Serotonin systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors and induce anorexia via a leptin-independent pathway in mice

NEFA/nucleobindin2 (NUCB2), a novel satiety molecule, is associated with leptin-independent melanocortin signaling in the central nervous system. Here, we show that systemic administration of m-chlorophenylpiperazine (mCPP), a serotonin 5-HT1B/2C receptor agonist, significantly increased the expression of hypothalamic NUCB2 in wild-type mice. The increases in hypothalamic NUCB2 expression induced by mCPP were attenuated in 5-HT2C receptor mutant mice. Systemic administration of mCPP suppressed food intake in db/db mice with leptin receptor mutation as well as lean control mice. On the other hand, the expression of hypothalamic NUCB2 and proopiomelanocortin (POMC) was significantly decreased in hyperphagic and non-obese 5-HT2C receptor mutants compared with age-matched wild-type mice. Interestingly, despite increased expression of hypothalamic POMC, hypothalamic NUCB2 expression was decreased in 5-HT2C receptor mutant mice with heterozygous mutation of β-endorphin gene. These findings suggest that 5-HT systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors, and induce anorexia via a leptin-independent pathway in mice.     

Production of Nurr-1 Specific Polyclonal Antibodies Free of Cross-reactivity Against Its Close Homologs,Nor1 and Nur77

The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson’s disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.     

The contribution of serotonin 5-HT2C and melanocortin-4 receptors to the satiety signaling of glucagon-like peptide 1 and liragultide, a glucagon-like peptide 1 receptor agonist, in mice

Glucagon-like peptide 1 (GLP-1), an insulinotropic gastrointestinal peptide produced mainly from intestinal endocrine L-cells, and liraglutide, a GLP-1 receptor (GLP-1R) agonist, induce satiety. The serotonin 5-HT2C receptor (5-HT2CR) and melanoroctin-4 receptor (MC4R) are involved in the regulation of food intake. Here we show that systemic administration of GLP-1 (50 and 200 μg/kg)-induced anorexia was blunted in mice with a 5HT2CR null mutation, and was attenuated in mice with a heterozygous MC4R mutation. On the other hand, systemic administration of liraglutide (50 and 100 μg/kg) suppressed food intake in mice lacking 5-HT2CR, mice with a heterozygous mutation of MC4R and wild-type mice matched for age. Moreover, once-daily consecutive intraperitoneal administration of liraglutide (100 μg/kg) over 3 days significantly suppressed daily food intake and body weight in mice with a heterozygous mutation of MC4R as well as wild-type mice. These findings suggest that GLP-1 and liraglutide induce anorexia via different central pathways.     

Urocortin1-induced anorexia is regulated by activation of the serotonin 2C receptor in the brain

This study was conducted to determine the mechanisms by which serotonin (5-hydroxytryptamine, 5-HT) receptors are involved in the suppression of food intake in a rat stress model and to observe the degree of activation in the areas of the brain involved in feeding. In the stress model, male Sprague–Dawley rats (8 weeks old) were given intracerebroventricular injections of urocortin (UCN) 1. To determine the role of the 5-HT2c receptor (5-HT2cR) in the decreased food intake in UCN1-treated rats, specific 5-HT2cR or 5-HT2b receptor (5-HT2bR) antagonists were administered. Food intake was markedly reduced in UCN1-injected rats compared with phosphate buffered saline treated control rats. Intraperitoneal administration of a 5-HT2cR antagonist, but not a 5-HT2bR antagonist, significantly inhibited the decreased food intake. To assess the involvement of neural activation, we tracked the expression of c-fos mRNA as a neuronal activation marker. Expression of the c-fos mRNA in the arcuate nucleus, ventromedial hypothalamic nucleus (VMH) and rostral ventrolateral medulla (RVLM) in UNC1-injected rats showed significantly higher expression than in the PBS-injected rats. Increased c-fos mRNA was also observed in the paraventricular nucleus (PVN), the nucleus of the solitary tract (NTS), and the amygdala (AMG) after injection of UCN1. Increased 5-HT2cR protein expression was also observed in several areas. However, increased coexpression of 5-HT2cR and c-fos was observed in the PVN, VMH, NTS, RVLM and AMG. Whereas, pro-opiomelanocortin mRNA expression was not changed. In an UNC1-induced stress model, 5-HT2cR expression and activation was found in brain areas involved in feeding control.     

Parathyroid hormone induces the nuclear orphan receptor NOR-1 in osteoblasts

Parathyroid hormone (PTH) significantly affects osteoblast function by altering gene expression. We have identified neuron-derived orphan receptor-1 (NOR-1) as a PTH-induced primary gene in osteoblastic cells. NOR-1, Nurr1, and Nur77 comprise the NGFI-B nuclear orphan receptor family and Nurr1 and Nur77 are PTH-induced primary osteoblastic genes. Ten nM PTH maximally induced NOR-1 mRNA at 2h in primary mouse osteoblasts and at 1h in mouse calvariae. Cycloheximide pretreatment did not inhibit PTH-induced NOR-1 mRNA. PTH activates cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling. Forskolin (PKA activator) and PMA (PKC activator) mimicked PTH-induced NOR-1 mRNA. Ionomycin (calcium ionophore) and PTH(3-34), which do not activate PKA, failed to induce NOR-1 mRNA. PKA inhibition with H89 blocked PTH- and FSK-induced NOR-1 mRNA. PMA pretreatment to deplete PKC inhibited PMA-induced, but not PTH-induced, NOR-1 mRNA. We conclude that NOR-1 is a PTH-regulated primary osteoblastic gene that is induced mainly through cAMP-PKA signaling.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

Lack of CB1 receptor activity impairs serotonergic negative feedback

Serotonergic and endocannabinoid systems are important substrates for the control of emotional behaviour and growing evidence show an involvement in the pathophysiology of mood disorders. In the present study, the absence of the activity of the CB₁ cannabinoid receptor impaired serotonergic negative feedback in mice. Thus, in vivo microdialysis experiments revealed increased basal 5-HT extracellular levels and attenuated fluoxetine-induced increase of 5-HT extracellular levels in the prefrontal cortex of CB₁ knockout compared with wild-type mice. These observations could be related to the significant reduction in the 5-HT transporter binding site density detected in frontal cortex and hippocampus of CB₁ knockout mice. The lack of CB₁ receptor also altered some 5-HT receptors related to the 5-HT feedback. Extracellular recordings in the dorsal raphe nucleus (DRN) revealed that the genetic and pharmacological blockade of CB₁ receptor induced a 5-HT_1A autoreceptor functional desensitization. In situ hybridization studies showed a reduction in the expression of the 5-HT_2C receptor within several brain areas related to the control of the emotional responses, such as the DRN, the nucleus accumbens and the paraventricular nucleus of the hypothalamus, whereas an over-expression was observed in the CA3 area of the ventral hippocampus. These results reveal that the lack of CB₁ receptor induces a facilitation of the activity of serotonergic neurons in the DRN by altering different components of the 5-HT feedback as well as an increase in 5-HT extracellular levels in the prefrontal cortex in mice.     

Serotonin,Eating Behavior,and Fat Intake

There is an intimate relationship between nutritional intake (eating) and serotonin activity. Experimental manipulations (mainly neuropharmacological) of serotonin influence the pattern of eating behavior, subjective feelings of appetite motivation, and the response to nutritional challenges. Similarly, nutritional manipulations (food restriction, dieting, or altered nutrient supply) change the sensitivity of the serotonin network. Traditionally, serotonin has been linked to the macronutrient carbohydrate via the intermediary step of plasma amino acid ratios. However, it has also been demonstrated that 5-HT drugs will reduce energy intake and reverse body weight gain in rats exposed to weight increasing high fat diets. 5-HT drugs can also reduce food intake and block weight gain of rats on a high fat cafeteria diet. Some diet selection studies in rats indicate that the most prominent reduction of macronutrient intake is for fat. These data indicate that 5-HT activity can bring about a reduction in fat consumption. In turn, different types of dietary fat can alter brain 5-HT activity. In human studies the methodology of food choice experiments has often precluded the detection of an effect of 5-HT manipulation on fat intake. However, there is evidence that in obese and lean subjects some 5-HT drugs can readily reduce the intake of high fat foods. Data also suggest that 5-HT activation can lead to a selective avoidance of fat in the diet. These effects of 5-HT on the intake of dietary fat may involve a pre-absorptive mechanism and there is evidence that 5-HT is linked to cholecystokinin and enterostatin. These proposals have theoretical and practical implications and suggest possible strategies to intensify or advance fat-induced satiety signals.     

Hypothalamic Ahi1 mediates feeding behavior through interaction with 5-HT2C receptor

It is indicated that there are important molecules interacting with brain nervous systems to regulate feeding and energy balance by influencing the signaling pathways of these systems, but relatively few of the critical players have been identified. In the present study, we provide the evidence for the role of Abelson helper integration site 1 (Ahi1) protein as a mediator of feeding behavior through interaction with serotonin receptor 2C (5-HT(2C)R), known for its critical role in feeding and appetite control. First, we demonstrated the co-localization and interaction between hypothalamic Ahi1 and 5-HT(2C)R. Ahi1 promoted the degradation of 5-HT(2C)R through the lysosomal pathway. Then, we investigated the effects of fasting on the expression of hypothalamic Ahi1 and 5-HT(2C)R. Fasting resulted in an increased Ahi1 expression and a concomitant decreased expression of 5-HT(2C)R. Knockdown of hypothalamic Ahi1 led to a concomitant increased expression of 5-HT(2C)R and a decrease of food intake and body weight. Last, we found that Ahi1 could regulate the expression of neuropeptide Y and proopiomelanocortin. Taken together, our results indicate that Ahi1 mediates feeding behavior by interacting with 5-HT(2C)R to modulate the serotonin signaling pathway.