Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization

Leptin plays a pivotal role in the regulation of energy homeostasis and metabolism, primarily by acting on neurons in the hypothalamus that control food intake. However, leptin receptors are more widely expressed in the brain suggesting additional, as yet unknown, functions of leptin. Here we show that both embryonic and adult hippocampal neurons express leptin receptors coupled to activation of STAT3 and phosphatidylinositol 3-kinase-Akt signaling pathways. Leptin protects hippocampal neurons against cell death induced by neurotrophic factor withdrawal and excitotoxic and oxidative insults. The neuroprotective effect of leptin is antagonized by the JAK2-STAT3 inhibitor AG-490, STAT3 decoy DNA, and phosphatidylinositol 3-kinase/Akt inhibitors but not by an inhibitor of MAPK. Leptin induces the production of manganese superoxide dismutase and the anti-apoptotic protein Bcl-xL, and stabilizes mitochondrial membrane potential and lessens mitochondrial oxidative stress. Leptin receptor-deficient mice (db/db mice) are more vulnerable to seizure-induced hippocampal damage, and intraventricular administration of leptin protects neurons against seizures. By enhancing mitochondrial resistance to apoptosis and excitotoxicity, our findings suggest that leptin signaling serves a neurotrophic function in the developing and adult hippocampus.     

Hypothalamic IKKbeta/NF-kappaB and ER stress link overnutrition to energy imbalance and obesity

Overnutrition is associated with chronic inflammation in metabolic tissues. Whether metabolic inflammation compromises the neural regulatory systems and therefore promotes overnutrition-associated diseases remains unexplored. Here we show that a mediator of metabolic inflammation, IKKbeta/NF-kappaB, normally remains inactive although enriched in hypothalamic neurons. Overnutrition atypically activates hypothalamic IKKbeta/NF-kappaB at least in part through elevated endoplasmic reticulum stress in the hypothalamus. While forced activation of hypothalamic IKKbeta/NF-kappaB interrupts central insulin/leptin signaling and actions, site- or cell-specific suppression of IKKbeta either broadly across the brain or locally within the mediobasal hypothalamus, or specifically in hypothalamic AGRP neurons significantly protects against obesity and glucose intolerance. The molecular mechanisms involved include regulation by IKKbeta/NF-kappaB of SOCS3, a core inhibitor of insulin and leptin signaling. Our results show that the hypothalamic IKKbeta/NF-kappaB program is a general neural mechanism for energy imbalance underlying obesity and suggest that suppressing hypothalamic IKKbeta/NF-kappaB may represent a strategy to combat obesity and related diseases.     

运动改善肥胖症瘦素抵抗及其机制研究进展

大部分肥胖患者体内出现瘦素抵抗,表现为血清瘦素水平异常升高,但机体对瘦素不敏感或无反应,使瘦素抑制食欲、增加能量消耗和降低血糖等功能不能有效发挥.减轻瘦素抵抗被认为是治疗肥胖及肥胖相关疾病的有效途径.运动减轻肥胖、改善糖脂代谢和增强胰岛素敏感性的作用与运动降低瘦素水平、改善瘦素抵抗密切相关.本文在概述瘦素实现生理功能的机制、肥胖症的中枢及外周瘦素抵抗的基础上,主要综述近年来运动减轻肥胖症瘦素抵抗机制的研究进展,包括减轻高瘦素血症、改善中枢和外周瘦素抵抗,以期为运动防治肥胖机制的研究提供新视角.     

Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling

Phosphatidylinositol 3-OH-kinase (PI3K) and STAT3 are signal transduction molecules activated by leptin in brain areas controlling food intake. To investigate their role in leptin-mediated inhibition of hypothalamic neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression, male Sprague-Dawley rats (n = 5/group) were either fed ad libitum or subjected to a 52-h fast. At 12-h intervals, the PI3K inhibitor LY-294002 (LY, 1 nmol) or vehicle was injected intracerebroventricularly (ICV) as a pretreatment, followed 1 h later by leptin (3 microg icv) or vehicle. Fasting increased hypothalamic Npy and Agrp mRNA levels (P < 0.05), and ICV leptin administration prevented this increase. As predicted, LY pretreatment blocked this inhibitory effect of leptin, such that Npy and Agrp levels in LY-leptin-treated animals were similar to fasted controls. By comparison, leptin-mediated activation of hypothalamic STAT3 signaling, as measured by induction of both phospho-STAT3 immunohistochemistry and suppressor of cytokine signaling-3 (Socs3) mRNA, was not significantly attenuated by ICV LY pretreatment. Because NPY/AgRP neurons project to the hypothalamic paraventricular nucleus (PVN), we next investigated whether leptin activation of PVN neurons is similarly PI3K dependent. Compared with vehicle, leptin increased the number of c-Fos positive cells within the parvocellular PVN (P = 0.001), and LY pretreatment attenuated this effect by 35% (P = 0.043). We conclude that leptin requires intact PI3K signaling both to inhibit hypothalamic Npy and Agrp gene expression and activate neurons within the PVN. In addition, these data suggest that leptin activation of STAT3 is insufficient to inhibit expression of Npy or Agrp in the absence of PI3K signaling.     

Synergistic interaction between leptin and cholecystokinin in the rat nodose ganglia is mediated by PI3K and STAT3 signaling pathways: implications for leptin as a regulator of short term satiety

Research has shown that the synergistic interaction between vagal cholecystokinin-A receptors (CCKARs) and leptin receptors (LRbs) mediates short term satiety. We hypothesize that this synergistic interaction is mediated by cross-talk between signaling cascades used by CCKARs and LRbs, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. Whole cell patch clamp recordings were performed on isolated rat nodose ganglia neurons. Western immunoblots elucidated the intracellular signaling pathways that modulate leptin/CCK synergism. In addition, STAT3, PI3K, Src, and MAPK genes were silenced by lentiviral infection and transient Lipofectamine transfection of cultured rat nodose ganglia to determine the effect of these molecules on leptin/CCK synergism. Patch clamp studies showed that a combination of leptin and CCK-8 caused a significant increase in membrane input resistance compared with leptin or CCK-8 alone. Silencing the STAT3 gene abolished the synergistic action of leptin/CCK-8 on neuronal firing. Leptin/CCK-8 synergistically stimulated a 7.7-fold increase in phosphorylated STAT3 (pSTAT3), which was inhibited by AG490, C3 transferase, PP2, LY294002, and wortmannin, but not PD98059. Silencing the Src and PI3K genes resulted in a loss of leptin/CCK-stimulated pSTAT3. We conclude that the synergistic interaction between vagal CCKARs and LRbs is mediated by the phosphorylation of STAT3, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/Src/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways. Malfunctioning of these signaling molecules may result in eating disorders.     

Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis

Neuroanatomical and electrophysiological studies have shown that hypothalamic POMC neurons are targets of the adipostatic hormone leptin. However, the physiological relevance of leptin signaling in these neurons has not yet been directly tested. Here, using the Cre/loxP system, we critically test the functional importance of leptin action on POMC neurons by deleting leptin receptors specifically from these cells in mice. Mice lacking leptin signaling in POMC neurons are mildly obese, hyperleptinemic, and have altered expression of hypothalamic neuropeptides. In summary, leptin receptors on POMC neurons are required but not solely responsible for leptin's regulation of body weight homeostasis.     

Critical role of STAT3 in leptin's metabolic actions

Leptin has pleiotropic effects on glucose homeostasis and feeding behavior. Here, we validate the use of a cell-permeable phosphopeptide that blocks STAT3 activation in vivo. The combination of this biochemical approach with stereotaxic surgical techniques allowed us to pinpoint the contribution of hypothalamic STAT3 to the acute effects of leptin on food intake and glucose homeostasis. Leptin's ability to acutely reduce food intake critically depends on intact STAT3 signaling. Likewise, hypothalamic signaling of leptin through STAT3 is required for the acute effects of leptin on liver glucose fluxes. Lifelong obliteration of STAT3 signaling via the leptin receptor in mice (s/s mice) results in severe hepatic insulin resistance that is comparable to that observed in db/db mice, devoid of leptin receptor signaling. Our results demonstrate that the activation of the hypothalamic STAT3 pathway is an absolute requirement for the effects of leptin on food intake and hepatic glucose metabolism.     

Glucose enhances leptin signaling through modulation of AMPK activity

Leptin exerts its action by binding to and activating the long form of leptin receptors (LEPRb). LEPRb activates JAK2 that subsequently phosphorylates and activates STAT3. The JAK2/STAT3 pathway is required for leptin control of energy balance and body weight. Defects in leptin signaling lead to leptin resistance, a primary risk factor for obesity. Body weight is also regulated by nutrients, including glucose. Defects in glucose sensing also contribute to obesity. Here we report crosstalk between leptin and glucose. Glucose starvation blocked the ability of leptin to stimulate tyrosyl phosphorylation and activation of JAK2 and STAT3 in a variety of cell types. Glucose dose-dependently enhanced leptin signaling. In contrast, glucose did not enhance growth hormone-stimulated phosphorylation of JAK2 and STAT5. Glucose starvation or 2-deoxyglucose-induced inhibition of glycolysis activated AMPK and inhibited leptin signaling; pharmacological inhibition of AMPK restored the ability of leptin to stimulate STAT3 phosphorylation. Conversely, pharmacological activation of AMPK was sufficient to inhibit leptin signaling and to block the ability of glucose to enhance leptin signaling. These results suggest that glucose and/or its metabolites play a permissive role in leptin signaling, and that glucose enhances leptin sensitivity at least in part by attenuating the ability of AMPK to inhibit leptin signaling.     

Insulin and Leptin Signaling Interact in the Mouse Kiss1 Neuron during the Peripubertal Period

Reproduction requires adequate energy stores for parents and offspring to survive. Kiss1 neurons, which are essential for fertility, have the potential to serve as the central sensors of metabolic factors that signal to the reproductive axis the presence of stored calories. Paradoxically, obesity is often accompanied by infertility. Despite excess circulating levels of insulin and leptin, obese individuals exhibit resistance to both metabolic factors in many neuron types. Thus, resistance to insulin or leptin in Kiss1 neurons could lead to infertility. Single deletion of the receptors for either insulin or the adipokine leptin from Kiss1 neurons does not impair adult reproductive dysfunction. However, insulin and leptin signaling pathways may interact in such a way as to obscure their individual functions. We hypothesized that in the presence of genetic or obesity-induced concurrent insulin and leptin resistance, Kiss1 neurons would be unable to maintain reproductive function. We therefore induced a chronic hyperinsulinemic and hyperleptinemic state in mice lacking insulin receptors in Kiss1 neurons through high fat feeding and examined the impact on fertility. In an additional, genetic model, we ablated both leptin and insulin signaling in Kiss1 neurons (IR/LepR^Kiss mice). Counter to our hypothesis, we found that the addition of leptin insensitivity did not alter the reproductive phenotype of IR^Kiss mice. We also found that weight gain, body composition, glucose and insulin tolerance were normal in mice of both genders. Nonetheless, leptin and insulin receptor deletion altered pubertal timing as well as LH and FSH levels in mid-puberty in a reciprocal manner. Our results confirm that Kiss1 neurons do not directly mediate the critical role that insulin and leptin play in reproduction. However, during puberty kisspeptin neurons may experience a critical window of susceptibility to the influence of metabolic factors that can modify the onset of fertility.     

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.     

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.     

PTP1B regulates leptin signal transduction in vivo

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.     

Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.     

Induction of leptin resistance by activation of cAMP-Epac signaling

Leptin regulates energy balance and glucose homeostasis. Shortly after leptin was identified, it was established that obesity is commonly associated with leptin resistance, though the molecular mechanisms remain to be identified. To explore potential mechanisms of leptin resistance, we employed organotypic brain slices to identify candidate signaling pathways that negatively regulate leptin sensitivity. We found that elevation of adenosine 3', 5'-monophosphate (cAMP) levels impairs multiple signaling cascades activated by leptin within the hypothalamus. Notably, this effect is independent of protein kinase A activation. In contrast, activation of Epac, a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap1, was sufficient to impair leptin signaling with concomitant induction of SOCS-3 expression. Epac activation also blunted leptin-induced depolarization of hypothalamic POMC neurons. Finally, central infusion of an Epac activator blunted the anorexigenic actions of leptin. Thus, activation of hypothalamic cAMP-Epac pathway is sufficient to induce multiple indices of leptin resistance.     

Expression of Mutant Huntingtin in Leptin Receptor-Expressing Neurons Does Not Control the Metabolic and Psychiatric Phenotype of the BACHD Mouse

Metabolic and psychiatric disturbances occur early on in the clinical manifestation of Huntington’s disease (HD), a neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. Hypothalamus has emerged as an important site of pathology and alterations in this area and its neuroendocrine circuits may play a role in causing early non-motor symptoms and signs in HD. Leptin is a hormone that controls energy homeostasis by signaling through leptin receptors in the hypothalamus. Disturbed leptin action is implicated in both obesity and depression and altered circulating levels of leptin have been reported in both clinical HD and rodent models of the disease. Pathological leptin signaling may therefore be involved in causing the metabolic and psychiatric disturbances of HD. Here we tested the hypothesis that expression of mutant HTT in leptin receptor carrying neurons plays a role in the development of the non-motor phenotype in the BACHD mouse model. Our results show that inactivation of mutant HTT in leptin receptor-expressing neurons in the BACHD mouse using cross-breeding based on a cre-loxP system did not have an effect on the metabolic phenotype or anxiety-like behavior. The data suggest that mutant HTT disrupts critical hypothalamic pathways by other mechanisms than interfering with intracellular leptin signaling.