Deletion of ghrelin impairs neither growth nor appetite

Abstract

Pharmacological studies show that ghrelin stimulates growth hormone release, appetite, and fat deposition, but ghrelin's physiological role in energy homeostasis has not been established. Ghrelin was also proposed to regulate leptin and insulin release and to be important for the normal function of stomach, heart, kidney, lung, testis, and placenta. To help determine a definable physiological role for ghrelin, we generated ghrelin-null mice. In contrast to predictions made from the pharmacology of ghrelin, ghrelin-null mice are not anorexic dwarfs; their size, growth rate, food intake, body composition, reproduction, gross behavior, and tissue pathology are indistinguishable from wild-type littermates. Fasting produces identical decreases in serum leptin and insulin in null and wild-type mice. Ghrelin-null mice display normal responses to starvation and diet-induced obesity. As in wild-type mice, the administration of exogenous ghrelin stimulates appetite in null mice. Our data show that ghrelin is not critically required for viability, fertility, growth, appetite, bone density, and fat deposition and not likely to be a direct regulator of leptin and insulin. Therefore, antagonists of ghrelin are unlikely to have broad utility as antiobesity agents.

FIG. 1.

(A) Schematic diagram of the derivation of ghrelin^−/− mice by homologous recombination. The wild-type allele is shown at the top, and five exons are indicated by a vertical line or rectangular boxes. The targeting construct is shown below. (B) Southern blot analysis of DNA from mice derived from mating heterozygotes.

FIG. 2.

(A) RT-PCR analysis of ghrelin and lacZ mRNA expression. Lanes: S, stomach; B, brain. (B) Immunofluorescence analysis of ghrelin and expression of β-galactosidase activity in the stomach of 8-week-old mice. Magnification, ×8.

FIG. 3.

Bone density (A) and body composition (percent fat) (B) in 8-week-old mice (n = 6, P > 0.05 [ghrelin^+/+ versus ghrelin^−/− mice]).

FIG. 4.

ghrelin^−/− mice exhibit normal growth rate and normal food intake. (A) Body weight; (B) cumulative food intake. Mice were 16 weeks old at the beginning of the study, and data were collected every other week (n = 8, P > 0.05 [ghrelin^+/+ versus ghrelin^−/− mice]).

FIG. 5.

Changes in body weight (A) and food intake (B) during fasting and refeeding. Twenty-week-old mice were fasted for 24 h and then allowed to eat. Body weight was measured before and after fasting and at 24 and 48 h after food was given. Cumulative food intake was measured 2, 4, 6, 24, and 48 h after food was given. The results were plotted according to the mean of each datum point (n = 5, P > 0.05 (ghrelin^+/+ versus ghrelin^−/− mice]).

FIG. 6.

ghrelin^−/− mice exhibit a normal orexigenic response to exogenous ghrelin. Cumulative food intake was measured in 12-week-old mice every 30 min for the duration of the experiment. Saline alone (100 μl) or saline containing 10 μg of ghrelin (Phoenix Pharmaceuticals, Inc.) was injected intraperitoneally into each mouse. Mice were first treated with saline and then 0.5 and 1.5 h later with saline containing ghrelin. For each gender group (n = 5), P < 0.05 (✽) in males and P < 0.001 in females (✽✽) (saline versus ghrelin).

FIG. 7.

Serum leptin and insulin concentrations in 18-week-old fed or fasted male mice (n = 5). Blood was collected between 8 and 9 a.m. Mice were fasted for 48 h, which produced a fall in leptin and insulin in each genotype (✽, P < 0.05 [fed versus fasted] for both ghrelin^+/+ and ghrelin^−/− mice). There was no significant difference in the levels of leptin and insulin between each genotype whether they were fed or fasted (P > 0.05 [ghrelin^+/+ versus ghrelin^−/− mice]).

FIG. 8.

ghrelin^−/− mice exhibit normal susceptibility to diet-induced obesity. Twenty-week-old mice were fed either a high-fat (A) or a high-protein (B) diet for 10 weeks. M, male (n = 7); F, female (n = 7). Body weight, P > 0.05 for +/+ versus −/− animals of the same sex on each diet. (C) Body composition after 10 weeks on normal, high-fat, and high-protein diets. Males, **, P < 0.001 high-fat versus normal and high-protein diets; females, *, P < 0.05 high-fat versus normal and high-protein diets. P > 0.05 for +/+ versus −/− animals within each diet.