Role of POMC and AgRP neuronal activities on glycaemia in mice.

Leptin regulates both feeding and glycaemia primarily through its receptors expressed on agouti-related peptide (AgRP) and pro-opiomelanocortin-expressing (POMC) neurons; however, it is unknown whether activity of these neuronal populations mediates the regulation of these processes. To determine this, we injected Cre-dependent designer receptors exclusively activated by designer drugs (DREADD) viruses into the hypothalamus of normoglycaemic and diabetic AgRP-ires-cre and POMC-cre mice to chemogenetically activate or inhibit these neuronal populations. Despite robust changes in food intake, activation or inhibition of AgRP neurons did not affect glycaemia, while activation caused significant (P = 0.014) impairment in insulin sensitivity. Stimulation of AgRP neurons in diabetic mice reversed leptin's ability to inhibit feeding but did not counter leptin's ability to lower blood glucose levels. Notably, the inhibition of POMC neurons stimulated feeding while decreasing glucose levels in normoglycaemic mice. The findings suggest that leptin's effects on feeding by AgRP neurons are mediated by changes in neuronal firing, while the control of glucose balance by these cells is independent of chemogenetic activation or inhibition. The firing-dependent glucose lowering mechanism within POMC neurons is a potential target for the development of novel anti-diabetic medicines.

The role of GluN2A and GluN2B NMDA receptor subunits in AgRP and POMC neurons on body weight and glucose homeostasis.

OBJECTIVE: Hypothalamic agouti-related peptide (AgRP) and pro-opiomelanocortin (POMC) expressing neurons play critical roles in control of energy balance. Glutamatergic input via n-methyl-d-aspartate receptors (NMDARs) is pivotal for regulation of neuronal activity and is required in AgRP neurons for normal body weight homeostasis. NMDARs typically consist of the obligatory GluN1 subunit and different GluN2 subunits, the latter exerting crucial differential effects on channel activity and neuronal function. Currently, the role of specific GluN2 subunits in AgRP and POMC neurons on whole body energy and glucose balance is unknown. METHODS: We used the cre-lox system to genetically delete GluN2A or GluN2B only from AgRP or POMC neurons in mice. Mice were then subjected to metabolic analyses and assessment of AgRP and POMC neuronal function through morphological studies. RESULTS: We show that loss of GluN2B from AgRP neurons reduces body weight, fat mass, and food intake, whereas GluN2B in POMC neurons is not required for normal energy balance control. GluN2A subunits in either AgRP or POMC neurons are not required for regulation of body weight. Deletion of GluN2B reduces the number of AgRP neurons and decreases their dendritic length. In addition, loss of GluN2B in AgRP neurons of the morbidly obese and severely diabetic leptin-deficient Lep (ob/ob) mice does not affect body weight and food intake but, remarkably, leads to full correction of hyperglycemia. Lep (ob/ob) mice lacking GluN2B in AgRP neurons are also more sensitive to leptin's anti-obesity actions. CONCLUSIONS: GluN2B-containing NMDA receptors in AgRP neurons play a critical role in central control of body weight homeostasis and blood glucose balance via mechanisms that likely involve regulation of AgRP neuronal survival and structure, and modulation of hypothalamic leptin action.

Hypothalamic agouti-related peptide neurons and the central melanocortin system are crucial mediators of leptin's antidiabetic actions.

Leptin has beneficial effects on glucose metabolism via actions in the hypothalamus, but the roles of specific subgroups of neurons responsible for these antidiabetic effects remain unresolved. We generated diabetic Lep(ob/ob) or Lepr(db/db) mice lacking or re-expressing leptin receptors (LepRb) in subgroups of neurons to explore their contributions to leptin's glucose-lowering actions. We show that agouti-related peptide (AgRP)-expressing neurons are both required and sufficient to correct hyperglycemia by leptin. LepRb in pro-opiomelanocortin (POMC) neurons or steroidogenic factor-1 (SF1) neurons are not required. Furthermore, normalization of blood glucose by leptin is blunted in Lep(ob/ob)/MC4R-null mice, but not in Lep(ob/ob) mice lacking neuropeptide Y (NPY) or gamma-aminobutyric acid (GABA) in AgRP neurons. Leptin's ability to improve glucose balance is accompanied by a reduction in circulating glucagon. We conclude that AgRP neurons play a crucial role in glucose-lowering actions by leptin and that this requires the melanocortin system, but not NPY and GABA.

Leptin modulates the intrinsic excitability of AgRP/NPY neurons in the arcuate nucleus of the hypothalamus.

The hypothalamic arcuate nucleus (ARH) is a brain region critical for regulation of food intake and a primary area for the action of leptin in the CNS. In lean mice, the adipokine leptin inhibits neuropeptide Y (NPY) and agouti-related peptide (AgRP) neuronal activity, resulting in decreased food intake. Here we show that diet-induced obesity in mice is associated with persistent activation of NPY neurons and a failure of leptin to reduce the firing rate or hyperpolarize the resting membrane potential. However, the molecular mechanism whereby diet uncouples leptin's effect on neuronal excitability remains to be fully elucidated. In NPY neurons from lean mice, the Kv channel blocker 4-aminopyridine inhibited leptin-induced changes in input resistance and spike rate. Consistent with this, we found that ARH NPY neurons have a large, leptin-sensitive delayed rectifier K(+) current and that leptin sensitivity of this current is blunted in neurons from diet-induced obese mice. This current is primarily carried by Kv2-containing channels, as the Kv2 channel inhibitor stromatoxin-1 significantly increased the spontaneous firing rate in NPY neurons from lean mice. In HEK cells, leptin induced a significant hyperpolarizing shift in the voltage dependence of Kv2.1 but had no effect on the function of the closely related channel Kv2.2 when these channels were coexpressed with the long isoform of the leptin receptor LepRb. Our results suggest that dynamic modulation of somatic Kv2.1 channels regulates the intrinsic excitability of NPY neurons to modulate the spontaneous activity and the integration of synaptic input onto these neurons in the ARH.

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 revisited: its mechanism of action and potential for treating diabetes.

Since the discovery of leptin in 1994, we now have a better understanding of the cellular and molecular mechanisms underlying its biological effects. In addition to its established anti-obesity effects, leptin exerts antidiabetic actions that are independent of its regulation of body weight and food intake. In particular, leptin can correct diabetes in animal models of type 1 and type 2 diabetes. In addition, long-term leptin replacement therapy improves glycaemic control, insulin sensitivity and plasma triglycerides in patients with severe insulin resistance due to lipodystrophy. These results have spurred enthusiasm for the use of leptin therapy to treat diabetes. Here, we review the current understanding of the glucoregulatory functions of leptin, emphasizing its central mechanisms of action and lessons learned from clinical studies, and discuss possible therapeutic applications of leptin in the treatment of type 1 and type 2 diabetes.

Rho-kinase regulates energy balance by targeting hypothalamic leptin receptor signaling.

Leptin regulates energy balance. However, knowledge of the critical intracellular transducers of leptin signaling remains incomplete. We found that Rho-kinase 1 (ROCK1) regulates leptin action on body weight homeostasis by activating JAK2, an initial trigger of leptin receptor signaling. Leptin promoted the physical interaction of JAK2 and ROCK1, thereby increasing phosphorylation of JAK2 and downstream activation of Stat3 and FOXO1. Mice lacking ROCK1 in either pro-opiomelanocortin (POMC) or agouti-related protein neurons, mediators of leptin action, displayed obesity and impaired leptin sensitivity. In addition, deletion of ROCK1 in the arcuate nucleus markedly enhanced food intake, resulting in severe obesity. Notably, ROCK1 was a specific mediator of leptin, but not insulin, regulation of POMC neuronal activity. Our data identify ROCK1 as a key regulator of leptin action on energy homeostasis.

Over-expression of leptin receptors in hypothalamic POMC neurons increases susceptibility to diet-induced obesity.

Diet-induced obesity (DIO) in rodents is characterized by impaired activation of signal-transducer and activator of transcription 3 (STAT3) by leptin receptors (LepRb) within the hypothalamic arcuate nucleus. This signaling defect likely plays an important role in development of DIO. However, the neuro-chemical identity of the leptin-STAT3 resistant arcuate neurons has not been determined and the underlying mechanisms responsible for development of cellular leptin resistance remain unclear. To investigate this, we first measured arcuate gene expression of known key signaling components of the LepRb signaling pathway and tested whether specifically the critical arcuate pro-opiomelanocortin (POMC) neurons are resistant to LepRb-STAT3 signaling in mice given a high-fat-diet (HFD) compared to mice provided a low-fat control diet (LFD). We found that leptin-dependent STAT3 phosphorylation was decreased within POMC neurons of HFD mice. In addition, Leprb mRNA and suppressor of cytokine signaling 3 (Socs3) mRNA were elevated in the arcuate of HFD mice. To investigate whether increased LepRb expression per se in POMC neurons can influence development of DIO and Socs3 expression, we created mice that over-express LepRb selectively in POMC neurons (POMC-LepRb). No differences in body weight, fat mass or food intake were found between LFD POMC-LepRb mice and LFD controls. Surprisingly, body weight, fat mass and caloric intake of HFD POMC-LepRb mice was markedly higher than HFD control mice. In addition, arcuate Socs3 mRNA was increased in HFD POMC-LepRb mice compared to HFD controls. These data show that specifically POMC neurons of DIO mice are resistant to STAT3 activation by leptin, indicating that those cells might play a role in development of DIO. Furthermore, over-expression of LepRb selectively in POMC neurons increases susceptibility to the development of DIO. We propose a model where over-reactivity of the leptin-LepRb signaling system in arcuate neurons may play causal a role in development of diet-induced obesity.

Melanocortins mimic the effects of leptin to restore reproductive function in lean hypogonadotropic ewes.

BACKGROUND/AIMS: Leptin restores gonadotropic function in lean hypogonadotropic animals by an unknown mechanism. We aimed to test the hypothesis that restoration of gonadotropic function is a result of an upregulation of central acetylated melanocortin production. METHODS AND RESULTS: Lean ovariectomised (OVX) ewes received intracerebroventricular (i.c.v.) infusions of leptin (or vehicle) for 3 days, which upregulated proopiomelanocortin (POMC) mRNA and restored pulsatile luteinizing hormone (LH) secretion. A melanocortin agonist (MTII), but not naloxone treatment, reinstated pulsatile LH secretion in lean OVX ewes. We treated (i.c.v.) lean OVX ewes with leptin (or vehicle) and measured peptide levels and post-translational modification in the arcuate nucleus (ARC). Levels of beta-endorphin (beta-END) were lower in lean animals, with no effect of leptin treatment. Desacetyl-alpha-MSH was the predominant form of alpha-melanocyte-stimulating hormone (alpha-MSH) in the ARC and levels were similar in all groups. In another group of lean and normal-weight OVX ewes, we measured the different forms of alpha-MSH in ARC, hypothalamus (ARC-removed) and the preoptic area (POA). Acetylated alpha-MSH levels were lower in lean animals in the terminal beds of the hypothalamus and POA but not the ARC. CONCLUSIONS: Leptin corrects the hypogonadotropic state in the lean condition by upregulation of POMC gene expression, and may increase transport and acetylation of melanocortins to target cells in the brain. Melanocortin treatment restores LH secretion in lean animals.

Central leptin receptor action and resistance in obesity.

The discovery of leptin in 1994 has led to remarkable advances in obesity research. We now know that leptin is a cytokinelike hormone that is produced in adipose tissue and plays a pivotal role in regulation of energy balance and in a variety of additional processes via actions in the central nervous system. This symposium review covers current understandings of neuronal leptin receptor signaling and mechanisms of obesity-related leptin resistance in the central nervous system and provides recent insights into the regulation of peripheral glucose balance by central leptin action in rodents.

Leptin-dependent control of glucose balance and locomotor activity by POMC neurons.

Leptin plays a pivotal role in regulation of energy balance. Via unknown central pathways, leptin also affects peripheral glucose homeostasis and locomotor activity. We hypothesized that, specifically, pro-opiomelanocortin (POMC) neurons mediate those actions. To examine this possibility, we applied Cre-Lox technology to express leptin receptors (ObRb) exclusively in POMC neurons of the morbidly obese, profoundly diabetic, and severely hypoactive leptin receptor-deficient Lepr(db/db) mice. Here, we show that expression of ObRb only in POMC neurons leads to a marked decrease in energy intake and a modest reduction in body weight in Lepr(db/db) mice. Remarkably, blood glucose levels are entirely normalized. This normalization occurs independently of changes in food intake and body weight. In addition, physical activity is greatly increased despite profound obesity. Our results suggest that leptin signaling exclusively in POMC neurons is sufficient to stimulate locomotion and prevent diabetes in the severely hypoactive and hyperglycemic obese Lepr(db/db) mice.

Divergent leptin signaling in proglucagon neurons of the nucleus of the solitary tract in mice and rats.

The central targets mediating the anorectic and other actions of leptin have yet to be fully identified. Although previous studies focused on the hypothalamus, leptin also acts on neurons in extrahypothalamic sites, including the nucleus of the solitary tract (NTS). Moreover, injection of leptin into the NTS of rats suppresses food intake. Within the central nervous system, glucagon-like peptide (GLP-1), a product of proglucagon, is synthesized almost exclusively in neurons of the NTS. Intracerebroventricular administration of GLP-1 inhibits energy intake, and GLP-1 receptor antagonists attenuate the anorexic effects of leptin in rats. To examine whether NTS proglucagon neurons are directly regulated by leptin, we performed double GLP-1 and phosphorylation of signal transducer and activator of transcription-3 immunohistochemistry on brain sections from ip leptin-treated mice and rats. Leptin induced phosphorylation of signal transducer and activator of transcription-3 in 100% of GLP-1 cells in the caudal brainstem of mice. In striking contrast, 0% of GLP-1-positive neurons in rats responded to leptin. We then measured regulation of NTS proglucagon mRNA using real-time RT-PCR in mice and rats fed ad libitum, fasted, or fasted and treated ip with leptin. In mice, proglucagon mRNA fell by fasting, and this was prevented by leptin administration. In rats, by contrast, proglucagon mRNA was unaffected by either fasting or leptin. Taken together, our studies reveal direct regulation of proglucagon neurons by leptin in mice but not rats along with corresponding species differences in the regulation of proglucagon mRNA expression. These data, combined with previous results, suggest a different mechanism of interaction between leptin and NTS proglucagon neurons in mice and rats.

Urocortin trafficking in cerebral microvessel endothelial cells.

Urocortin, a potent peptide inhibitor of feeding behavior, can enter the brain from blood by leptin-facilitated permeation across the blood-brain barrier. Here, we show in cultured RBE4 cerebral microvessel endothelial cells that urocortin endocytosis is increased by leptin in a time- and dose-dependent manner. Fluorescently labeled urocortin (Alexa488-urocortin) shows vesicular trafficking localized in early endosomes at 1 min and the Golgi complex at 20 min. The endocytosis at 20 min was increased by 10 microg/mL, but not 2 microg/mL, of leptin. The facilitating effect of leptin at the dose of 10 microg/mL was seen at 20 and 30 min but not at 10 min. This increase could be abolished by excess unlabeled urocortin in radio-tracer uptake studies, indicating selective rather than nonsaturable entry. The specificity of the effect was further supported by the lack of changes in gamma-glutamyl transpeptidase activity and endothelial nitric oxide synthase upon stimulation by high doses of leptin and urocortin. Leptin did not affect the level of expression of the urocortin corticotropin-releasing hormone receptor (CRHR) after 30 min of treatment but appeared to slow the turnover of CRHRs induced by urocortin. In MDCK cells overexpressing CRHR2, leptin facilitated urocortin uptake, whereas ObRa coexpression did not exert an additional effect. Thus, urocortin endocytosis is a saturable process leading to vesicular intracellular transport that can be enhanced by cell-surface leptin.

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.

Leptin and the control of food intake: neurons in the nucleus of the solitary tract are activated by both gastric distension and leptin.

Leptin reduces food intake by an unspecified mechanism. Studies show that forebrain ventricular leptin delivery increases the inhibitory effects of gastrointestinal (GI) stimulation on intake and amplifies the electrophysiological response to gastric distension in neurons of the medial subnucleus of the nucleus tractus solitarius (mNTS). However, forebrain ventricular delivery leaves unspecified the neuroanatomical site(s) mediating leptin's effect on intake. Detailed anatomical analysis in rats and mice by phosphorylated signal transducer and activator of transcription 3 immunohistochemistry shows that hindbrain leptin-responsive neurons are located exclusively within the mNTS. Here, we investigate 1) whether leptin and gastric distension affect the same mNTS neurons and 2) whether the intake-inhibitory action of gastric distension is potentiated by hindbrain leptin delivery. Twenty-five minutes after gastric balloon distension or sham distension, rats were injected with leptin or vehicle and killed 35 min later. Double-fluorescent immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 and c-Fos revealed that about 40% of leptin-responsive cells also respond to gastric distension. A paradigm was then developed to examine the relationship between leptin and gastric distension volume on intake inhibition. At subthreshold levels, hindbrain ventricular leptin or distension volume were without effect. When combined, an interaction occurred that significantly reduced food intake. We conclude that 1) leptin-responsive neurons in the hindbrain are primarily located in the mNTS at the level of the area postrema, a key vagal afferent projection zone of the GI system; 2) a significant proportion of leptin-responsive neurons in the mNTS are activated by stomach distension; and 3) leptin delivered to the hindbrain is sufficient to potentiate the intake-suppressive effects of an otherwise ineffective volume of gastric distension. These results are consistent with the hypothesis that leptin acts directly on neurons within the mNTS to reduce food intake through an interaction with GI signal processing.

Divergent regulation of proopiomelanocortin neurons by leptin in the nucleus of the solitary tract and in the arcuate hypothalamic nucleus.

Proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus are activated by leptin and mediate part of leptin's central actions to influence energy balance. However, little is known about potential leptin signaling in POMC neurons located in the nucleus of the solitary tract (NTS), the only other known population of POMC neurons. Leptin-responsive neurons do exist in the NTS, but their neurochemical phenotype is largely unknown. The contribution of NTS POMC neurons versus ARC POMC neurons in leptin action is thus undetermined. We show here that in contrast to POMC neurons in the ARC, leptin does not stimulate phosphorylation of signal-transducer and activator of transcription 3 in NTS POMC neurons of POMC-EGFP reporter mice. In addition, leptin does not induce c-Fos expression in NTS POMC neurons unlike ARC POMC neurons. Fasting induces a fall in POMC mRNA in both the ARC and the NTS, but different from the ARC, the reduction in NTS POMC mRNA is not reversed by leptin. We conclude that POMC neurons in the NTS do not respond to leptin unlike ARC POMC neurons. POMC neurons in the hypothalamus may therefore mediate all of leptin's signaling via POMC-derived peptides in the central nervous system.

Overexpression of suppressor of cytokine signaling 3 in adipose tissue causes local but not systemic insulin resistance.

In adipocytes, suppressor of cytokine signaling (SOCS)3 deficiency increases insulin-stimulated insulin receptor substrate (IRS)-1 and -2 phosphorylation, IRS-associated phosphatidylinositol 3 kinase activity, and insulin-stimulated glucose uptake. Moreover, SOCS3 is required for tumor necrosis factor-alpha full inhibition of insulin-stimulated IRS-1 and -2 phosphorylation, phosphatidylinositol 3 kinase activity, and glucose uptake. Whether SOCS3 also inhibits adipocyte insulin signaling in vivo and whether this action further affects systemic insulin sensitivity is not clear. We therefore generated a transgenic mouse (aP2-SOCS3 mouse) overexpressing SOCS3 in adipose tissue. Overexpression of SOCS3 in adipocytes decreases IRS1 protein levels and subsequent insulin-stimulated IRS-1 and -2 phosphorylation, decreases p85 binding to IRS-1, and leads to decreased insulin-stimulated glucose uptake in adipocytes. This impaired insulin signaling in adipose tissue of aP2-SOCS3 mice causes decreased lipogenesis and blocks insulin's antilipolytic action. However, because of decreased energy partitioning in adipose tissue, aP2-SOCS3 mice are resistant to diet-induced obesity and are protected against systemic insulin resistance caused by a high-fat diet. Therefore, overexpression of SOCS3 in adipocytes causes local adipocyte insulin resistance, but it is not sufficient to cause systemic insulin resistance.

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

BACKGROUND INFORMATION: Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. RESULTS: The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. CONCLUSIONS: Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

Regulation of hypothalamic prohormone convertases 1 and 2 and effects on processing of prothyrotropin-releasing hormone.

Regulation of energy balance by leptin involves regulation of several neuropeptides, including thyrotropin-releasing hormone (TRH). Synthesized from a larger inactive precursor, its maturation requires proteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). Since this maturation in response to leptin requires prohormone processing, we hypothesized that leptin might regulate hypothalamic PC1 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Using hypothalamic neurons, we found that leptin stimulated PC1 and PC2 mRNA and protein expression and also increased PC1 and PC2 promoter activities in transfected 293T cells. Starvation of rats, leading to low serum leptin levels, decreased PC1 and PC2 gene and protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Exogenous administration of leptin to fasted animals restored PC1 levels in the median eminence (ME) and the PVN to approximately the level found in fed control animals. Consistent with this regulation of PCs in the PVN, concentrations of TRH in the PVN and ME were substantially reduced in the fasted animals relative to the fed animals, and leptin reversed this decrease. Further analysis showed that proteolytic cleavage of pro-thyrotropin-releasing hormone (proTRH) at known PC cleavage sites was reduced by fasting and increased in animals given leptin. Combined, these findings suggest that leptin-dependent stimulation of hypothalamic TRH expression involves both activation of trh transcription and stimulation of PC1 and PC2 expression, which lead to enhanced processing of proTRH into mature TRH.

Region-specific leptin resistance within the hypothalamus of diet-induced obese mice.

Leptin resistance in diet-induced obese (DIO) mice is characterized by elevated serum leptin and a decreased response to exogenous leptin and is caused by unknown defects in the central nervous system. Leptin normally acts on several brain nuclei, but a detailed description of leptin resistance within individual brain regions has not been reported. We first mapped leptin-responsive cells in brains from DIO mice using phospho-signal transducer and activator of transcription (P-STAT3) immunohistochemistry. After 16 wk of high-fat-diet feeding, leptin-activated P-STAT3 staining within the arcuate nucleus (ARC) was dramatically decreased. In contrast, other hypothalamic and extrahypothalamic nuclei remained leptin sensitive. Reduced leptin-induced P-STAT3 in the ARC could also be detected after 4 wk and as early as 6 d of a high-fat diet. To examine potential mechanisms for leptin-resistant STAT3 activation in the ARC of DIO mice, we measured mRNA levels of candidate signaling molecules in the leptin receptor-STAT3 pathway. We found that the level of suppressor of cytokine signaling 3 (SOCS-3), an inhibitor of leptin signaling, is specifically increased in the ARC of DIO mice. The study suggests that the ARC is selectively leptin resistant in DIO mice and that this may be caused by elevated suppressor of cytokine signaling 3 in this hypothalamic nucleus. Defects in leptin action in the ARC may play a role in the pathogenesis of leptin-resistant obesity.