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.

Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3.

Leptin is an adipocyte-derived hormone that regulates energy balance and neuroendocrine function primarily by acting on specific hypothalamic pathways. Resistance to the weight reducing effects of leptin is a feature of most cases of human and rodent obesity, yet the molecular basis of leptin resistance is poorly understood. We have previously identified suppressor of cytokine signaling-3 (Socs3) as a leptin-induced negative regulator of leptin receptor signaling and potential mediator of leptin resistance. However, due to the non-viability of mice with targeted disruption of Socs3 (ref. 6), the importance of Socs3 in leptin action in vivo was unclear. To determine the functional significance of Socs3 in energy balance in vivo we undertook studies in mice with heterozygous Socs3 deficiency (Socs3(+/-)). We report here that Socs3(+/-) mice display greater leptin sensitivity than wild-type control mice: Socs3(+/-) mice show both enhanced weight loss and increased hypothalamic leptin receptor signaling in response to exogenous leptin administration. Furthermore, Socs3(+/-) mice are significantly protected against the development of diet-induced obesity and associated metabolic complications. The level of Socs3 expression is thus a critical determinant of leptin sensitivity and obesity susceptibility in vivo and this molecule is a potential target for therapeutic intervention.

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.

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.

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.

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.

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.

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.

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.

Insulin resistance and lipodystrophy in mice lacking ribosomal S6 kinase 2.

The p90 ribosomal S6 kinase 2 (RSK2) is a serine/threonine kinase with high expression levels in adipose tissue. Numerous in vitro studies show that RSK2 is activated by a broad number of cellular stimuli and suggest that RSK2 is involved in the regulation of a variety of cellular processes. However, the physiological role of RSK2 still remains elusive. We therefore generated rsk2 knockout (KO) mice to better understand the function of RSK2 in vivo. Birth weights of RSK2 KO mice are normal, but the body weight is reduced with age, as compared with wild-type littermates. We found that the difference in body weight was largely caused by a specific loss of white adipose tissue that is accompanied by reduced serum levels of the adipocyte-derived peptide, leptin. KO mice also have impaired glucose tolerance and elevated fasting insulin and glucose levels that are restored following administration of low amounts of leptin, which do not affect food intake. We conclude that RSK2 plays a novel and an important role in regulation of adipose mass in mice and speculate that the reduction in fat tissue may negatively affect insulin sensitivity, as observed in human lipodystrophy, through reduced levels of adipocyte-derived factors, such as leptin.

Ciliary neurotrophic factor and leptin induce distinct patterns of immediate early gene expression in the brain.

Ciliary neurotrophic factor (CNTF) and leptin decrease food intake and body weight. Lipopolysaccharide (LPS) is a potent exogenous pyrogen and produces anorexia via cytokine production. CNTF-, leptin-, and LPS-induced cytokines all act on type I cytokine receptors. However, it is not known if these cytokines engage similar central nervous system (CNS) pathways to exert their effects. To assess mechanisms by which these cytokines act, we examined the patterns of immediate early gene expression (SOCS-3, c-fos, and tis-11) in the brain following intravenous administration. CNTF and LPS induced gene expression in circumventricular organs; ependymal cells of the ventricles, meninges, and choroid plexus; and the arcuate nucleus of the hypothalamus. CNTF administration also induced fever and cyclooxygenase-2 mRNA expression. In contrast, we found no evidence of leptin-induced inflammation. CNTF and leptin are being assessed as potential therapeutic anti-obesity agents, and both potently reduce food intake. Our findings support the hypothesis that CNTF and leptin engage distinct CNS sites and CNTF possesses inflammatory properties distinct from leptin.

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.

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.

Role of signal transducer and activator of transcription 3 in regulation of hypothalamic trh gene expression by leptin.

During starvation in rodents, the hypothalamic-pituitary-thyroid axis is down-regulated, resulting in low circulating thyroid hormone levels. This involves a reduction in hypothalamic TRH mRNA that is caused in part by a fall in serum leptin levels, which is sensed by neurons within the hypothalamus. The mechanism by which this regulation occurs is not fully understood. Here we show transfection data and in vivo evidence, suggesting that leptin can regulate trh gene expression via activation of intracellular signal transducer and activator of transcription 3 (STAT3) proteins in TRH neurons. In trh promoter assays using transfected cells, functional STAT3 proteins are required for maximal activation of the trh promoter by leptin. Consistent with this, the STAT3-binding site on the leptin receptor is also required for this regulation. Using double immunohistochemistry, we show that peripherally administered leptin rapidly stimulates STAT3 phosphorylation in approximately 40% of TRH neurons in the paraventricular nucleus of the hypothalamus (PVN) in rats. Detailed anatomical analyses reveal that the leptin-responsive TRH neurons are concentrated in the caudal region of the medial and periventricular parvocellular subnucleus of the PVN. Combined, our data show that only a subpopulation of TRH neurons in the PVN is leptin responsive and suggest that stimulation of hypothalamic trh gene expression by leptin involves activation of STAT3 and that this signaling pathway is important for regulation of the hypothalamic-pituitary-thyroid axis by leptin.

Role of signal transducer and activator of transcription 3 in regulation of hypothalamic proopiomelanocortin gene expression by leptin.

Leptin acts on the brain to regulate body weight and neuroendocrine function. Proopiomelanocortin (POMC) neurons in the hypothalamus are important targets of leptin. These cells express the leptin receptor ObRb, and leptin can regulate POMC mRNA levels, but the cellular mechanisms by which this occurs is unknown. Here we show evidence that leptin stimulates pomc gene transcription via activation of intracellular signal transducer and activator of transcription 3 (STAT3) proteins. In pomc-promoter assays using transfected cells, leptin induces pomc promoter activity. Expression of dominant negative STAT3 strongly suppresses this effect. Furthermore, maximal activation requires the presence of the STAT3-binding site, tyrosine 1138, of ObRb. Mutational analysis identifies a 30-bp promoter element that is required for regulation by leptin. In rats, robust leptin-dependent induction of STAT3 phosphorylation is demonstrated in hypothalamic POMC neurons using double immunohistochemistry. In total, approximately 37% of POMC cells are positive for phospho-STAT3 after leptin treatment. Furthermore, leptin-responsive POMC neurons are concentrated in the rostral region of the hypothalamus. Combined, our data show that a subpopulation of POMC neurons is leptin-responsive and suggest that stimulation of hypothalamic pomc gene expression in these cells requires STAT3 activation. We speculate that STAT3 is critical for leptin-dependent effects on energy homeostasis that are mediated by the central melanocortin system.

Characterizaton of short isoforms of the leptin receptor in rat cerebral microvessels and of brain uptake of leptin in mouse models of obesity.

Leptin deficiency causes obesity in rodents and humans, but circulating levels of leptin are paradoxically elevated in obesity. The mechanisms underlying this leptin resistance are unknown, but may involve reduced leptin transport across the blood-brain barrier via short isoforms of the leptin receptor (Ob-R). Here, we first quantified short Ob-R mRNA expression in isolated rat cerebral microvessels constituting the blood-brain barrier and found that Ob-Ra and Ob-Rc mRNA were abundantly expressed in similar amounts. Second, brain uptake of leptin was reduced in mice lacking Ob-R. Third, brain uptake of leptin in New Zealand Obese mice, a strain that responds to central, but not peripheral, leptin, was reduced, suggesting that their obesity is at least partly due to deficient leptin transport into the brain. Fourth, brain uptake of leptin was significantly reduced in diet-induced obese mice. Neither New Zealand Obese mice nor diet-induced obese mice exhibited significant decreases in Ob-R mRNA expression in isolated cerebral microvessels. These data support the ideas that short isoforms of Ob-R are involved in brain uptake of leptin and that impaired blood-brain barrier function contributes to the pathogenesis of obesity. However, the mechanisms by which obesity-related deficits in brain uptake of leptin occur remain to be defined.

Identification and characterization of melanocortin-4 receptor gene mutations in morbidly obese finnish children and adults.

Two Finnish cohorts, comprising 56 children with severe early-onset obesity (relative weight for height greater than or equal to +70% before age 10) and 252 morbidly obese adults (body mass index, > or = 40 kg/m(2)), were screened for melanocortin-4 receptor (MC4R) mutations. We identified a pathogenic mutation (S127L) in one child, causing severe early-onset obesity. We describe the phenotype of this particular mutation for the first time. We also identified a novel (I226T) polymorphism in the coding and two new variations (-439delGC and 1059C>T) outside the coding region of the MC4R gene. Three previously described polymorphisms (V103I, T112M, and I125L) were identified. In vitro functional studies of variants T112M, S127L, and I226T supported a pathogenic role of the S127L mutation, because signaling properties of the receptor in response to the MC4R agonists alpha-MSH, beta-MSH, and gamma(1)-MSH were impaired. The S127L mutation did not affect receptor inhibition by the antagonist agouti-related protein. Localization of the three variant receptors was similar to that of wild type. In conclusion, a pathogenic MC4R mutation was found among subjects with severe early-onset obesity but not among morbidly obese adults. Impaired function of the S127L receptor was due to reduced activation, not a defect of protein transport to the cell membrane.

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.