Neurochemical Characterization of Body Weight-Regulating Leptin Receptor Neurons in the Nucleus of the Solitary Tract

Abstract The action of peripherally released leptin at long-form leptin receptors (LepRb) within the brain represents a fundamental axis in the regulation of energy homeostasis and body weight. Efforts to delineate the neuronal mediators of leptin action have recently focused on extrahypothalamic populations and have revealed that leptin action within the nucleus of the solitary tract (NTS) is critical for normal appetite and body weight regulation. To elucidate the neuronal circuits that mediate leptin action within the NTS, we employed multiple transgenic reporter lines to characterize the neurochemical identity of LepRb-expressing NTS neurons. LepRb expression was not detected in energy balance-associated NTS neurons that express cocaine- and amphetamine-regulated transcript, brain-derived neurotrophic factor, neuropeptide Y, nesfatin, catecholamines, γ-aminobutyric acid, prolactin-releasing peptide, or nitric oxide synthase. The population of LepRb-expressing NTS neurons was comprised of subpopulations marked by a proopiomelanocortin-enhanced green fluorescent protein (EGFP) transgene and distinct populations that express proglucagon and/or cholecystokinin. The significance of leptin action on these three populations of NTS neurons was assessed in leptin-deficient Ob/Ob mice, revealing increased NTS proglucagon and cholecystokinin, but not proopiomelanocortin, expression. These data provide new insight into the appetitive brainstem circuits engaged by leptin.

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Leptin receptor neurons in the mouse hypothalamus are colocalized with the neuropeptide galanin and mediate anorexigenic leptin action

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00643.2012 ◽

2013 ◽

Vol 304 (9) ◽

pp. E999-E1011 ◽

Cited By ~ 55

Author(s):

Amanda Laque ◽

Yan Zhang ◽

Sarah Gettys ◽

Tu-Anh Nguyen ◽

Kelly Bui ◽

...

Keyword(s):

Energy Homeostasis ◽

Leptin Receptor ◽

Fluorescent Protein ◽

Inhibitory Function ◽

Reporter Mice ◽

Brainstem Nucleus ◽

Melanin Concentrating Hormone ◽

Regulate Food Intake ◽

Receptor Neurons ◽

Green Fluorescent

Leptin acts centrally via leptin receptor (LepRb)-expressing neurons to regulate food intake, energy expenditure, and other physiological functions. LepRb neurons are found throughout the brain, and several distinct populations contribute to energy homeostasis control. However, the function of most LepRb populations remains unknown, and their contribution to regulate energy homeostasis has not been studied. Galanin has been hypothesized to interact with the leptin signaling system, but literature investigating colocalization of LepRb and galanin has been inconsistent, which is likely due to technical difficulties to visualize both. We used reporter mice with green fluorescent protein expression from the galanin locus to recapitulate the colocalization of galanin and leptin-induced p-STAT3 as a marker for LepRb expression. Here, we report the existence of two populations of galanin-expressing LepRb neurons (Gal-LepRb neurons): in the hypothalamus overspanning the perifornical area and adjacent dorsomedial and lateral hypothalamus [collectively named extended perifornical area (exPFA)] and in the brainstem (nucleus of the solitary tract). Surprisingly, despite the known orexigenic galanin action, leptin induces galanin mRNA expression and stimulates LepRb neurons in the exPFA, thus conflicting with the expected anorexigenic leptin action. However, we confirmed that intra-exPFA leptin injections were indeed sufficient to mediate anorexic responses. Interestingly, LepRb and galanin-expressing neurons are distinct from orexin or melanin-concentrating hormone (MCH)-expressing neurons, but exPFA galanin neurons colocalized with the anorexigenic neuropeptides neurotensin and cocaine- and amphetamine-regulated transcript (CART). Based on galanin's known inhibitory function, we speculate that in exPFA Gal-LepRb neurons galanin acts inhibitory rather than orexigenic.

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Leptin Signaling Is Not Required for Anorexigenic Estradiol Effects in Female Mice

Endocrinology ◽

10.1210/en.2015-1594 ◽

2016 ◽

Vol 157 (5) ◽

pp. 1991-2001 ◽

Cited By ~ 9

Author(s):

Joon S. Kim ◽

Mohammed Z. Rizwan ◽

Deborah J. Clegg ◽

Greg M. Anderson

Keyword(s):

Body Weight ◽

Cross Talk ◽

Metabolic Regulation ◽

Leptin Receptor ◽

Fluorescent Protein ◽

Estrogen Receptor Α ◽

Female Mice ◽

Leptin Sensitivity ◽

Stat3 Phosphorylation ◽

Green Fluorescent

Abstract Estradiol and leptin are critical hormones in the regulation of body weight. The aim of this study was to determine whether this cross talk between leptin receptor (LepRb) and estrogen receptor-α (ERα) signaling is critical for estradiol's anorexigenic effects. Leprb-Cre mice were crossed with Cre-dependent Tau-green fluorescent protein (GFP) reporter, Stat3-flox or Erα-flox mice to generate female mice with GFP expression, signal transducer and activator of transcription 3 (STAT3) knockout (KO), or ERα KO, specifically in LepRb-expressing cells. The proportion of Leprb-GFP cells colocalizing ERα was high (∼80%) in the preoptic area but low (∼10%) in the mediobasal hypothalamus, suggesting that intracellular cross talk between these receptors is minimal for metabolic regulation. To test whether estradiol enhanced arcuate leptin sensitivity, ovarectomized mice received varying levels of estradiol replacement. Increasing estrogenic states did not increase the degree of leptin-induced STAT3 phosphorylation. LepRb-specific STAT3 KO mice and controls were ovarectomized and given either chronic estradiol or vehicle treatment to test whether STAT3 is required for estrogen-induced body weight suppression. Both groups of estradiol-treated mice showed an equivalent reduction in body weight and fat content compared with vehicle controls. Finally, mice lacking ERα specifically in LepRb-expressing neurons also showed no increase in body weight or impairments in metabolic function compared with controls, indicating that estradiol acts independently of leptin-responsive cells to regulate body weight. However, fecundity was impaired in in Leprb-ERα KO females. Contrary to the current dogma, we report that estradiol has minimal direct actions on LepRb cells in the mediodasal hypothalamus and that its anorexigenic effects can occur entirely independently of LepRb-STAT3 signaling in female mice.

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Characterization of Leptin-Responsive Neurons in the Caudal Brainstem

Endocrinology ◽

10.1210/en.2005-0877 ◽

2006 ◽

Vol 147 (7) ◽

pp. 3190-3195 ◽

Cited By ~ 86

Author(s):

Kate L. J. Ellacott ◽

Ilia G. Halatchev ◽

Roger D. Cone

Keyword(s):

Green Fluorescent Protein ◽

Energy Homeostasis ◽

Leptin Receptor ◽

Fluorescent Protein ◽

Physiological Role ◽

Cell Group ◽

Melanocortin System ◽

Peptide Precursor ◽

Green Fluorescent

The central melanocortin system plays a key role in the regulation of energy homeostasis. Neurons containing the peptide precursor proopiomelanocortin (POMC) are found at two sites in the brain, the arcuate nucleus of the hypothalamus (ARC) and the caudal region of the nucleus of the solitary tract (NTS). ARC POMC neurons, which also express cocaine- and amphetamine-regulated transcript (CART), are known to mediate part of the response to factors regulating energy homeostasis, such as leptin and ghrelin. In contrast, the physiological role(s) of the POMC neurons in the caudal brainstem are not well characterized. However, development of a transgenic mouse expressing green fluorescent protein under the control of the POMC promoter [POMC-enhanced green fluorescent protein (EGFP) mouse] has aided the study of these neurons. Indeed, recent studies have shown significant activation of NTS POMC-EGFP cells by the gut released satiety factor cholecystokinin (CCK). Here we show that peripheral leptin administration induces the expression of phospho-signal transducer and activator of transcription 3 immunoreactivity (pSTAT3-IR), a marker of leptin receptor signaling, in more than 50% of NTS POMC-EGFP neurons. Furthermore, these POMC-EGFP neurons comprise 30% of all pSTAT3-IR cells in the NTS. Additionally, we also show that in contrast to the ARC population, NTS POMC-EGFP neurons do not coexpress CART immunoreactivity. These data suggest that NTS POMC neurons may participate with ARC POMC cells in mediating some of the effects of leptin and thus comprise a novel cell group regulated by both long-term adipostatic signals and satiety factors such as CCK.

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Abstract 016: Bbs1 Gene Deletion From The Leptin Receptor Neurons Causes Obesity And Hypertension In Mice

Hypertension ◽

10.1161/hyp.64.suppl_1.016 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Deng F Guo ◽

Donald A Morgan ◽

Justin L Grobe ◽

Darryl Nishimura ◽

Charles Searby ◽

...

Keyword(s):

Body Weight ◽

Energy Expenditure ◽

Arterial Pressure ◽

Energy Homeostasis ◽

Leptin Receptor ◽

Autosomal Recessive Disorder ◽

Obesity Phenotype ◽

Receptor Neurons ◽

Depressor Effect ◽

Old Mice

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder associated with several features including obesity and hypertension. Deletion of BBS genes globally or in the nervous system recapitulated many of the BBS phenotypes including obesity and hypertension. Here, we assessed the effect of ablating the Bbs1 gene from the neurons expressing the long signaling form of the leptin receptor (LepRb). Breeding Bbs1 flox with LepRb Cre mice created mice deficient in the Bbs1 gene only in LepRb-positive neurons (visualized by tdTomato expression) as indicated by loss of leptin activation of Stat3. Importantly, Bbs1 flox /LepR Cre mice display an obesity phenotype as indicated by the increased (P<0.05) body weight (39±2 vs. 30±1 g in controls) and fat mass measured by MRI (14±3 vs. 4±1 g in controls) associated with increased (P<0.05) food intake (3.4±0.1 vs. 2.9±0.1 g in controls) in 25 weeks old mice. However, body weight and fat pads of pair-fed LRb Cre /Bbs1 fl/fl mice remained significantly elevated compared to controls suggesting that LRb Cre /Bbs1 fl/fl mice have reduced energy expenditure. Consistent with this possibility, LRb Cre /Bbs1 fl/fl mice displayed decreased (P<0.05) O 2 consumption (2.6±0.1 vs. 3.1±0.1 mL/100g/min in controls) and heat production (8.1±0.3 vs. 9.6±0.3 kcal/kg/h in controls). These results indicate that hyperphagia and decreased energy expenditure contribute to the development of obesity in Bbs1 flox /LepR Cre mice. Next, we assessed the effect on arterial pressure (AP) and sympathetic nerve activity (SNA) of ablating the Bbs1 gene from the LepR-containing neurons. Interestingly, deletion of the Bbs1 gene in LepR neurons recapitulates the hypertension phenotype of BBS as indicated by elevated mean AP (125±4 vs 109±3 mmHg in controls, P=0.03). Conscious renal SNA was also elevated in LRb Cre /Bbs1 fl/fl mice relative to controls (97±8 vs 62±10 spikes/sec, P<0.05). Finally, the depressor effect of ganglionic blockade (hexamethonium) was exaggerated in Bbs1 flox /LepR Cre mice (-57±5 vs -38±5 mmHg in control, P=0.01). These findings demonstrate that the Bbs1 gene in LepR neurons is critical for energy homeostasis and arterial pressure regulation.

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Targeted leptin receptor blockade: role of ventral tegmental area and nucleus of the solitary tract leptin receptors in body weight homeostasis

Journal of Endocrinology ◽

10.1530/joe-13-0455 ◽

2014 ◽

Vol 222 (1) ◽

pp. 27-41 ◽

Cited By ~ 3

Author(s):

M Matheny ◽

K Y E Strehler ◽

M King ◽

N Tümer ◽

P J Scarpace

Keyword(s):

Body Weight ◽

Weight Gain ◽

Food Intake ◽

Ventral Tegmental Area ◽

Food Consumption ◽

Leptin Receptor ◽

Body Weight Gain ◽

Solitary Tract ◽

Leptin Receptors ◽

Ventral Tegmental

The present investigation examined whether leptin stimulation of ventral tegmental area (VTA) or nucleus of the solitary tract (NTS) has a role in body weight homeostasis independent of the medial basal hypothalamus (MBH). To this end, recombinant adeno-associated viral techniques were employed to target leptin overexpression or overexpression of a dominant negative leptin mutant (leptin antagonist). Leptin antagonist overexpression in MBH or VTA increased food intake and body weight to similar extents over 14 days in rats. Simultaneous overexpression of leptin in VTA with antagonist in MBH resulted in food intake and body weight gain that were less than with control treatment but greater than with leptin alone in VTA. Notably, leptin overexpression in VTA increased P-STAT3 in MBH along with VTA, and leptin antagonist overexpression in the VTA partially attenuated P-STAT3 levels in MBH. Interestingly, leptin antagonist overexpression elevated body weight gain, but leptin overexpression in the NTS failed to modulate either food intake or body weight despite increased P-STAT3. These data suggest that leptin function in the VTA participates in the chronic regulation of food consumption and body weight in response to stimulation or blockade of VTA leptin receptors. Moreover, one component of VTA-leptin action appears to be independent of the MBH, and another component appears to be related to leptin receptor-mediated P-STAT3 activation in the MBH. Finally, leptin receptors in the NTS are necessary for normal energy homeostasis, but mostly they appear to have a permissive role. Direct leptin activation of NTS slightly increases UCP1 levels, but has little effect on food consumption or body weight.

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Leptin resistance: unsolved diagnostic issues

Problems of Endocrinology ◽

10.14341/probl201864162-66 ◽

2018 ◽

Vol 64 (1) ◽

pp. 62-66

Author(s):

Daria A. Borodkina ◽

Olga V. Gruzdeva ◽

Olga E. Akbasheva ◽

Ekaterina V. Belik ◽

Elena I. Palicheva ◽

...

Keyword(s):

Insulin Resistance ◽

Body Weight ◽

Skeletal Muscles ◽

Energy Homeostasis ◽

Leptin Receptor ◽

Leptin Resistance ◽

Fatty Tissue ◽

Leptin Receptors ◽

Secretion Regulation ◽

Tissue Sensitivity

Leptin and its receptors are key regulators of body weight and energy homeostasis. A decrease in tissue sensitivity to leptin leads to the development of obesity, insulin resistance, dyslipidemia, etc. Currently, the phenomenon of leptin resistance is explained by a number of mechanisms, including impairment of gene structure, leptin transport through the blood-brain barrier, and leptin receptor signaling. However, it is not known, a decrease in the number of receptors of which area leads to the development of leptin resistance. No relationship has been found between the basal leptin level in obesity and expression of leptin receptors in the skeletal muscles. It is also important to investigate the contribution of fatty tissue of different localization to leptin secretion regulation and activity of its receptors. The term «leptin resistence» reflects a complex pathophysiological phenomenon with broad perspectives for study. In this review, we analyze methods of diagnosing leptin resistance.

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Leptin resistance: unsolved diagnostic issues

Problems of Endocrinology ◽

10.14341/probl8740 ◽

2018 ◽

Vol 64 (1) ◽

pp. 62-66

Author(s):

Daria A. Borodkina ◽

Olga V. Gruzdeva ◽

Olga E. Akbasheva ◽

Ekaterina V. Belik ◽

Elena I. Palicheva ◽

...

Keyword(s):

Insulin Resistance ◽

Body Weight ◽

Skeletal Muscles ◽

Energy Homeostasis ◽

Leptin Receptor ◽

Leptin Resistance ◽

Fatty Tissue ◽

Leptin Receptors ◽

Secretion Regulation ◽

Tissue Sensitivity

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Adiponectin receptors are expressed in hypothalamus and colocalized with proopiomelanocortin and neuropeptide Y in rodent arcuate neurons

Journal of Endocrinology ◽

10.1677/joe-08-0348 ◽

2008 ◽

Vol 200 (1) ◽

pp. 93-105 ◽

Cited By ~ 95

Author(s):

E Guillod-Maximin ◽

A F Roy ◽

C M Vacher ◽

A Aubourg ◽

V Bailleux ◽

...

Keyword(s):

Neuropeptide Y ◽

Energy Homeostasis ◽

Fluorescent Protein ◽

Peripheral Tissues ◽

Rat Hypothalamus ◽

Ampk Phosphorylation ◽

Hypothalamic Nuclei ◽

Green Fluorescent ◽

Amp Activated Protein Kinase

Adiponectin is involved in the control of energy homeostasis in peripheral tissues through Adipor1 and Adipor2 receptors. An increasing amount of evidence suggests that this adipocyte-secreted hormone may also act at the hypothalamic level to control energy homeostasis. In the present study, we observed the gene and protein expressions of Adipor1 and Adipor2 in rat hypothalamus using different approaches. By immunohistochemistry, Adipor1 expression was ubiquitous in the rat brain. By contrast, Adipor2 expression was more limited to specific brain areas such as hypothalamus, cortex, and hippocampus. In arcuate and paraventricular hypothalamic nuclei, Adipor1, and Adipor2 were expressed by neurons and astrocytes. Furthermore, using transgenic green fluorescent protein mice, we showed that Adipor1 and Adipor2 were present in pro–opiomelanocortin (POMC) and neuropeptide Y (NPY) neurons in the arcuate nucleus. Finally, adiponectin treatment by intracerebroventricular injection induced AMP-activated protein kinase (AMPK) phosphorylation in the rat hypothalamus. This was confirmed byin vitrostudies using hypothalamic membrane fractions. In conclusion, Adipor1 and Adipor2 are both expressed by neurons (including POMC and NPY neurons) and astrocytes in the rat hypothalamic nuclei. Adiponectin is able to increase AMPK phosphorylation in the rat hypothalamus. These data reinforced a potential role of adiponectin and its hypothalamic receptors in the control of energy homeostasis.

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Isolation and Immortalization of MIP-GFP Neurons From the Hypothalamus

Endocrinology ◽

10.1210/en.2013-2128 ◽

2014 ◽

Vol 155 (6) ◽

pp. 2314-2319 ◽

Cited By ~ 5

Author(s):

Zi Chen Wang ◽

Michael B. Wheeler ◽

Denise D. Belsham

Keyword(s):

Green Fluorescent Protein ◽

Energy Homeostasis ◽

Fluorescent Protein ◽

Transgenic Animals ◽

Unintended Effects ◽

Hypothalamic Neurons ◽

Related Peptide ◽

Promoter Construct ◽

Insulin Responsiveness ◽

Green Fluorescent

The mouse insulin I promoter (MIP) construct was developed to eliminate the promoter activity detected with the rat insulin II promoter in specific hypothalamic neurons that may have unintended effects on glucose and energy homeostasis in transgenic models. Thus, the specificity of this novel construct must be validated prior to the widespread availability of derived Cre models. Although limited validation efforts have indicated a lack of MIP activity within neuronal tissue, the global immunohistochemical methodology used may not be specific enough to rule out the possibility of specific populations of neurons with MIP activity. To investigate possible MIP activity within the hypothalamus, primary hypothalamic isolates from MIP-green fluorescent protein reporter mice were analyzed after fluorescent-activated cell sorting. Primary hypothalamic neurons isolated from the MIP-green fluorescent protein mice were immortalized. Characterization detected the presence of hypothalamic neuropeptide Y (NPY) and agouti-related peptide, involved in the control of energy homeostasis, as well as confirmed insulin responsiveness in the cell lines. Moreover, because insulin was demonstrated to differentially regulate NPY expression within these MIP neurons, the promoter construct may be active in multiple hypothalamic NPY/agouti-related peptide subpopulations with unique physiological functions. MIP transgenic animals may therefore face similar limitations seen previously with rat insulin II promoter-based models.

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Increased Hypothalamic Signal Transducer and Activator of Transcription 3 Phosphorylation after Hindbrain Leptin Injection

Endocrinology ◽

10.1210/en.2009-0854 ◽

2010 ◽

Vol 151 (4) ◽

pp. 1509-1519 ◽

Cited By ~ 20

Author(s):

Marieke Ruiter ◽

Patricia Duffy ◽

Steven Simasko ◽

Robert C. Ritter

Keyword(s):

Body Weight ◽

Food Intake ◽

Fourth Ventricle ◽

Leptin Receptor ◽

Janus Kinase ◽

Signal Transducer ◽

Solitary Tract ◽

Stat3 Signaling ◽

Stat3 Phosphorylation ◽

Transcription 3

Reduction of food intake and body weight by leptin is attributed largely to its action in the hypothalamus. However, the signaling splice variant of the leptin receptor, LRb, also is expressed in the hindbrain, and leptin injections into the fourth cerebral ventricle or dorsal vagal complex are associated with reductions of feeding and body weight comparable to those induced by forebrain leptin administration. Although these observations suggest direct hindbrain action of leptin on feeding and body weight, the possibility that hindbrain leptin administration also activates the Janus kinase/signal transducer and activator of transcription 3 (STAT3) signaling in the hypothalamus has not been investigated. Confirming earlier work, we found that leptin produced comparable reductions of feeding and body weight when injected into the lateral ventricle or the fourth ventricle. We also found that lateral and fourth ventricle leptin injections produced comparable increases of STAT3 phosphorylation in both the hindbrain and the hypothalamus. Moreover, injection of 50 ng of leptin directly into the nucleus of the solitary tract also increased STAT3 phosphorylation in the hypothalamic arcuate and ventromedial nuclei. Increased hypothalamic STAT3 phosphorylation was not due to elevation of blood leptin concentrations and the pattern of STAT3 phosphorylation did not overlap distribution of the retrograde tracer, fluorogold, injected via the same cannula. Our observations indicate that even small leptin doses administered to the hindbrain can trigger leptin-related signaling in the forebrain, and raise the possibility that STAT3 phosphorylation in the hypothalamus may contribute to behavioral and metabolic changes observed after hindbrain leptin injections.

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