Hypothalamic LepRb Research Articles

Suppression of food intake by Glp1r/Lepr-coexpressing neurons prevents obesity in mouse models.

The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A potentially unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-Seq of enriched mouse hypothalamic LepRb cells, identifying several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations displayed strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons, which expressed more Lepr than other LepRb cell populations. Ablating Lepr from LepRbGlp1r cells provoked hyperphagic obesity without impairing energy expenditure. Similarly, improvements in energy balance caused by Lepr reactivation in GABA neurons of otherwise Lepr-null mice required Lepr expression in GABAergic Glp1r-expressing neurons. Furthermore, restoration of Glp1r expression in LepRbGlp1r neurons in otherwise Glp1r-null mice enabled food intake suppression by the GLP1R agonist, liraglutide. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the suppression of food intake by leptin and GLP1R agonists.

LepRb+ cell-specific deletion of Slug mitigates obesity and nonalcoholic fatty liver disease in mice.

Leptin exerts its biological actions by activating the long-form leptin receptor (LepRb). LepRb signaling impairment and leptin resistance are believed to cause obesity. The transcription factor Slug - also known as Snai2 - recruits epigenetic modifiers and regulates gene expression by an epigenetic mechanism; however, its epigenetic action has not been explored in leptin resistance. Here, we uncover a proobesity function of neuronal Slug. Hypothalamic Slug was upregulated in obese mice. LepRb+ cell-specific Slug-knockout (SlugΔLepRb) mice were resistant to diet-induced obesity, type 2 diabetes, and liver steatosis and experienced decreased food intake and increased fat thermogenesis. Leptin stimulated hypothalamic Stat3 phosphorylation and weight loss to a markedly higher level in SlugΔLepRb than in Slugfl/fl mice, even before their body weight divergence. Conversely, hypothalamic LepRb+ neuron-specific overexpression of Slug, mediated by AAV-hSyn-DIO-Slug transduction, induced leptin resistance, obesity, and metabolic disorders in mice on a chow diet. At the genomic level, Slug bound to and repressed the LepRb promoter, thereby inhibiting LepRb transcription. Consistently, Slug deficiency decreased methylation of LepRb promoter H3K27, a repressive epigenetic mark, and increased LepRb mRNA levels in the hypothalamus. Collectively, these results unravel what we believe to be a previously unrecognized hypothalamic neuronal Slug/epigenetic reprogramming/leptin resistance axis that promotes energy imbalance, obesity, and metabolic disease.

Prenatal exercise reverses high-fat-diet-induced placental alterations and alters male fetal hypothalamus during late gestation in rats†

Maternal high-fat (HF) diet negatively affects maternal metabolism and placental function. This study aimed to determine whether gestational exercise prevents the effect of HF diet on placental amino acid transporter expression and nutrient-sensing signaling and the fetal response. Pregnant Sprague-Dawley rats were either fed with a CHOW (13.5% fat) or HF (60% fat) diet during gestation and further divided into two subgroups: voluntary exercised and sedentary. Placentae were collected on gestational day (GD) 14 and GD20, and male placentae were used in this study. We found that gestational exercise ameliorated the detrimental effects of HF diet on dams' adiposity, plasma leptin, and insulin concentrations. Maternal exercise did not influence fetoplacental growth but affected male fetal hypothalamic Leprb, Stat3, Insr, Agrp, and Pomc expressions on GD20. Maternal HF diet decreased placental labyrinth thickness and increased system A amino acid transporter SNAT2 expression, while these changes were normalized by exercise. The activation of placental mechanistic target of rapamycin complex 1/4E-BP1 and LepRb/STAT3 signaling might contribute to the increased placental SNAT2 expression in HF-fed dams, which were reversed by exercise on GD20. These data highlight that gestational exercise reverses HF-diet-induced placental alterations during late gestation without influencing fetal growth. However, maternal exercise altered fetal hypothalamic gene expression, which may affect long-term offspring health.

Specific subpopulations of hypothalamic leptin receptor-expressing neurons mediate the effects of early developmental leptin receptor deletion on energy balance

ObjectiveTo date, early developmental ablation of leptin receptor (LepRb) expression from circumscribed populations of hypothalamic neurons (e.g., arcuate nucleus (ARC) Pomc- or Agrp-expressing cells) has only minimally affected energy balance. In contrast, removal of LepRb from at least two large populations (expressing vGat or Nos1) spanning multiple hypothalamic regions produced profound obesity and metabolic dysfunction. Thus, we tested the notion that the total number of leptin-responsive hypothalamic neurons (rather than specific subsets of cells with a particular molecular or anatomical signature) subjected to early LepRb deletion might determine energy balance. MethodsWe generated new mouse lines deleted for LepRb in ARC GhrhCre neurons or in Htr2cCre neurons (representing roughly half of all hypothalamic LepRb neurons, distributed across many nuclei). We compared the phenotypes of these mice to previously-reported models lacking LepRb in Pomc, Agrp, vGat or Nos1 cells. ResultsThe early developmental deletion of LepRb from vGat or Nos1 neurons produced dramatic obesity, but deletion of LepRb from Pomc, Agrp, Ghrh, or Htr2c neurons minimally altered energy balance. ConclusionsAlthough early developmental deletion of LepRb from known populations of ARC neurons fails to substantially alter body weight, the minimal phenotype of mice lacking LepRb in Htr2c cells suggests that the phenotype that results from early developmental LepRb deficiency depends not simply upon the total number of leptin-responsive hypothalamic LepRb cells. Rather, specific populations of LepRb neurons must play particularly important roles in body energy homeostasis; these as yet unidentified LepRb cells likely reside in the DMH.

Defining the Transcriptional Targets of Leptin Reveals a Role for Atf3 in Leptin Action.

Leptin acts via its receptor (LepRb) to modulate gene expression in hypothalamic LepRb-expressing neurons, thereby controlling energy balance and glucose homeostasis. Despite the importance of the control of gene expression in hypothalamic LepRb neurons for leptin action, the transcriptional targets of LepRb signaling have remained undefined because LepRb cells contribute a small fraction to the aggregate transcriptome of the brain regions in which they reside. We thus employed translating ribosome affinity purification followed by RNA sequencing to isolate and analyze mRNA from the hypothalamic LepRb neurons of wild-type or leptin-deficient (Lepob/ob) mice treated with vehicle or exogenous leptin. Although the expression of most of the genes encoding the neuropeptides commonly considered to represent the main targets of leptin action were altered only following chronic leptin deprivation, our analysis revealed other transcripts that were coordinately regulated by leptin under multiple treatment conditions. Among these, acute leptin treatment increased expression of the transcription factor Atf3 in LepRb neurons. Furthermore, ablation of Atf3 from LepRb neurons (Atf3LepRbKO mice) decreased leptin efficacy and promoted positive energy balance in mice. Thus, this analysis revealed the gene targets of leptin action, including Atf3, which represents a cellular mediator of leptin action.

Setting the TRAP for central leptin targets.

Setting the TRAP for central leptin targets.

Leptin action through hypothalamic nitric oxide synthase-1–expressing neurons controls energy balance

SummaryFew effective measures exist to combat the worldwide obesity epidemic1, and the identification of potential therapeutic targets requires a deeper understanding of the mechanisms that control energy balance. Leptin, an adipocyte hormone that signals the status of cellular energy stores, acts via multiple types of leptin receptor (LepR-b)-expressing neurons in the brain to control feeding, energy expenditure and endocrine function2–4. The modest contributions to energy balance attributable to leptin action via many previously-studied LepR-b populations5–9 suggest that other, heretofore unidentified, hypothalamic LepR-b neurons play important roles. Here, we examine the role of LepR-b in neuronal nitric oxide synthase (NOS1)-expressing (LepR-bNOS1) neurons that comprise approximately 20% of hypothalamic LepR-b neurons. Nos1cre-mediated ablation of LepR-b (LeprNOS1KO mice) produces hyperphagic obesity, decreased energy expenditure and hyperglycemia approaching that of LepR-b-null mice. In contrast, endocrine functions in LeprNOS1KO mice are relatively spared. Thus, hypothalamic LepR-bNOS1 neurons are essential for the control of energy balance by leptin.

Direct Innervation and Modulation of Orexin Neurons by Lateral Hypothalamic LepRb Neurons

Leptin, the adipose-derived hormonal signal of body energy stores, acts via the leptin receptor (LepRb) on neurons in multiple brain regions. We previously identified LepRb neurons in the lateral hypothalamic area (LHA), which are distinct from neighboring leptin-regulated melanin-concentrating hormone (MCH)- or orexin (OX)-expressing cells. Neither the direct synaptic targets of LHA LepRb neurons nor their potential role in the regulation of other LHA neurons has been determined, however. We thus generated several adenoviral and transgenic systems in which cre recombinase promotes the expression of the tracer, WGA (wheat germ agglutinin), and used these in combination with LepRb(cre) mice to determine the neuronal targets of LHA LepRb neurons. This analysis revealed that, although some LHA LepRb neurons project to dopamine neurons in the ventral tegmental area, LHA LepRb neurons also densely innervate the LHA where they directly synapse with OX, but not MCH, neurons. Indeed, few other LepRb neurons in the brain project to the OX-containing region of the mouse LHA, and direct leptin action via LHA LepRb neurons regulates gene expression in OX neurons. These findings thus reveal a major role for LHA leptin action in the modulation of OX neurons, suggesting the importance of LHA LepRb neurons in the regulation of OX signaling that is crucial to leptin action and metabolic control.

Distribution and neurochemical characterization of protein kinase C-theta and -delta in the rodent hypothalamus

PKC-theta (PKC-θ), a member of the novel protein kinase C family (nPKC), regulates a wide variety of functions in the periphery. However, its presence and role in the CNS has remained largely unknown. Recently, we demonstrated the presence of PKC-θ in the arcuate hypothalamic nucleus (ARC) and knockdown of PKC-θ from the ARC protected mice from developing diet-induced obesity. Another isoform of the nPKC group, PKC-delta (PKC-δ), is expressed in several non-hypothalamic brain sites including the thalamus and hippocampus. Although PKC-δ has been implicated in regulating hypothalamic glucose homeostasis, its distribution in the hypothalamus has not previously been described. In the current study, we used immunohistochemistry to examine the distribution of PKC-θ and -δ immunoreactivity in rat and mouse hypothalamus. We found PKC-θ immunoreactive neurons in several hypothalamic nuclei including the ARC, lateral hypothalamic area, perifornical area and tuberomammillary nucleus. PKC-δ immunoreactive neurons were found in the paraventricular and supraoptic nuclei. Double-label immunohistochemisty in mice expressing green fluorescent protein either with the long form of leptin receptor (LepR-b) or in orexin (ORX) neurons indicated that PKC-θ is highly colocalized in lateral hypothalamic ORX neurons but not in lateral hypothalamic LepR-b neurons. Double-label immunohistochemistry in oxytocin-enhanced yellow fluorescent protein mice or arginine vasopressin-enhanced green fluorescent protein (AVP-EGFP) transgenic rats revealed a high degree of colocalization of PKC-δ within paraventricular and supraoptic oxytocin neurons but not the vasopressinergic neurons. We conclude that PKC-θ and -δ are expressed in different hypothalamic neuronal populations.

Excessive nitric oxide attenuates leptin-mediated signal transducer and activator of transcription 3 activation

The mechanisms of leptin resistance observed in most cases of human obesity are poorly understood. Therefore, we evaluated the effects of nitric oxide (NO) on the leptin-induced activation of Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathways and on the leptin receptor (LEPR) expression using SH-SY5Y cells. Here, we show that the NO donor spermine/NONOate inhibited leptin-induced activation of STAT3 in vitro. The inhibition of leptin-mediated STAT3 phosphorylation caused by excessive NO was partially prevented by a sulfhydryl reducing agent, ascorbic acid. Cellular experiments show that reduced expression of long form leptin receptor (LEPR-b) and STAT3 protein instability induced by NO may be mechanisms of the NO-mediated inhibition of leptin-STAT3 signaling. We also present data showing that the hypothalamic NO content of high-fat (HF)-diet-induced obese mice was higher than that of control mice; this is likely caused by decreased caveolin-1 expression and increased nNOS expression induced by HF diet over 19 weeks. Concurrently with the overproduction of NO, the decrease of hypothalamic LEPR-b in obese mice also supports these in vitro data. Combined results suggest that excess of NO can induce the attenuation of leptin-mediated STAT3 activation through reduced expression of LEPR-b mRNA and instability of STAT3 protein at least in part. Furthermore, our in vivo data indicate that long-term HF diet induces hypothalamic overproduction of NO, which may be related with leptin insensitivity. However, further study is required to warrant direct in vivo evidence of a causal relationship between endogenous excess of hypothalamic NO and central leptin resistance.

Increased expression of hypothalamic leptin receptor and adiponectin accompany resistance to dietary-induced obesity and infertility in female C57BL/6J mice

Obesity is strongly associated with female infertility, but the mechanisms underlying this relationship are largely unknown. We investigated the effect of increasing dietary fat percentage upon body mass, hypothalamic neuropeptide gene expression, adipose hormone secretion and fertility in females of the inbred mouse strains C57BL/6J and DBA/2J. To assess the effect of obesity independent of dietary influence, we also compared these parameters in wild-type female C57BL/6J mice to those congenic for the obesogenic mutations ob/ob and A(y)/a. After 24 weeks, rather than exhibiting an obese, leptin-resistant phenotype like their female DBA/2J counterparts, wild-type female C57BL/6J mice remained lean, fertile and manifested increased hypothalamic LEPR-B expression. Although both mutant genotypes were associated with obesity and subfertility, ob/ob mice demonstrated significantly increased hypothalamic LEPR-B expression, whereas A(y)/a mice had a significant reduction. Interestingly, wild-type female C57BL/6J mice were noted to manifest significantly higher and lower levels of adiponectin and tissue plasminogen activator inhibitor-1 (tPAI-1), respectively, than weight-matched wild-type female DBA/2J mice. We conclude that (1) resistance to the obese-infertile phenotype in female C57BL/6J mice is associated with increased hypothalamic leptin receptor expression and alterations in adipokine levels consistent with decreased adipose tissue inflammation and (2) that long-standing hyperleptinemic obesity in mice is associated with a downregulation of the hypothalamic leptin receptor.