Toll-like receptors (TLRs) are traditionally associated with immune-mediated host defense. Here, ... more Toll-like receptors (TLRs) are traditionally associated with immune-mediated host defense. Here, we ascribe a novel extra-immune, hypothalamic-associated function to TLR2, a TLR-family member known to recognize lipid components, in the protection against obesity. We found that TLR2-deficient mice exhibited mature-onset obesity and susceptibility to high-fat diet (HFD)-induced weight gain, via modulation of food intake. Age-related obesity was still evident in chimeric mice, carrying comparable TLR2(+) immune cells, suggesting a non-hematopoietic-related involvement of this receptor. TLR2 was up-regulated with age or HFD in pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus, a brain area participating in central-metabolic regulation, possibly modulating the hypothalamic-anorexigenic peptide, α-melanocyte-stimulating hormone (α-MSH). Direct activation of TLR2 in a hypothalamic-neuronal cell-line via its known ligands, further supports its capacity to mediat...
Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, ... more Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.
All domains of life feature diverse molecular clock machineries that synchronize physiological pr... more All domains of life feature diverse molecular clock machineries that synchronize physiological processes to diurnal environmental fluctuations. However, no mechanisms are known to cross-regulate prokaryotic and eukaryotic circadian rhythms in multikingdom ecosystems. Here, we show that the intestinal microbiota, in both mice and humans, exhibits diurnal oscillations that are influenced by feeding rhythms, leading to time-specific compositional and functional profiles over the course of a day. Ablation of host molecular clock components or induction of jet lag leads to aberrant microbiota diurnal fluctuations and dysbiosis, driven by impaired feeding rhythmicity. Consequently, jet-lag-induced dysbiosis in both mice and humans promotes glucose intolerance and obesity that are transferrable to germ-free mice upon fecal transplantation. Together, these findings provide evidence of coordinated metaorganism diurnal rhythmicity and offer a microbiome-dependent mechanism for common metabolic disturbances in humans with aberrant circadian rhythms, such as those documented in shift workers and frequent flyers.
Corticotropin-releasing factor type 2 receptors (CRFR2) are suggested to facilitate successful re... more Corticotropin-releasing factor type 2 receptors (CRFR2) are suggested to facilitate successful recovery from stress to maintain mental health. They are abundant in the midbrain raphe nuclei, where they regulate serotonergic neuronal activity and have been demonstrated to mediate behavioural consequences of stress. Here, we describe behavioural and serotonergic responses consistent with maladaptive recovery from stressful challenge in CRFR2-null mice. CRFR2-null mice showed similar anxiety levels to control mice before and immediately after acute restraint stress, and also after cessation of chronic stress. However, they showed increased anxiety by 24 hours after restraint, whether or not they had been chronically stressed.Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents were quantified and the level of 5-HIAA in the caudal dorsal raphe nucleus (DRN) was increased under basal conditions in CRFR2-null mice, indicating increased 5-HT turnover. Twenty-four hours followi...
The effects of stress on energy balance and the involvement of the neuropeptide corticotropin rel... more The effects of stress on energy balance and the involvement of the neuropeptide corticotropin releasing factor in modulating the anorexia of stress and sympathetic nervous system tone are well recognized. Currently, studies centered on the roles of the more recently described members of this family of ligands, the urocortins, and their preferred receptor, the corticotropin releasing factor type 2 receptor, suggest that they are important modulators of centrally controlled metabolic functions. In addition, urocortins also regulate fuel utilization in the periphery by acting locally within key metabolic tissues through autocrine and/or paracrine mechanisms. Recent findings have demonstrated that urocortin 2 and urocortin 3, by acting through their specific receptor in peripheral tissues, are novel modulators of glucose homeostasis and metabolic functions.
Corticotropin-releasing factor receptor type 1 (CRFR1) plays a major role in the regulation of ne... more Corticotropin-releasing factor receptor type 1 (CRFR1) plays a major role in the regulation of neuroendocrine and behavioral responses to stress and is considered a key mediator of anxiety behavior. The globus pallidus external (GPe), a main relay center within the basal ganglia that is primarily associated with motor and associative functions, is one of the brain nuclei with the highest levels of CRFR1 expression in the rodent brain. However, the role of CRFR1 in the GPe is yet unknown. In the present study, we used a lentiviral-based system of RNA interference to show that knockdown of CRFR1 mRNA expression in the GPe of adult mice induces a significant increase in anxiety-like behavior, as revealed by the light-dark transfer, open-field, and elevated plus-maze tests. This effect was further confirmed by pharmacological administration of the selective CRFR1 antagonist NBI 30775 (1.75 g/side) directly into the GPe. In the marble-burying test, blockade of CRFR1 in the GPe increased the percentage of marbles buried and the duration of burying behavior. Additionally, we present evidence suggesting that the enkephalin system is involved in the effect of GPe-CRFR1 on anxiety-like behavior. In contrast to the well established anxiogenic role of CRFR1 in the extended amygdala, our data reveal a novel anxiolytic role for CRFR1 in the GPe.
Posttraumatic stress disorder (PTSD) is a debilitating disease, which affects 8 -10% of the popul... more Posttraumatic stress disorder (PTSD) is a debilitating disease, which affects 8 -10% of the population exposed to traumatic events. The factors that make certain individuals susceptible to PTSD and others resilient are currently unknown. Corticotropin-releasing factor receptor type 2 (CRFR2) has been implicated in mediating stress coping mechanisms. Here, we use a physiological PTSD-like animal model and an in-depth battery of tests that reflect the symptomology of PTSD to separate mice into subpopulations of "PTSD-like" and "Resilient" phenotypes. PTSD-like mice are hypervigilant, hyperalert, insomniac, have impaired attention and risk assessment, as well as accompanying attenuated corticosterone levels. Intriguingly, PTSD-like mice show long-term robust upregulation of BNST-CRFR2 mRNA levels, and BNST-CRFR2-specific lentiviral knockdown reduces susceptibility to PTSD-like behavior. Additionally, using a BNST mRNA expression array, PTSD-like mice exhibit a general transcriptional attenuation profile, which was associated with upregulation of the BNST-deacetylation enzyme, HDAC5. We suggest PTSD to be a disease of maladaptive coping.
A growing body of experimental and clinical studies supports a strong association between psychol... more A growing body of experimental and clinical studies supports a strong association between psychological stress and cardiovascular disease. An important endogenous cardioprotective role in heart physiology has been attributed to corticotropin-releasing factor receptor type 2beta (CRFR2beta). Here, we report the isolation of cDNA from mouse (m) heart encoding a novel CRFR2beta splice variant. Translation of this insertion variant (iv)-mCRFR2beta isoform produces a 421-aa protein that includes a unique C-terminal cytoplasmic tail. Our functional analysis and cellular localization studies demonstrated that when coexpressed with wild-type mCRFR2beta, iv-mCRFR2beta significantly inhibited the wild-type mCRFR2beta membrane expression and its functional signaling by ER-Golgi complex retention, suggesting a dose-dependent dominant negative effect. Interestingly, mice exposed to a 4-wk paradigm of chronic variable stress, a model of chronic psychological stress in humans, presented significantly lower levels of mCRFR2beta and higher levels of iv-mCRFR2beta mRNA expression in their hearts, compared to nonstressed control mice. The dominant-negative effect of iv-mCRFR2beta and its up-regulation by psychological stress suggest a new form of regulation of the mCRFR2beta cardioprotective effect and a potential role for this novel isoform in stress-induced heart disease.
Proceedings of the National Academy of Sciences, 2006
Skeletal muscle is the principal tissue responsible for insulinstimulated glucose disposal and is... more Skeletal muscle is the principal tissue responsible for insulinstimulated glucose disposal and is a major site of peripheral insulin resistance. Urocortin 2 (Ucn 2), a member of the corticotropinreleasing factor (CRF) family, and its cognate type 2 CRF receptor (CRFR2) are highly expressed in skeletal muscle. To determine the physiological role of Ucn 2, we generated mice that are deficient in this peptide. Using glucose-tolerance tests (GTTs), insulin-tolerance tests (ITTs), and hyperinsulinemic euglycemic glucose clamp studies, we demonstrated that mice lacking Ucn 2 exhibited increased insulin sensitivity and were protected against fat-induced insulin resistance. Administration of synthetic Ucn 2 to mutant mice before the GTTs and ITTs restored blood glucose to WT levels.
Proceedings of the National Academy of Sciences, 2010
In response to physiological or psychological challenges, the brain activates behavioral and neur... more In response to physiological or psychological challenges, the brain activates behavioral and neuroendocrine systems linked to both metabolic and emotional outputs designed to adapt to the demand. However, dysregulation of integration of these physiological responses to challenge can have severe psychological and physiological consequences, and inappropriate regulation, disproportional intensity, or chronic or irreversible activation of the stress response is linked to the etiology and pathophysiology of mood and metabolic disorders. Using a transgenic mouse model and lentiviral approach, we demonstrate the involvement of the hypothalamic neuropeptide Urocortin-3, a specific ligand for the type-2 corticotropin-releasing factor receptor, in modulating septal and hypothalamic nuclei responsible for anxiety-like behaviors and metabolic functions, respectively. These results position Urocortin-3 as a neuromodulator linking stress-induced anxiety and energy homeostasis and pave the way toward better understanding of the mechanisms that mediate the reciprocal relationships between stress, mood and metabolic disorders. metabolic disorders | mood disorders | corticotropin-releasing factor (CRF) | CRF receptor type 2 | stress response I n modern Western societies, the high stress load correlates with an increasing incidence of mood disorders and metabolic syndrome, both of which have reached epidemic proportions over the past decades (1, 2). Exposure to acute or chronic stress is associated with derangement of metabolic and behavioral homeostasis that contributes to the clinical presentation of visceral obesity, type 2 diabetes, atherosclerosis, and metabolic syndrome (2-4). The corticotropin-releasing factor (CRF) neuropeptide system is the primary central mediator of the stress response and contributes to the etiology of stress-related psychiatric illness (5-8). Studies conducted using CRF receptor type 2 (CRFR2)-null mice or Urocortin-2 (Ucn2)-null mice provided evidence that, in addition to its role in mediating stress-related behavior, central CRFR2 is important in modulating metabolic rate, appetite, and feeding behaviors (9-12).
Environmental enrichment (EE) is known to have an anxiolytic effect in several animal models; how... more Environmental enrichment (EE) is known to have an anxiolytic effect in several animal models; however, the molecular mechanisms underlying these behavioral changes are not understood. In this study, we have shown that the anxiolytic effect of EE is associated with alterations in the corticotropin-releasing factor receptor type 1 (CRFR1) expression levels in the limbic system. We found that the decrease in anxiety-like behavior after housing in enriched conditions was associated with very low levels of CRFR1 mRNA expression in the basolateral amygdala of C57BL/6 mice. We further showed using a lentiviral-based system of RNA interference, that knockdown of CRFR1 mRNA expression in the basolateral amygdala induces a significant decrease in anxiety levels, similar to those achieved by EE nurture. Our data strongly suggest that reduced expression of CRFR1 mRNA levels in the basolateral amygdala mediates the effect of EE on anxiety-like behavior.
Corticotropin-releasing factor (CRF) has a key role in the central stress response, and altered l... more Corticotropin-releasing factor (CRF) has a key role in the central stress response, and altered levels of this neuropeptide are linked to stress-related psychopathologies such as anxiety and depression. These disorders are associated with the inability to properly regulate stress response, specifically following exposure to prolonged stressful stimuli. Therefore, the current study assessed the effects of prolonged and site-specific over-expression of CRF, which mimics the state of chronic production, in extended amygdala nuclei that are known to be involved in mediating anxiety-like states. We first constructed and generated lentiviruses that overexpress (OE) CRF in a robust and stable manner, and then generated two male mouse models continuously over-expressing CRF, either at the central nucleus of the amygdala (CeA), or at the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNSTdl). After 4 months, behavioral assessments were conducted for anxiety and depressive indices on these mice. Surprisingly, prolonged CRF OE at the CeA attenuated stress-induced anxiety-like behaviors, whereas prolonged CRF OE in the BNSTdl increased depressive-like behaviors, without affecting anxiety levels. These results show possible differential roles for CRF expressed by distinct loci of the extended amygdala, in mediating stress-induced emotional behaviors. Prolonged and site-specific over-expression of CRF L Regev et al Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp) Prolonged and site-specific over-expression of CRF L Regev et al
Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus are central compone... more Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus are central components of systems regulating appetite and energy homeostasis. Here we report on the establishment of a mouse model in which the ribonuclease III ribonuclease Dicer-1 has been specifically deleted from POMC-expressing neurons (POMC(ΔDCR)), leading to postnatal cell death. Mice are born phenotypically normal, at the expected genetic ratio and with normal hypothalamic POMC-mRNA levels. At 6 weeks of age, no POMC neurons/cells could be detected either in the arcuate nucleus or in the pituitary of POMC(ΔDCR) mice. POMC(ΔDCR) develop progressive obesity secondary to decreased energy expenditure but unrelated to food intake, which was surprisingly lower than in control mice. Reduced expression of AgRP and ghrelin receptor in the hypothalamus and reduced uncoupling protein 1 expression in brown adipose tissue can potentially explain the decreased food intake and decreased heat production, respectively, in these mice. Fasting glucose levels were dramatically elevated in POMC(ΔDCR) mice and the glucose tolerance test revealed marked glucose intolerance in these mice. Secondary to corticotrope ablation, basal and stress-induced corticosterone levels were undetectable in POMC(ΔDCR) mice. Despite this lack of activation of the neuroendocrine stress response, POMC(ΔDCR) mice exhibited an anxiogenic phenotype, which was accompanied with elevated levels of hypothalamic corticotropin-releasing factor and arginine-vasopressin transcripts. In conclusion, postnatal ablation of POMC neurons leads to enhanced anxiety and the development of obesity despite decreased food intake and glucocorticoid deficiency.
Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM... more Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM) tissue where it is suggested to inhibit interactions between insulin signaling pathway components affecting whole-body glucose homeostasis. However, little is known about factors regulating SM CRFR2 expression. Here, we demonstrate the exclusive expression of CRFR2, and not CRFR1, in mature SM tissue using RT-PCR and ribonuclease protection assays and report a differential expression of CRF receptors during C2C12 myogenic differentiation. Whereas C2C12 myoblasts exclusively express CRFR1, the C2C12 myotubes solely express CRFR2. Using cAMP luciferase assays and calcium mobilization measurements, we further demonstrate the functionality of these differentially expressed receptors. Using luciferase reporter assays we show a differential activation of CRFR promoters during myogenic differentiation. Transfections with different fragments of the 5Ј-flanking region of the mCRFR2 gene fused to a luciferase reporter gene show a promoterdependent expression of the reporter gene and reveal the importance of the myocyte enhancer factor 2 consensus sequence located at the 3Ј-proximal region of CRFR2 promoter. Furthermore, we demonstrate that CRFR2 gene transcription in the mature mouse is stimulated by both high-fat diet and chronic variable stress conditions. Performing a whole-genome expression microarray analysis of SM tissues obtained from CRFR2-null mice or wild-type littermates revealed a robust reduction in retinol-binding protein 4 expression levels, an adipokine whose serum levels are elevated in insulin-resistant states. In correlation with the SM CRFR2 levels, the SM retinolbinding protein 4 levels were also elevated in mice subjected to high-fat diet and chronic variable stress conditions. The current findings further position the SM CRFR2 pathways as a relevant physiological system that may affect the known reciprocal relationship between psychological and physiological challenges and the metabolic syndrome. (Molecular Endocrinology 25: 157-169, 2011)
Aims/hypothesis Urocortins are the endogenous ligands for the corticotropin-releasing factor rece... more Aims/hypothesis Urocortins are the endogenous ligands for the corticotropin-releasing factor receptor type 2 (CRFR2), which is implicated in regulating energy balance and/or glucose metabolism. We determined the effects of chronic CRFR2 activation on metabolism in vivo, by generating and phenotyping transgenic mice overproducing the specific CRFR2 ligand urocortin 3. Methods Body composition, glucose metabolism, insulin sensitivity, energy efficiency and expression of key metabolic genes were assessed in adult male urocortin 3 transgenic mice (Ucn3 + ) under control conditions and following an obesogenic high-fat diet (HFD) challenge.
Molecular-level understanding of body weight control is essential for combating obesity. We show ... more Molecular-level understanding of body weight control is essential for combating obesity. We show that female mice lacking tyrosine phosphatase epsilon (RPTPe) are protected from weight gain induced by high-fat food, ovariectomy, or old age and exhibit increased whole-body energy expenditure and decreased adiposity. RPTPe-deficient mice, in particular males, exhibit improved glucose homeostasis. Female nonobese RPTPe-deficient mice are leptin hypersensitive and exhibit reduced circulating leptin concentrations, suggesting that RPTPe inhibits hypothalamic leptin signaling in vivo. Leptin hypersensitivity persists in aged, ovariectomized, and high-fat-fed RPTPe-deficient mice, indicating that RPTPe helps establish obesity-associated leptin resistance. RPTPe associates with and dephosphorylates JAK2, thereby downregulating leptin receptor signaling. Leptin stimulation induces phosphorylation of hypothalamic RPTPe at its C-terminal Y695, which drives RPTPe to downregulate JAK2. RPTPe is therefore an inhibitor of hypothalamic leptin signaling in vivo, and provides controlled negative-feedback regulation of this pathway following its activation.
The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role... more The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role in aiding recovery from acute stress, but any chronic effects of CRFR2 activation are unknown. CRFR2 in the midbrain raphé nuclei modulate serotonergic activity of this key source of serotonin (5-HT) forebrain innervation.
The Journal of Clinical Endocrinology & Metabolism, 2013
Context: Adipose tissue macrophages (ATMs) are thought to engulf the remains of dead adipocytes i... more Context: Adipose tissue macrophages (ATMs) are thought to engulf the remains of dead adipocytes in obesity, potentially resulting in increased intracellular neutral lipid content. Lipid-laden macrophages (foam cells [FCs]) have been described in atherosclerotic lesions and have been proposed to contribute to vascular pathophysiology, which is enhanced in obesity.
Toll-like receptors (TLRs) are traditionally associated with immune-mediated host defense. Here, ... more Toll-like receptors (TLRs) are traditionally associated with immune-mediated host defense. Here, we ascribe a novel extra-immune, hypothalamic-associated function to TLR2, a TLR-family member known to recognize lipid components, in the protection against obesity. We found that TLR2-deficient mice exhibited mature-onset obesity and susceptibility to high-fat diet (HFD)-induced weight gain, via modulation of food intake. Age-related obesity was still evident in chimeric mice, carrying comparable TLR2(+) immune cells, suggesting a non-hematopoietic-related involvement of this receptor. TLR2 was up-regulated with age or HFD in pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus, a brain area participating in central-metabolic regulation, possibly modulating the hypothalamic-anorexigenic peptide, α-melanocyte-stimulating hormone (α-MSH). Direct activation of TLR2 in a hypothalamic-neuronal cell-line via its known ligands, further supports its capacity to mediat...
Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, ... more Non-caloric artificial sweeteners (NAS) are among the most widely used food additives worldwide, regularly consumed by lean and obese individuals alike. NAS consumption is considered safe and beneficial owing to their low caloric content, yet supporting scientific data remain sparse and controversial. Here we demonstrate that consumption of commonly used NAS formulations drives the development of glucose intolerance through induction of compositional and functional alterations to the intestinal microbiota. These NAS-mediated deleterious metabolic effects are abrogated by antibiotic treatment, and are fully transferrable to germ-free mice upon faecal transplantation of microbiota configurations from NAS-consuming mice, or of microbiota anaerobically incubated in the presence of NAS. We identify NAS-altered microbial metabolic pathways that are linked to host susceptibility to metabolic disease, and demonstrate similar NAS-induced dysbiosis and glucose intolerance in healthy human subjects. Collectively, our results link NAS consumption, dysbiosis and metabolic abnormalities, thereby calling for a reassessment of massive NAS usage.
All domains of life feature diverse molecular clock machineries that synchronize physiological pr... more All domains of life feature diverse molecular clock machineries that synchronize physiological processes to diurnal environmental fluctuations. However, no mechanisms are known to cross-regulate prokaryotic and eukaryotic circadian rhythms in multikingdom ecosystems. Here, we show that the intestinal microbiota, in both mice and humans, exhibits diurnal oscillations that are influenced by feeding rhythms, leading to time-specific compositional and functional profiles over the course of a day. Ablation of host molecular clock components or induction of jet lag leads to aberrant microbiota diurnal fluctuations and dysbiosis, driven by impaired feeding rhythmicity. Consequently, jet-lag-induced dysbiosis in both mice and humans promotes glucose intolerance and obesity that are transferrable to germ-free mice upon fecal transplantation. Together, these findings provide evidence of coordinated metaorganism diurnal rhythmicity and offer a microbiome-dependent mechanism for common metabolic disturbances in humans with aberrant circadian rhythms, such as those documented in shift workers and frequent flyers.
Corticotropin-releasing factor type 2 receptors (CRFR2) are suggested to facilitate successful re... more Corticotropin-releasing factor type 2 receptors (CRFR2) are suggested to facilitate successful recovery from stress to maintain mental health. They are abundant in the midbrain raphe nuclei, where they regulate serotonergic neuronal activity and have been demonstrated to mediate behavioural consequences of stress. Here, we describe behavioural and serotonergic responses consistent with maladaptive recovery from stressful challenge in CRFR2-null mice. CRFR2-null mice showed similar anxiety levels to control mice before and immediately after acute restraint stress, and also after cessation of chronic stress. However, they showed increased anxiety by 24 hours after restraint, whether or not they had been chronically stressed.Serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents were quantified and the level of 5-HIAA in the caudal dorsal raphe nucleus (DRN) was increased under basal conditions in CRFR2-null mice, indicating increased 5-HT turnover. Twenty-four hours followi...
The effects of stress on energy balance and the involvement of the neuropeptide corticotropin rel... more The effects of stress on energy balance and the involvement of the neuropeptide corticotropin releasing factor in modulating the anorexia of stress and sympathetic nervous system tone are well recognized. Currently, studies centered on the roles of the more recently described members of this family of ligands, the urocortins, and their preferred receptor, the corticotropin releasing factor type 2 receptor, suggest that they are important modulators of centrally controlled metabolic functions. In addition, urocortins also regulate fuel utilization in the periphery by acting locally within key metabolic tissues through autocrine and/or paracrine mechanisms. Recent findings have demonstrated that urocortin 2 and urocortin 3, by acting through their specific receptor in peripheral tissues, are novel modulators of glucose homeostasis and metabolic functions.
Corticotropin-releasing factor receptor type 1 (CRFR1) plays a major role in the regulation of ne... more Corticotropin-releasing factor receptor type 1 (CRFR1) plays a major role in the regulation of neuroendocrine and behavioral responses to stress and is considered a key mediator of anxiety behavior. The globus pallidus external (GPe), a main relay center within the basal ganglia that is primarily associated with motor and associative functions, is one of the brain nuclei with the highest levels of CRFR1 expression in the rodent brain. However, the role of CRFR1 in the GPe is yet unknown. In the present study, we used a lentiviral-based system of RNA interference to show that knockdown of CRFR1 mRNA expression in the GPe of adult mice induces a significant increase in anxiety-like behavior, as revealed by the light-dark transfer, open-field, and elevated plus-maze tests. This effect was further confirmed by pharmacological administration of the selective CRFR1 antagonist NBI 30775 (1.75 g/side) directly into the GPe. In the marble-burying test, blockade of CRFR1 in the GPe increased the percentage of marbles buried and the duration of burying behavior. Additionally, we present evidence suggesting that the enkephalin system is involved in the effect of GPe-CRFR1 on anxiety-like behavior. In contrast to the well established anxiogenic role of CRFR1 in the extended amygdala, our data reveal a novel anxiolytic role for CRFR1 in the GPe.
Posttraumatic stress disorder (PTSD) is a debilitating disease, which affects 8 -10% of the popul... more Posttraumatic stress disorder (PTSD) is a debilitating disease, which affects 8 -10% of the population exposed to traumatic events. The factors that make certain individuals susceptible to PTSD and others resilient are currently unknown. Corticotropin-releasing factor receptor type 2 (CRFR2) has been implicated in mediating stress coping mechanisms. Here, we use a physiological PTSD-like animal model and an in-depth battery of tests that reflect the symptomology of PTSD to separate mice into subpopulations of "PTSD-like" and "Resilient" phenotypes. PTSD-like mice are hypervigilant, hyperalert, insomniac, have impaired attention and risk assessment, as well as accompanying attenuated corticosterone levels. Intriguingly, PTSD-like mice show long-term robust upregulation of BNST-CRFR2 mRNA levels, and BNST-CRFR2-specific lentiviral knockdown reduces susceptibility to PTSD-like behavior. Additionally, using a BNST mRNA expression array, PTSD-like mice exhibit a general transcriptional attenuation profile, which was associated with upregulation of the BNST-deacetylation enzyme, HDAC5. We suggest PTSD to be a disease of maladaptive coping.
A growing body of experimental and clinical studies supports a strong association between psychol... more A growing body of experimental and clinical studies supports a strong association between psychological stress and cardiovascular disease. An important endogenous cardioprotective role in heart physiology has been attributed to corticotropin-releasing factor receptor type 2beta (CRFR2beta). Here, we report the isolation of cDNA from mouse (m) heart encoding a novel CRFR2beta splice variant. Translation of this insertion variant (iv)-mCRFR2beta isoform produces a 421-aa protein that includes a unique C-terminal cytoplasmic tail. Our functional analysis and cellular localization studies demonstrated that when coexpressed with wild-type mCRFR2beta, iv-mCRFR2beta significantly inhibited the wild-type mCRFR2beta membrane expression and its functional signaling by ER-Golgi complex retention, suggesting a dose-dependent dominant negative effect. Interestingly, mice exposed to a 4-wk paradigm of chronic variable stress, a model of chronic psychological stress in humans, presented significantly lower levels of mCRFR2beta and higher levels of iv-mCRFR2beta mRNA expression in their hearts, compared to nonstressed control mice. The dominant-negative effect of iv-mCRFR2beta and its up-regulation by psychological stress suggest a new form of regulation of the mCRFR2beta cardioprotective effect and a potential role for this novel isoform in stress-induced heart disease.
Proceedings of the National Academy of Sciences, 2006
Skeletal muscle is the principal tissue responsible for insulinstimulated glucose disposal and is... more Skeletal muscle is the principal tissue responsible for insulinstimulated glucose disposal and is a major site of peripheral insulin resistance. Urocortin 2 (Ucn 2), a member of the corticotropinreleasing factor (CRF) family, and its cognate type 2 CRF receptor (CRFR2) are highly expressed in skeletal muscle. To determine the physiological role of Ucn 2, we generated mice that are deficient in this peptide. Using glucose-tolerance tests (GTTs), insulin-tolerance tests (ITTs), and hyperinsulinemic euglycemic glucose clamp studies, we demonstrated that mice lacking Ucn 2 exhibited increased insulin sensitivity and were protected against fat-induced insulin resistance. Administration of synthetic Ucn 2 to mutant mice before the GTTs and ITTs restored blood glucose to WT levels.
Proceedings of the National Academy of Sciences, 2010
In response to physiological or psychological challenges, the brain activates behavioral and neur... more In response to physiological or psychological challenges, the brain activates behavioral and neuroendocrine systems linked to both metabolic and emotional outputs designed to adapt to the demand. However, dysregulation of integration of these physiological responses to challenge can have severe psychological and physiological consequences, and inappropriate regulation, disproportional intensity, or chronic or irreversible activation of the stress response is linked to the etiology and pathophysiology of mood and metabolic disorders. Using a transgenic mouse model and lentiviral approach, we demonstrate the involvement of the hypothalamic neuropeptide Urocortin-3, a specific ligand for the type-2 corticotropin-releasing factor receptor, in modulating septal and hypothalamic nuclei responsible for anxiety-like behaviors and metabolic functions, respectively. These results position Urocortin-3 as a neuromodulator linking stress-induced anxiety and energy homeostasis and pave the way toward better understanding of the mechanisms that mediate the reciprocal relationships between stress, mood and metabolic disorders. metabolic disorders | mood disorders | corticotropin-releasing factor (CRF) | CRF receptor type 2 | stress response I n modern Western societies, the high stress load correlates with an increasing incidence of mood disorders and metabolic syndrome, both of which have reached epidemic proportions over the past decades (1, 2). Exposure to acute or chronic stress is associated with derangement of metabolic and behavioral homeostasis that contributes to the clinical presentation of visceral obesity, type 2 diabetes, atherosclerosis, and metabolic syndrome (2-4). The corticotropin-releasing factor (CRF) neuropeptide system is the primary central mediator of the stress response and contributes to the etiology of stress-related psychiatric illness (5-8). Studies conducted using CRF receptor type 2 (CRFR2)-null mice or Urocortin-2 (Ucn2)-null mice provided evidence that, in addition to its role in mediating stress-related behavior, central CRFR2 is important in modulating metabolic rate, appetite, and feeding behaviors (9-12).
Environmental enrichment (EE) is known to have an anxiolytic effect in several animal models; how... more Environmental enrichment (EE) is known to have an anxiolytic effect in several animal models; however, the molecular mechanisms underlying these behavioral changes are not understood. In this study, we have shown that the anxiolytic effect of EE is associated with alterations in the corticotropin-releasing factor receptor type 1 (CRFR1) expression levels in the limbic system. We found that the decrease in anxiety-like behavior after housing in enriched conditions was associated with very low levels of CRFR1 mRNA expression in the basolateral amygdala of C57BL/6 mice. We further showed using a lentiviral-based system of RNA interference, that knockdown of CRFR1 mRNA expression in the basolateral amygdala induces a significant decrease in anxiety levels, similar to those achieved by EE nurture. Our data strongly suggest that reduced expression of CRFR1 mRNA levels in the basolateral amygdala mediates the effect of EE on anxiety-like behavior.
Corticotropin-releasing factor (CRF) has a key role in the central stress response, and altered l... more Corticotropin-releasing factor (CRF) has a key role in the central stress response, and altered levels of this neuropeptide are linked to stress-related psychopathologies such as anxiety and depression. These disorders are associated with the inability to properly regulate stress response, specifically following exposure to prolonged stressful stimuli. Therefore, the current study assessed the effects of prolonged and site-specific over-expression of CRF, which mimics the state of chronic production, in extended amygdala nuclei that are known to be involved in mediating anxiety-like states. We first constructed and generated lentiviruses that overexpress (OE) CRF in a robust and stable manner, and then generated two male mouse models continuously over-expressing CRF, either at the central nucleus of the amygdala (CeA), or at the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNSTdl). After 4 months, behavioral assessments were conducted for anxiety and depressive indices on these mice. Surprisingly, prolonged CRF OE at the CeA attenuated stress-induced anxiety-like behaviors, whereas prolonged CRF OE in the BNSTdl increased depressive-like behaviors, without affecting anxiety levels. These results show possible differential roles for CRF expressed by distinct loci of the extended amygdala, in mediating stress-induced emotional behaviors. Prolonged and site-specific over-expression of CRF L Regev et al Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp) Prolonged and site-specific over-expression of CRF L Regev et al
Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus are central compone... more Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus are central components of systems regulating appetite and energy homeostasis. Here we report on the establishment of a mouse model in which the ribonuclease III ribonuclease Dicer-1 has been specifically deleted from POMC-expressing neurons (POMC(ΔDCR)), leading to postnatal cell death. Mice are born phenotypically normal, at the expected genetic ratio and with normal hypothalamic POMC-mRNA levels. At 6 weeks of age, no POMC neurons/cells could be detected either in the arcuate nucleus or in the pituitary of POMC(ΔDCR) mice. POMC(ΔDCR) develop progressive obesity secondary to decreased energy expenditure but unrelated to food intake, which was surprisingly lower than in control mice. Reduced expression of AgRP and ghrelin receptor in the hypothalamus and reduced uncoupling protein 1 expression in brown adipose tissue can potentially explain the decreased food intake and decreased heat production, respectively, in these mice. Fasting glucose levels were dramatically elevated in POMC(ΔDCR) mice and the glucose tolerance test revealed marked glucose intolerance in these mice. Secondary to corticotrope ablation, basal and stress-induced corticosterone levels were undetectable in POMC(ΔDCR) mice. Despite this lack of activation of the neuroendocrine stress response, POMC(ΔDCR) mice exhibited an anxiogenic phenotype, which was accompanied with elevated levels of hypothalamic corticotropin-releasing factor and arginine-vasopressin transcripts. In conclusion, postnatal ablation of POMC neurons leads to enhanced anxiety and the development of obesity despite decreased food intake and glucocorticoid deficiency.
Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM... more Corticotropin-releasing factor receptor type 2 (CRFR2) is highly expressed in skeletal muscle (SM) tissue where it is suggested to inhibit interactions between insulin signaling pathway components affecting whole-body glucose homeostasis. However, little is known about factors regulating SM CRFR2 expression. Here, we demonstrate the exclusive expression of CRFR2, and not CRFR1, in mature SM tissue using RT-PCR and ribonuclease protection assays and report a differential expression of CRF receptors during C2C12 myogenic differentiation. Whereas C2C12 myoblasts exclusively express CRFR1, the C2C12 myotubes solely express CRFR2. Using cAMP luciferase assays and calcium mobilization measurements, we further demonstrate the functionality of these differentially expressed receptors. Using luciferase reporter assays we show a differential activation of CRFR promoters during myogenic differentiation. Transfections with different fragments of the 5Ј-flanking region of the mCRFR2 gene fused to a luciferase reporter gene show a promoterdependent expression of the reporter gene and reveal the importance of the myocyte enhancer factor 2 consensus sequence located at the 3Ј-proximal region of CRFR2 promoter. Furthermore, we demonstrate that CRFR2 gene transcription in the mature mouse is stimulated by both high-fat diet and chronic variable stress conditions. Performing a whole-genome expression microarray analysis of SM tissues obtained from CRFR2-null mice or wild-type littermates revealed a robust reduction in retinol-binding protein 4 expression levels, an adipokine whose serum levels are elevated in insulin-resistant states. In correlation with the SM CRFR2 levels, the SM retinolbinding protein 4 levels were also elevated in mice subjected to high-fat diet and chronic variable stress conditions. The current findings further position the SM CRFR2 pathways as a relevant physiological system that may affect the known reciprocal relationship between psychological and physiological challenges and the metabolic syndrome. (Molecular Endocrinology 25: 157-169, 2011)
Aims/hypothesis Urocortins are the endogenous ligands for the corticotropin-releasing factor rece... more Aims/hypothesis Urocortins are the endogenous ligands for the corticotropin-releasing factor receptor type 2 (CRFR2), which is implicated in regulating energy balance and/or glucose metabolism. We determined the effects of chronic CRFR2 activation on metabolism in vivo, by generating and phenotyping transgenic mice overproducing the specific CRFR2 ligand urocortin 3. Methods Body composition, glucose metabolism, insulin sensitivity, energy efficiency and expression of key metabolic genes were assessed in adult male urocortin 3 transgenic mice (Ucn3 + ) under control conditions and following an obesogenic high-fat diet (HFD) challenge.
Molecular-level understanding of body weight control is essential for combating obesity. We show ... more Molecular-level understanding of body weight control is essential for combating obesity. We show that female mice lacking tyrosine phosphatase epsilon (RPTPe) are protected from weight gain induced by high-fat food, ovariectomy, or old age and exhibit increased whole-body energy expenditure and decreased adiposity. RPTPe-deficient mice, in particular males, exhibit improved glucose homeostasis. Female nonobese RPTPe-deficient mice are leptin hypersensitive and exhibit reduced circulating leptin concentrations, suggesting that RPTPe inhibits hypothalamic leptin signaling in vivo. Leptin hypersensitivity persists in aged, ovariectomized, and high-fat-fed RPTPe-deficient mice, indicating that RPTPe helps establish obesity-associated leptin resistance. RPTPe associates with and dephosphorylates JAK2, thereby downregulating leptin receptor signaling. Leptin stimulation induces phosphorylation of hypothalamic RPTPe at its C-terminal Y695, which drives RPTPe to downregulate JAK2. RPTPe is therefore an inhibitor of hypothalamic leptin signaling in vivo, and provides controlled negative-feedback regulation of this pathway following its activation.
The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role... more The corticotropin-releasing factor type 2 receptor (CRFR2) is suggested to play an important role in aiding recovery from acute stress, but any chronic effects of CRFR2 activation are unknown. CRFR2 in the midbrain raphé nuclei modulate serotonergic activity of this key source of serotonin (5-HT) forebrain innervation.
The Journal of Clinical Endocrinology & Metabolism, 2013
Context: Adipose tissue macrophages (ATMs) are thought to engulf the remains of dead adipocytes i... more Context: Adipose tissue macrophages (ATMs) are thought to engulf the remains of dead adipocytes in obesity, potentially resulting in increased intracellular neutral lipid content. Lipid-laden macrophages (foam cells [FCs]) have been described in atherosclerotic lesions and have been proposed to contribute to vascular pathophysiology, which is enhanced in obesity.
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Papers by Yael Kuperman