leptin action (leptin resistance) in obese states, as with diet-induced obesity in rodents (9-11). Potential mechanisms to explain this leptin resistance include alterations in leptin transport into the CNS and inhibition of leptin signaling (12, 13).

The prevalence of obesity continues to increase at alarming rates throughout the world, fostering the rise in obesity-related comorbidities, such as diabetes and cardiovascular disease (1-3). Whereas body energy homeostasis is tightly regulated, only recently have we begun to understand the physiologic mechanisms that regulate feeding and body weight to effect this balance (4, 5). One important mediator of body energy homeostasis is leptin, which is produced by adipocytes as a signal of the repletion of body energy (fat) stores (6, 7). Leptin acts in the central nervous system to promote satiety and enable neuroendocrine energy expenditure (8 -14). The lack of leptin action due to mutations in leptin (e.g. ob/ob mice) or LepRb (e.g. db/db mice) or as a consequence of lowered fat stores results in increased appetite and an energy-sparing neuroendocrine starvation response that includes infertility and growth retardation (6, 10). In ob/ob and db/db animals, hyperphagia paired with decreased energy expenditure results in morbid obesity and a propensity to Type 2 diabetes (12). Conversely, in normal leptin-sensitive animals, high leptin levels tend to reduce appetite and permit neuroendocrine energy expenditure, and leptin administration decreases feeding and body weight while preserving metabolic energy utilization (10). The failure of elevated leptin levels to mediate weight loss in common forms of human obesity suggests the attenuation of leptin action ("leptin resistance") in obese states, as with diet-induced obesity in rodents (15-17). Potential mechanisms to explain this leptin resistance include alterations in leptin signaling, among others (18,19).Leptin binding activates the constitutively LepRb-associated Janus kinase (Jak) 3 2 tyrosine kinase to mediate tyrosine phosphorylation-dependent leptin signaling via several pathways (8, 20 -23). Phosphorylated LepRb Tyr 1138 recruits the latent transcription factor, signal transducer and activator of transcription (STAT) 3 to mediate its tyrosine phosphorylation (20,22,23). The tyrosine phosphorylation of STAT proteins, including STAT3, promotes their nuclear translocation and ability to mediate transcriptional regulation (24, 25); hence, STAT3 recruitment by LepRb Tyr 1138 mediates its activation. Phosphorylated Tyr 985 of LepRb binds SH2-containing tyrosine phosphatase-2 (SHP2; aka PTPN11), which participates in extracellular signal-regulated kinase (ERK) activation during leptin signaling in cultured cells (22,26). Tyr 985 additionally binds the suppressor of cytokine signaling (SOCS) 3, and contributes to the attenuation of LepRb signaling (22,26,27). LepRb-associated Jak2 may also mediate signals independently of LepRb tyrosine phosphorylation sites (8,22), in addition to providing a second, lower affinity binding site for SOCS3 (28,29 3 The abbreviations used are: Jak, Janus kinase; STAT, signal transducers and activators of transcription; SH2, Src homology domain 2; ERK, extracellular signal-regulated kinase; SOCS3, suppressor of cytokine signaling 3; RS...

The medial basal hypothalamus, including the arcuate nucleus (ARC) and the ventromedial hypothalamic nucleus (VMH), integrates signals of energy status to modulate metabolism and energy balance. Leptin and feeding regulate the mammalian target of rapamycin complex 1 (mTORC1) in the hypothalamus, and hypothalamic mTORC1 contributes to the control of feeding and energy balance. To determine the mechanisms by which leptin modulates mTORC1 in specific hypothalamic neurons, we immunohistochemically assessed the mTORC1-dependent phosphorylation of ribosomal protein S6 (pS6). In addition to confirming the modulation of ARC mTORC1 activity by acute leptin treatment, this analysis revealed the robust activation of mTORC1-dependent ARC pS6 in response to fasting and leptin deficiency in leptin receptor-expressing Agouti-related protein neurons. In contrast, fasting and leptin deficiency suppress VMH mTORC1 signaling. The appropriate regulation of ARC mTORC1 by mutant leptin receptor isoforms correlated with their ability to suppress the activity of Agouti-related protein neurons, suggesting the potential stimulation of mTORC1 by the neuronal activity. Indeed, fasting- and leptin deficiency-induced pS6-immunoreactivity (IR) extensively colocalized with c-Fos-IR in ARC and VMH neurons. Furthermore, ghrelin, which activates orexigenic ARC neurons, increased ARC mTORC1 activity and induced colocalized pS6- and c-Fos-IR. Thus, neuronal activity promotes mTORC1/pS6 in response to signals of energy deficit. In contrast, insulin, which activates mTORC1 via the phosphatidylinositol 3-kinase pathway, increased ARC and VMH pS6-IR in the absence of neuronal activation. The regulation of mTORC1 in the basomedial hypothalamus thus varies by cell and stimulus type, as opposed to responding in a uniform manner to nutritional and hormonal perturbations.

The leptin receptor, LRb, and other cytokine receptors are devoid of intrinsic enzymatic activity and rely upon the activity of constitutively associated Jak family tyrosine kinases to mediate intracellular signaling. In order to clarify mechanisms by which Jak2, the cognate LRb-associated Jak kinase, is regulated and mediates downstream signaling, we employed tandem mass spectroscopic analysis to identify phosphorylation sites on Jak2. We identified Ser 523 as the first-described site of Jak2 serine phosphorylation and demonstrated that this site is phosphorylated on Jak2 from intact cells and mouse spleen. Ser 523 was highly phosphorylated in HEK293 cells independently of LRb-Jak2 activation, suggesting a potential role for the phosphorylation of Ser 523 in the regulation of LRb by other pathways. Indeed, mutation of Ser 523 sensitized and prolonged signaling by Jak2 following activation by the intracellular domain of LRb. The effect of Ser 523 on Jak2 function was independent of Tyr 570 -mediated inhibition. Thus, the phosphorylation of Jak2 on Ser 523 inhibits Jak2 activity and represents a novel mechanism for the regulation of Jak2-dependent cytokine signaling.Type I cytokines mediate a plethora of physiologic processes, ranging from hematopoietic and immune functions (such as those mediated by erythropoietin [Epo] and the interleukins) to growth and neuroendocrine responses (such as those mediated by growth hormone and leptin) (11,15,16,23,31). These actions are mediated by the activation of cytokine receptor proteins found on the surface of target cells. Cytokine receptors each contain an extracellular domain that recognizes its specific cytokine ligand, a single transmembrane domain, and an intracellular domain that, although devoid of enzymatic activity, transmits intracellular signals by means of an associated Jak family tyrosine kinase. Ligand binding activates the associated intracellular Jak kinase, resulting in Jak kinase autophosphorylation and activation and the subsequent tyrosine phosphorylation of the intracellular domain of the cytokine receptor. These tyrosine phosphorylation events mediate downstream signaling by the LRb/Jak2 complex (11,16,19,23).The Jak kinase family contains four members: Jak1 to Jak3 and Tyk2 (11,16). Of these, Jak1 and -2 and Tyk2 are ubiquitously expressed, while Jak3 is found predominantly in immune and hematopoietic tissues. Jak kinases are composed of four conserved domains. The NH 2 -terminal FERM domain is required for interaction with cytokine receptors (32, 35), while the adjacent SH2-like fold has no known function. The COOHterminal portion of Jak kinases contains a kinase-like JH2 domain that is devoid of enzymatic activity but that regulates the activity of the COOH-terminal JH1 tyrosine kinase domain (9,21,28,33,34).Our laboratory studies signaling by the long form of the leptin receptor (LRb), which regulates feeding, neuroendocrine, and immune function in response to leptin, which is in turn regulated by nutritional cues (8,10,23,31). Stimulation of LRb ...

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