Leptin action via LepR-b Tyr1077 contributes to the control of energy balance and female reproduction.

Leptin action in the brain signals the repletion of adipose energy stores, suppressing feeding and permitting energy expenditure on a variety of processes, including reproduction. Leptin binding to its receptor (LepR-b) promotes the tyrosine phosphorylation of three sites on LepR-b, each of which mediates distinct downstream signals. While the signals mediated by LepR-b Tyr1138 and Tyr985 control important aspects of energy homeostasis and LepR-b signal attenuation, respectively, the role of the remaining LepR-b phosphorylation site (Tyr1077) in leptin action has not been studied. To examine the function of Tyr1077, we generated a "knock-in" mouse model expressing LepR-b (F1077), which is mutant for LepR-b Tyr1077. Mice expressing LepR-b (F1077) demonstrate modestly increased body weight and adiposity. Furthermore, females display impairments in estrous cycling. Our results suggest that signaling by LepR-b Tyr1077 plays a modest role in the control of metabolism by leptin, and is an important link between body adiposity and the reproductive axis.

Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis.

IL-17 and IL-23 are known to be absolutely central to psoriasis pathogenesis because drugs targeting either cytokine are highly effective treatments for this disease. The efficacy of these drugs has been attributed to blocking the function of IL-17-producing T cells and their IL-23-induced expansion. However, we demonstrate that mast cells and neutrophils, not T cells, are the predominant cell types that contain IL-17 in human skin. IL-17(+) mast cells and neutrophils are found at higher densities than IL-17(+) T cells in psoriasis lesions and frequently release IL-17 in the process of forming specialized structures called extracellular traps. Furthermore, we find that IL-23 and IL-1ß can induce mast cell extracellular trap formation and degranulation of human mast cells. Release of IL-17 from innate immune cells may be central to the pathogenesis of psoriasis, representing a fundamental mechanism by which the IL-23-IL-17 axis mediates host defense and autoimmunity.

Critical roles for the TSC-mTOR pathway in ß-cell function.

TSC1 is a tumor suppressor that associates with TSC2 to inactivate Rheb, thereby inhibiting signaling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as translation, in response to growth factors and nutrient signals. To test roles for TSC1 and mTORC1 in ß-cell function, we utilized Rip2/Cre to generate mice lacking Tsc1 in pancreatic ß-cells (Rip-Tsc1cKO mice). Although obesity developed due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, young animals displayed a prominent gain-of-function ß-cell phenotype prior to the onset of obesity. The young Rip-Tsc1cKO animals displayed improved glycemic control due to mTOR-mediated enhancement of ß-cell size, mass, and insulin production but not determinants of ß-cell number (proliferation and apoptosis), consistent with an important anabolic role for mTOR in ß-cell function. Furthermore, mTOR mediated these effects in the face of impaired Akt signaling in ß-cells. Thus, mTOR promulgates a dominant signal to promote ß-cell/islet size and insulin production, and this pathway is crucial for ß-cell function and glycemic control.

Complex regulation of mammalian target of rapamycin complex 1 in the basomedial hypothalamus by leptin and nutritional status.

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.

Critical role for hypothalamic mTOR activity in energy balance.

The mammalian target of rapamycin (mTOR) promotes anabolic cellular processes in response to growth factors and metabolic cues. The TSC1 and TSC2 tumor suppressors are major upstream inhibitory regulators of mTOR signaling. Mice with Rip2/Cre-mediated deletion of Tsc1 (Rip-Tsc1cKO mice) developed hyperphagia and obesity, suggesting that hypothalamic disruption (for which Rip2/Cre is well known) of Tsc1 may dysregulate feeding circuits via mTOR activation. Indeed, Rip-Tsc1cKO mice displayed increased mTOR signaling and enlarged neuron cell size in a number of hypothalamic populations, including Pomc neurons. Furthermore, Tsc1 deletion with Pomc/Cre (Pomc-Tsc1cKO mice) resulted in dysregulation of Pomc neurons and hyperphagic obesity. Treatment with the mTOR inhibitor, rapamycin, ameliorated the hyperphagia, obesity, and the altered Pomc neuronal morphology in developing or adult Pomc-Tsc1cKO mice, and cessation of treatment reinstated these phenotypes. Thus, ongoing mTOR activation in Pomc neurons blocks the catabolic function of these neurons to promote nutrient intake and increased adiposity.

Loss of cytokine-STAT5 signaling in the CNS and pituitary gland alters energy balance and leads to obesity.

Signal transducers and activators of transcription (STATs) are critical components of cytokine signaling pathways. STAT5A and STAT5B (STAT5), the most promiscuous members of this family, are highly expressed in specific populations of hypothalamic neurons in regions known to mediate the actions of cytokines in the regulation of energy balance. To test the hypothesis that STAT5 signaling is essential to energy homeostasis, we used Cre-mediated recombination to delete the Stat5 locus in the CNS. Mutant males and females developed severe obesity with hyperphagia, impaired thermal regulation in response to cold, hyperleptinemia and insulin resistance. Furthermore, central administration of GM-CSF mediated the nuclear accumulation of STAT5 in hypothalamic neurons and reduced food intake in control but not in mutant mice. These results demonstrate that STAT5 mediates energy homeostasis in response to endogenous cytokines such as GM-CSF.

The long form of the leptin receptor regulates STAT5 and ribosomal protein S6 via alternate mechanisms.

The action of leptin via the long form of its receptor (LepRb) is central to the control of body energy homeostasis and neuroendocrine function, but the mechanisms by which LepRb regulates intracellular signaling have remained incompletely understood. Here we demonstrate that leptin stimulates the phosphorylation of STAT5 and ribosomal protein S6 in the hypothalamic arcuate nucleus in mice. In cultured cells, we investigate the mechanisms by which leptin regulates each of these pathways. Our analysis reveals a dominant role for LepRb Tyr(1077) (which we demonstrate to be phosphorylated during receptor activation) and a secondary role for LepRb Tyr(1138) in the acute phosphorylation of STAT5a and STAT5b. Tyr(1138) and STAT3 attenuate STAT5-dependent transcription over the long-term, however. In contrast, Tyr(985) (the LepRb phosphorylation site required for ERK activation) mediates the phosphorylation of the ribosomal S6 kinase (RSK) and S6, as well as cap-dependent translation. Thus, these data demonstrate the phosphorylation of Tyr(1077) on LepRb during receptor activation, substantiate the hypothalamic regulation of STAT5 and S6 by leptin, and define the alternate LepRb signaling pathways that mediate each of these signals and their effects in cultured cells. Dissecting the contributions of these individual pathways to leptin action will be important for our ultimate understanding of the processes that regulate energy balance in vivo.

Mice lacking inhibitory leptin receptor signals are lean with normal endocrine function.

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.