Ablations of Ghrelin and Ghrelin Receptor Exhibit Differential Metabolic Phenotypes and Thermogenic Capacity during Aging

Abstract

BackgroundObesity is a hallmark of aging in many Western societies, and is a precursor to numerous serious age-related diseases. Ghrelin (Ghrl), via its receptor (growth hormone secretagogue receptor, GHS-R), is shown to stimulate GH secretion and appetite. Surprisingly, our previous studies showed that Ghrl-/- mice have impaired thermoregulatory responses to cold and fasting stresses, while Ghsr-/- mice are adaptive.Methodology/Principal FindingsTo elucidate the mechanism, we analyzed the complete metabolic profiles of younger (3–4 months) and older (10–12 months) Ghrl-/- and Ghsr-/- mice. Food intake and locomotor activity were comparable for both null mice and their wild-type (WT) counterparts, regardless of age. There was also no difference in body composition between younger null mice and their WT counterparts. As the WT mice aged, as expected, the fat/lean ratio increased and energy expenditure (EE) decreased. Remarkably, however, older Ghsr-/- mice exhibited reduced fat/lean ratio and increased EE when compared to older WT mice, thus retaining a youthful lean and high EE phenotype; in comparison, there was no significant difference with EE in Ghrl-/- mice. In line with the EE data, the thermogenic regulator, uncoupling protein 1 (UCP1), was significantly up-regulated in brown adipose tissue (BAT) of Ghsr-/- mice, but not in Ghrl-/- mice.ConclusionsOur data therefore suggest that GHS-R ablation activates adaptive thermogenic function(s) in BAT and increases EE, thereby enabling the retention of a lean phenotype. This is the first direct evidence that the ghrelin signaling pathway regulates fat-burning BAT to affect energy balance during aging. This regulation is likely mediated through an as-yet-unidentified new ligand of GHS-R.

Characterization of a far-red analog of ghrelin for imaging GHS-R in P19-derived cardiomyocytes

“…1) with an in vitro binding assay using HEK 293 cells over-expressing GHSR1a that we generated. Table 1 shows that the binding affinity of Cy5-ghrelin to GHS-R1a was within an order of magnitude of that of wild-type ghrelin, and comparable to our previously reported value for fluorescein-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) [20]. In contrast, Cy5-des-acyl ghrelin did not show any appreciable binding to GHS-R1a.…”

“…HEK 293 cells were transiently transfected with the cDNA encoding GHS-R1a in order to visualize the binding of Cy5-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) in situ. Both wt HEK and HEK/GHS-R1a cells were plated at a density of 1 × 10 6 cells/well in a 6-well plate, and serum starved overnight.…”

“…Adult male or female C57BL/6 mice were sacrificed using CO 2 and the hearts were immediately removed and fixed in 4% paraformaldehyde (in PBS) overnight. Whole hearts were then equilibrated in 30% sucrose in PBS, snap-frozen in Tissue-Tek OCT using dry-ice cooled ethanol, and subsequently cryosectioned at 8 m. To assess binding of both acylated and non-acylated ghrelin, sections were incubated for 30 min at room temperature with various concentrations (0-20 M) of Cy5-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) or Cy5-des-acyl ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) alone. To determine the specificity of binding, sections were incubated with 10 M Cy5-ghrelin(1-19) and either 100 M ghrelin (1-28) or 100 M hexarelin.…”

“…Knockout mouse models have identified the importance of GHS-R signaling in energy homeostasis [12], feeding behavior leading to body weight control [29] and stressor-induced metabolic changes [22]. In addition to effects on whole-body metabolism, there is a growing body of evidence to support the hypothesis that ghrelin exerts effects on the cardiovascular system, and in particular, directly on cardiomyocytes.…”

“…These results indicate that GHS-R signaling is altered in cardiovascular disease, and point toward the use of ghrelin in pharmacologic therapy of heart disease. Therefore, given the importance of GHS-R signaling and expression in the heart, we are developing ghrelin analogs for the purpose of generating agents for in situ and in vivo imaging of cardiac GHS-R. We have previously designed a fluorescent ghrelin analog consisting of a fluorescein moiety attached through a lysine to the N-terminal 18 amino acids of ghrelin (fluorescein-ghrelin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)) [20] and have shown that it specifically binds to GHS-R1a in mouse heart tissue.…”