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1.
Cell Mol Life Sci ; 78(19-20): 6689-6708, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34559253

RESUMO

Ghrelin is a stomach-derived peptide hormone with salient roles in the regulation of energy balance and metabolism. Notably, ghrelin is recognized as the most powerful known circulating orexigenic hormone. Here, we systematically investigated the effects of ghrelin on energy homeostasis and found that ghrelin primarily induces a biphasic effect on food intake that has indirect consequences on energy expenditure and nutrient partitioning. We also found that ghrelin-induced biphasic effect on food intake requires the integrity of Agouti-related peptide/neuropeptide Y-producing neurons of the hypothalamic arcuate nucleus, which seem to display a long-lasting activation after a single systemic injection of ghrelin. Finally, we found that different autonomic, hormonal and metabolic satiation signals transiently counteract ghrelin-induced food intake. Based on our observations, we propose a heuristic model to describe how the orexigenic effect of ghrelin and the anorectic food intake-induced rebound sculpt a timely constrain feeding response to ghrelin.


Assuntos
Ingestão de Alimentos/efeitos dos fármacos , Grelina/farmacologia , Heurística/efeitos dos fármacos , Animais , Metabolismo Energético/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo
2.
Endocrinology ; 165(7)2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38815068

RESUMO

The growth hormone secretagogue receptor (GHSR), primarily known as the receptor for the hunger hormone ghrelin, potently controls food intake, yet the specific Ghsr-expressing cells mediating the orexigenic effects of this receptor remain incompletely characterized. Since Ghsr is expressed in gamma-aminobutyric acid (GABA)-producing neurons, we sought to investigate whether the selective expression of Ghsr in a subset of GABA neurons is sufficient to mediate GHSR's effects on feeding. First, we crossed mice that express a tamoxifen-dependent Cre recombinase in the subset of GABA neurons that express glutamic acid decarboxylase 2 (Gad2) enzyme (Gad2-CreER mice) with reporter mice, and found that ghrelin mainly targets a subset of Gad2-expressing neurons located in the hypothalamic arcuate nucleus (ARH) and that is predominantly segregated from Agouti-related protein (AgRP)-expressing neurons. Analysis of various single-cell RNA-sequencing datasets further corroborated that the primary subset of cells coexpressing Gad2 and Ghsr in the mouse brain are non-AgRP ARH neurons. Next, we crossed Gad2-CreER mice with reactivable GHSR-deficient mice to generate mice expressing Ghsr only in Gad2-expressing neurons (Gad2-GHSR mice). We found that ghrelin treatment induced the expression of the marker of transcriptional activation c-Fos in the ARH of Gad2-GHSR mice, yet failed to induce food intake. In contrast, food deprivation-induced refeeding was higher in Gad2-GHSR mice than in GHSR-deficient mice and similar to wild-type mice, suggesting that ghrelin-independent roles of GHSR in a subset of GABA neurons is sufficient for eliciting full compensatory hyperphagia in mice.


Assuntos
Núcleo Arqueado do Hipotálamo , Privação de Alimentos , Neurônios GABAérgicos , Grelina , Glutamato Descarboxilase , Hiperfagia , Receptores de Grelina , Animais , Masculino , Camundongos , Neurônios GABAérgicos/metabolismo , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Hiperfagia/metabolismo , Grelina/metabolismo , Grelina/farmacologia , Núcleo Arqueado do Hipotálamo/metabolismo , Privação de Alimentos/fisiologia , Glutamato Descarboxilase/metabolismo , Glutamato Descarboxilase/genética , Camundongos Transgênicos , Proteína Relacionada com Agouti/metabolismo , Proteína Relacionada com Agouti/genética , Camundongos Endogâmicos C57BL
3.
Commun Biol ; 7(1): 632, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38796563

RESUMO

The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions; therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here, we investigate the effects of a long-term (12-month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild-type (WT) Wistar male and female rats. Our main findings are that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increases thermogenesis and brain glucose uptake in male rats and modifies the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. We use RNA-sequencing to show that GHSR-KO rats have upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuates ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating is reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.


Assuntos
Dieta Hiperlipídica , Obesidade , Ratos Wistar , Receptores de Grelina , Caracteres Sexuais , Animais , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Dieta Hiperlipídica/efeitos adversos , Masculino , Feminino , Ratos , Obesidade/metabolismo , Obesidade/genética , Grelina/metabolismo , Termogênese/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/efeitos dos fármacos
4.
Mol Neurobiol ; 60(5): 2644-2660, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36694048

RESUMO

The dopamine receptor type 1 (D1R) and the dopamine receptor type 5 (D5R), which are often grouped as D1R-like due to their sequence and signaling similarities, exhibit high levels of constitutive activity. The molecular basis for this agonist-independent activation has been well characterized through biochemical and mutagenesis in vitro studies. In this regard, it was reported that many antipsychotic drugs act as inverse agonists of D1R-like constitutive activity. On the other hand, D1R is highly expressed in the medial prefrontal cortex (mPFC), a brain area with important functions such as working memory. Here, we studied the impact of D1R-like constitutive activity and chlorpromazine (CPZ), an antipsychotic drug and D1R-like inverse agonist, on various neuronal CaV conductances, and we explored its effect on calcium-dependent neuronal functions in the mouse medial mPFC. Using ex vivo brain slices containing the mPFC and transfected HEK293T cells, we found that CPZ reduces CaV2.2 currents by occluding D1R-like constitutive activity, in agreement with a mechanism previously reported by our lab, whereas CPZ directly inhibits CaV1 currents in a D1R-like activity independent manner. In contrast, CPZ and D1R constitutive activity did not affect CaV2.1, CaV2.3, or CaV3 currents. Finally, we found that CPZ reduces excitatory postsynaptic responses in mPFC neurons. Our results contribute to understanding CPZ molecular targets in neurons and describe a novel physiological consequence of CPZ non-canonical action as a D1R-like inverse agonist in the mouse brain.


Assuntos
Clorpromazina , Receptores Dopaminérgicos , Camundongos , Humanos , Animais , Clorpromazina/farmacologia , Agonismo Inverso de Drogas , Células HEK293 , Neurônios/metabolismo , Canais de Cálcio , Córtex Pré-Frontal/metabolismo , Cálcio/metabolismo
5.
Life Sci ; 293: 120284, 2022 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-35038454

RESUMO

AIMS: Angiotensin-converting enzyme 2 (ACE2) is a key regulator of the renin-angiotensin system (RAS) recently identified as the membrane receptor for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we aim to study whether two receptors from RAS, the angiotensin receptor type 1 (AT1R) and the bradykinin 2 receptor (B2R) modulate ACE2 internalization induced by a recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. Also, we investigated the impact of ACE2 coexpression on AT1R and B2R functionality. MATERIALS AND METHODS: To study ACE2 internalization, we assessed the distribution of green fluorescent protein (GFP) signal in HEK293T cells coexpressing GFP-tagged ACE2 and AT1R, or B2R, or AT1R plus B2R in presence of RBD alone or in combination with AT1R or B2R ligands. To estimate ACE2 internalization, we classified GFP signal distribution as plasma membrane uniform GFP (PMU-GFP), plasma membrane clustered GFP (PMC-GFP) or internalized GFP and calculated its relative frequency. Additionally, we investigated the effect of ACE2 coexpression on AT1R and B2R inhibitory action on voltage-gated calcium channels (CaV2.2) currents by patch-clamp technique. KEY FINDINGS: RBD induced ACE2-GFP internalization in a time-dependent manner. RBD-induced ACE2-GFP internalization was increased by angiotensin II and reduced by telmisartan in cells coexpressing AT1R. RBD-induced ACE2-GFP internalization was strongly inhibited by B2R co-expression. This effect was mildly modified by bradykinin and rescued by angiotensin II in presence of AT1R. ACE2 coexpression impacted on B2R- and AT1R-mediated inhibition of CaV2.2 currents. SIGNIFICANCE: Our work contributes to understand the role of RAS modulators in the susceptibility to SARS-CoV-2 infection and severity of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2/biossíntese , Receptor Tipo 1 de Angiotensina/biossíntese , Receptor B2 da Bradicinina/biossíntese , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Enzima de Conversão de Angiotensina 2/análise , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Células HEK293 , Humanos , Receptor Tipo 1 de Angiotensina/análise , Receptor B2 da Bradicinina/análise , Proteínas Recombinantes/administração & dosagem
6.
Biochem Pharmacol ; 202: 115114, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35659880

RESUMO

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that regulates essential physiological functions. In particular, activation of GHSR in response to its endogenous agonist ghrelin promotes food intake and blood glucose increase. Therefore, compounds aimed at blocking GHSR signaling constitute potential options against obesity-related metabolic disorders. We have previously developed potent ligands of GHSR based on a triazole scaffold. Here, we report a new 3,4,5-trisubstituted 1,2,4-triazole compound, named JMV 6616, that potently blocks GHSR activity in vitro and in vivo. Specifically, in HEK293T cells JMV 6616 behaves as an inverse agonist since it binds to GHSR and inhibits its ghrelin-independent signaling. Accordingly, using purified labeled GHSR assembled into lipid nanodiscs we found that JMV 6616 decreases GHSR-catalyzed G protein activation and stabilizes an inactive receptor conformation. Importantly, JMV 6616 also acts on native GHSR since it blocks the insulinostatic effect of ghrelin in pancreatic islets. In mice, JMV 6616 inhibits blood glucose-raising effects of ghrelin treatment and the orexigenic actions of acute ghrelin administration. Together, our data suggest that this triazole-derived modulator of GHSR holds promise to mitigate several pathological features associated with eating and metabolic disorders.


Assuntos
Grelina , Receptores de Grelina , Animais , Glicemia , Grelina/metabolismo , Grelina/farmacologia , Células HEK293 , Humanos , Camundongos , Triazóis/farmacologia
7.
Psychoneuroendocrinology ; 119: 104718, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32535402

RESUMO

Growth hormone secretagogue receptor (GHSR), the receptor for ghrelin, is expressed in key brain nuclei that regulate food intake. The dopamine (DA) pathways have long been recognized to play key roles mediating GHSR effects on feeding behaviors. Here, we aimed to determine the role of GHSR in DA neurons controlling appetitive and consummatory behaviors towards high fat (HF) diet. For this purpose, we crossed reactivable GHSR-deficient mice with DA transporter (DAT)-Cre mice, which express Cre recombinase under the DAT promoter that is active exclusively in DA neurons, to generate mice with GHSR expression limited to DA neurons (DAT-GHSR mice). We found that DAT-GHSR mice show an increase of c-Fos levels in brain areas containing DA neurons after ghrelin treatment, in a similar fashion as seen in wild-type mice; however, they did not increase food intake or locomotor activity in response to systemically- or centrally-administered ghrelin. In addition, we found that satiated DAT-GHSR mice displayed both anticipatory activity to scheduled HF diet exposure and HF intake in a binge-like eating protocol similar to those in wild-type mice, whereas GHSR-deficient mice displayed impaired responses. We conclude that GHSR expression in DA neurons is sufficient to both mediate increased anticipatory activity to a scheduled HF diet exposure and fully orchestrate binge-like HF intake, but it is insufficient to restore the acute orexigenic or locomotor effects of ghrelin treatment. Thus, GHSR in DA neurons affects appetitive and consummatory behaviors towards HF diet that take place in the absence of caloric needs.


Assuntos
Comportamento Consumatório/fisiologia , Dieta Hiperlipídica , Comportamento Alimentar/fisiologia , Receptores de Grelina/fisiologia , Animais , Regulação do Apetite/genética , Comportamento Animal/fisiologia , Neurônios Dopaminérgicos/metabolismo , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Preferências Alimentares/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Grelina/genética , Receptores de Grelina/metabolismo
8.
Mol Metab ; 39: 101004, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32339772

RESUMO

OBJECTIVE: Binding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR), stimulates GH release, induces eating, and increases blood glucose. These processes may also be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by findings in short people with naturally occurring GHSR-A204E mutations and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. In this study, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. METHODS: We generated mice with a GHSR mutation that replaces alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the previously described human GHSR-A204E mutation, and used them to conduct ex vivo neuronal electrophysiology and in vivo metabolic assessments. We also measured signaling within COS-7 and HEK293T cells transfected with wild-type GHSR (GHSR-WT) or GHSR-A203E constructs. RESULTS: In COS-7 cells, GHSR-A203E resulted in lower baseline IP3 accumulation than GHSR-WT; ghrelin-induced IP3 accumulation was observed in both constructs. In HEK293T cells co-transfected with voltage-gated CaV2.2 calcium channel complex, GHSR-A203E had no effect on basal CaV2.2 current density while GHSR-WT did; both GHSR-A203E and GHSR-WT inhibited CaV2.2 current in the presence of ghrelin. In cultured hypothalamic neurons from GHSR-A203E and GHSR-deficient mice, native calcium currents were greater than those in neurons from wild-type mice; ghrelin inhibited calcium currents in cultured hypothalamic neurons from both GHSR-A203E and wild-type mice. In brain slices, resting membrane potentials of arcuate NPY neurons from GHSR-A203E mice were hyperpolarized compared to those from wild-type mice; the same percentage of arcuate NPY neurons from GHSR-A203E and wild-type mice depolarized upon ghrelin exposure. The GHSR-A203E mutation did not significantly affect body weight, body length, or femur length in the first ∼6 months of life, yet these parameters were lower in GHSR-A203E mice after 1 year of age. During a 7-d 60% caloric restriction regimen, GHSR-A203E mice lacked the usual marked rise in plasma GH and demonstrated an exaggerated drop in blood glucose. Administered ghrelin also exhibited reduced orexigenic and GH secretagogue efficacies in GHSR-A203E mice. CONCLUSIONS: Our data suggest that the A203E mutation ablates constitutive GHSR activity and that constitutive GHSR activity contributes to the native depolarizing conductance of GHSR-expressing arcuate NPY neurons. Although the A203E mutation does not block ghrelin-evoked signaling as assessed using in vitro and ex vivo models, GHSR-A203E mice lack the usual acute food intake response to administered ghrelin in vivo. The GHSR-A203E mutation also blunts GH release, and in aged mice leads to reduced body length and femur length, which are consistent with the short stature of human carriers of the GHSR-A204E mutation.


Assuntos
Alelos , Substituição de Aminoácidos , Metabolismo Energético/genética , Mutação , Receptores de Grelina/genética , Animais , Pesos e Medidas Corporais , Sinalização do Cálcio , Linhagem Celular , Fenômenos Eletrofisiológicos , Regulação da Expressão Gênica , Marcação de Genes , Estudos de Associação Genética , Células HEK293 , Hormônios/metabolismo , Humanos , Hipotálamo/metabolismo , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Técnicas de Patch-Clamp , Receptores de Grelina/metabolismo
9.
J Neuroendocrinol ; 31(10): e12785, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31469195

RESUMO

The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that is highly expressed in the central nervous system. GHSR acts as a receptor for ghrelin and for liver-expressed antimicrobial peptide 2 (LEAP2), which blocks ghrelin-evoked activity. GHSR also displays ligand-independent activity, including a high constitutive activity that signals in the absence of ghrelin and is reduced by LEAP2. GHSR activity modulates a variety of food intake-related behaviours, including binge eating. Previously, we reported that GHSR-deficient mice daily and time-limited exposed to a high-fat (HF) diet display an attenuated binge-like HF intake compared to wild-type mice. In the present study, we aimed to determine whether ligand-independent GHSR activity affects binge-like HF intake in a 4-day binge-like eating protocol. We found that plasma levels of ghrelin and LEAP2 were not modified in mice exposed to this binge-like eating protocol. Moreover, systemic administration of ghrelin or LEAP2 did not alter HF intake in our experimental conditions. Interestingly, we found that central administration of LEAP2 or K-(D-1-Nal)-FwLL-NH2 , which are both blockers of constitutive GHSR activity, reduced binge-like HF intake, whereas central administration of ghrelin or the ghrelin-evoked GHSR activity blockers [D-Lys3]-GHRP-6 and JMV2959 did not modify binge-like HF intake. Taken together, current data indicate that GHSR activity in the brain affects binge-like HF intake in mice independently of plasma levels of ghrelin and LEAP2.


Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Bulimia/fisiopatologia , Grelina/fisiologia , Receptores de Grelina/agonistas , Receptores de Grelina/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/sangue , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bulimia/prevenção & controle , Dieta Hiperlipídica , Grelina/administração & dosagem , Grelina/sangue , Grelina/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Infusões Intraventriculares , Masculino , Camundongos , Oligopeptídeos/farmacologia , Receptores de Grelina/antagonistas & inibidores , Fatores de Tempo , Triazóis/farmacologia
10.
Neuroscience ; 392: 107-120, 2018 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-30268780

RESUMO

Ghrelin is a stomach-derived hormone that regulates rewarding behaviors and reinforcement by acting on the ventral tegmental area (VTA). The VTA is a complex midbrain structure mainly comprised of dopamine (DA) and gamma-aminobutiric acid (GABA) neurons that are distributed in several VTA sub-nuclei. Here, we investigated the neuroanatomical distribution and chemical phenotype of ghrelin-responsive neurons within the VTA. In wild-type mice, we found that: (1) ghrelin binding cells are present in most VTA sub-nuclei but not in its main target, the nucleus accumbens (Acb); (2) systemically injected ghrelin increases food intake but does neither affect locomotor activity nor the levels of the marker of neuronal activation c-Fos in the VTA sub-nuclei; (3) centrally injected ghrelin increases food intake, locomotor activity and c-Fos levels in non-DA neurons of all VTA sub-nuclei; (4) intra-VTA-injected ghrelin increases food intake, locomotor activity and c-Fos levels in non-DA neurons of all VTA sub-nuclei; (5) both centrally and intra-VTA-injected ghrelin increase c-Fos levels in DA neurons of the parabrachial pigmented VTA sub-nucleus. In genetically modified mice in which a subset of GABA neurons expresses the red fluorescent protein tdTomato, we found that centrally injected ghrelin increases c-Fos levels in GABA neurons of the interfascicular VTA sub-nucleus. These results suggest that ghrelin can recruit specific subsets of VTA neurons in order to modulate food intake and locomotor activity.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Neurônios GABAérgicos/fisiologia , Grelina/fisiologia , Neurônios/fisiologia , Área Tegmentar Ventral/fisiologia , Animais , Ingestão de Alimentos , Grelina/administração & dosagem , Locomoção/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos
11.
Brain Struct Funct ; 223(7): 3133-3147, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29761230

RESUMO

Ghrelin is a stomach-derived hormone that regulates a variety of biological functions such as food intake, gastrointestinal function and blood glucose metabolism, among others. Ghrelin acts via the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor located in key brain areas that mediate specific actions of the hormone. GHSR is highly expressed in the nucleus of the solitary tract (NTS), which is located in the medulla oblongata and controls essential functions, including orofacial, autonomic, neuroendocrine and behavioral responses. Here, we used a mouse model, in which the expression of enhanced green fluorescent protein (eGFP) is controlled by the promoter of GHSR (GHSR-eGFP mice), to gain neuroanatomical and functional insights of the GHSR-expressing neurons of the NTS. We found that GHSR-expressing neurons of the NTS are segregated in clusters that were symmetrically distributed to the midline: (1) a pair of rostral clusters, and (2) a caudal and medially located cluster. We also identified that a subset of GHSR neurons of the caudal NTS are GABAergic. Finally, we found that rostral NTS GHSR neurons increase the levels of the marker of neuronal activation c-Fos in mice exposed to fasting/refeeding or high-fat diet bingeing protocols, while caudal NTS GHSR neurons increase the levels of c-Fos in mice exposed to gastric distension or LiCl-induced malaise protocols. Thus, current data provide evidence that ghrelin receptor signaling seems to target segregated clusters of neurons within the NTS that, in turn, may be activated by different stimuli.


Assuntos
Neurônios GABAérgicos/metabolismo , Bulbo/metabolismo , Receptores de Grelina/metabolismo , Núcleo Solitário/metabolismo , Animais , Feminino , Fluorescência , Grelina/administração & dosagem , Grelina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais
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