Metabolic insights from a GHSR-A203E mutant mouse model

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.Fil: Torz, Lola J.. Universidad de Copenhagen; DinamarcaFil: Osborne Lawrence, Sherri. Ut Southwestern Medical Center; Estados UnidosFil: Rodriguez, Juan. Ut Southwestern Medical Center; Estados UnidosFil: He, Zhenyan. Ut Southwestern Medical Center; Estados UnidosFil: Cornejo, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Mustafá, Emilio Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Jin, Chunyu. Universidad de Copenhagen; DinamarcaFil: Petersen, Natalia. Universidad de Copenhagen; DinamarcaFil: Hedegaard, Morten A.. Universidad de Copenhagen; DinamarcaFil: Nybo, Maja. Universidad de Copenhagen; DinamarcaFil: Martínez Damonte, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Metzger, Nathan P.. Ut Southwestern Medical Center; Estados UnidosFil: Mani, Bharath K.. Ut Southwestern Medical Center; Estados UnidosFil: Williams, Kevin W.. Ut Southwestern Medical Center; Estados UnidosFil: Raingo, Jesica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Perello, Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Holst, Birgitte. Universidad de Copenhagen; DinamarcaFil: Zigman, Jeffrey M.. Ut Southwestern Medical Center; Estados Unido

NPFF Decreases Activity of Human Arcuate NPY Neurons:A Study in Embryonic-Stem-Cell-Derived Model

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons

Hypothalamic hormone-sensitive lipase regulates appetite and energy homeostasis

ObjectiveThe goal of this study was to investigate the importance of central hormone-sensitive lipase (HSL) expression in the regulation of food intake and body weight in mice to clarify whether intracellular lipolysis in the mammalian hypothalamus plays a role in regulating appetite.MethodsUsing pharmacological and genetic approaches, we investigated the role of HSL in the rodent brain in the regulation of feeding and energy homeostasis under basal conditions during acute stress and high-fat diet feeding.ResultsWe found that HSL, a key enzyme in the catabolism of cellular lipid stores, is expressed in the appetite-regulating centers in the hypothalamus and is activated by acute stress through a mechanism similar to that observed in adipose tissue and skeletal muscle. Inhibition of HSL in rodent models by a synthetic ligand, global knockout, or brain-specific deletion of HSL prevents a decrease in food intake normally seen in response to acute stress and is associated with the increased expression of orexigenic peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP). Increased food intake can be reversed by adeno-associated virus-mediated reintroduction of HSL in neurons of the mediobasal hypothalamus. Importantly, metabolic stress induced by a high-fat diet also enhances the hyperphagic phenotype of HSL-deficient mice. Specific deletion of HSL in the ventromedial hypothalamic nucleus (VMH) or AgRP neurons reveals that HSL in the VMH plays a role in both acute stress-induced food intake and high-fat diet-induced obesity.ConclusionsOur results indicate that HSL activity in the mediobasal hypothalamus is involved in the acute reduction in food intake during the acute stress response and sensing of a high-fat diet