Metabolic insights from a GHSR-A203E mutant mouse model

TORZ, LOLA J.; OSBORNE-LAWRENCE, SHERRI; RODRIGUEZ, JUAN; HE, ZHENYAN; CORNEJO, MARÍA PAULA; MUSTAFÁ, EMILIO ROMÁN; JIN, CHUNYU; PETERSEN, NATALIA; HEDEGAARD, MORTEN A.; NYBO, MAJA; DAMONTE, VALENTINA MARTÍNEZ; METZGER, NATHAN P.; MANI, BHARATH K.; WILLIAMS, KEVIN W.; RAINGO, JESICA; PERELLO, MARIO; HOLST, BIRGITTE; ZIGMAN, JEFFREY M.

Molecular Metabolism

ELSEVIER

Lugar: Amsterdam; Año: 2020

2212-8778

Objective Binding of ghrelin to its receptor, growth hormone secretagogue receptor (GHSR) stimulates GH release, induces eating, and raises blood glucose. These processes also may be influenced by constitutive (ghrelin-independent) GHSR activity, as suggested by the findings of short stature in people with the naturally-occurring GHSR-A204E mutation and reduced food intake and blood glucose in rodents administered GHSR inverse agonists, both of which impair constitutive GHSR activity. Here, we aimed to more fully determine the physiologic relevance of constitutive GHSR activity. Methods We generated mice with a GHSR mutation that replaces the alanine at position 203 with glutamate (GHSR-A203E), which corresponds to the above-referenced human GHSR-A204E mutation, and used them to perform ex vivo neuronal electrophysiology and in vivo metabolic assessments. Also, we 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 did GHSR-WT; ghrelin-induced IP3 accumulation was observed for both constructs. In HEK293T cells co-transfected with the 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 as 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 those parameters were lower in GHSR-A203E mice after 1 yr-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. Also, administered ghrelin 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. Also, the GHSR-A203E mutation 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.