Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice

Abstract

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.

FIG. 1.

Generation and validation of Agrp^−/− mice. (a) Schematic diagrams of the murine wild-type Agrp allele, targeting vector, and mutant allele. The mutant allele arises from a double reciprocal homologous recombination between the wild-type allele and targeting vector. R, EcoRI; B, BamHI; K, KpnI; TK, thymidine kinase gene; PGKneo, PGK-driven neomycin resistance gene. (b) Southern blot analysis of BamHI-digested tail DNAs. The coding-region probe (depicted in panel a) detects an 11-kb restriction fragment from wild-type and Agrp^+/− mice but not from Agrp^−/− mice. (c) In situ hybridization with a mixture of two oligonucleotide probes to Agrp reveals AgRP mRNA in the arcuate nucleus of wild-type mice but not in the same brain region of Agrp^−/− mice.

FIG. 2.

Agrp^−/− mice exhibit normal growth rates and feeding behavior. (a) Growth curves of male and female wild-type, Agrp^+/−, and Agrp^−/− littermate mice (males: +/+, n = 17; +/−, n = 17; −/−, n = 20; females: +/+, n = 17; +/−, n = 21; −/−, n = 23). (b) Body composition of 8-month-old wild-type and Agrp^−/− mice as determined by DEXA (males: +/+, n = 12; −/−, n = 10; females: +/+, n = 10; −/−, n = 13). (c) Average daily food intake of 10-week-old male wild-type, Agrp^+/−, and Agrp^−/− littermate mice (+/+, n = 12; +/−, n = 14; −/−, n = 10). (d) Male wild-type (n = 12) and Agrp^−/− (n = 10) littermate mice were fasted for 48 h and then refed with regular chow (refeeding). Cumulative food intake following the refeeding and changes in body weight during the fasting and refeeding are shown.

FIG. 2.

Agrp^−/− mice exhibit normal growth rates and feeding behavior. (a) Growth curves of male and female wild-type, Agrp^+/−, and Agrp^−/− littermate mice (males: +/+, n = 17; +/−, n = 17; −/−, n = 20; females: +/+, n = 17; +/−, n = 21; −/−, n = 23). (b) Body composition of 8-month-old wild-type and Agrp^−/− mice as determined by DEXA (males: +/+, n = 12; −/−, n = 10; females: +/+, n = 10; −/−, n = 13). (c) Average daily food intake of 10-week-old male wild-type, Agrp^+/−, and Agrp^−/− littermate mice (+/+, n = 12; +/−, n = 14; −/−, n = 10). (d) Male wild-type (n = 12) and Agrp^−/− (n = 10) littermate mice were fasted for 48 h and then refed with regular chow (refeeding). Cumulative food intake following the refeeding and changes in body weight during the fasting and refeeding are shown.

FIG. 2.

Agrp^−/− mice exhibit normal growth rates and feeding behavior. (a) Growth curves of male and female wild-type, Agrp^+/−, and Agrp^−/− littermate mice (males: +/+, n = 17; +/−, n = 17; −/−, n = 20; females: +/+, n = 17; +/−, n = 21; −/−, n = 23). (b) Body composition of 8-month-old wild-type and Agrp^−/− mice as determined by DEXA (males: +/+, n = 12; −/−, n = 10; females: +/+, n = 10; −/−, n = 13). (c) Average daily food intake of 10-week-old male wild-type, Agrp^+/−, and Agrp^−/− littermate mice (+/+, n = 12; +/−, n = 14; −/−, n = 10). (d) Male wild-type (n = 12) and Agrp^−/− (n = 10) littermate mice were fasted for 48 h and then refed with regular chow (refeeding). Cumulative food intake following the refeeding and changes in body weight during the fasting and refeeding are shown.

FIG. 2.

Agrp^−/− mice exhibit normal growth rates and feeding behavior. (a) Growth curves of male and female wild-type, Agrp^+/−, and Agrp^−/− littermate mice (males: +/+, n = 17; +/−, n = 17; −/−, n = 20; females: +/+, n = 17; +/−, n = 21; −/−, n = 23). (b) Body composition of 8-month-old wild-type and Agrp^−/− mice as determined by DEXA (males: +/+, n = 12; −/−, n = 10; females: +/+, n = 10; −/−, n = 13). (c) Average daily food intake of 10-week-old male wild-type, Agrp^+/−, and Agrp^−/− littermate mice (+/+, n = 12; +/−, n = 14; −/−, n = 10). (d) Male wild-type (n = 12) and Agrp^−/− (n = 10) littermate mice were fasted for 48 h and then refed with regular chow (refeeding). Cumulative food intake following the refeeding and changes in body weight during the fasting and refeeding are shown.

FIG. 3.

Agrp^−/− mice exhibit normal susceptibility to diet-induced obesity. (a) Male Agrp^−/− mice and wild-type littermates were given equal access to regular chow (Chow) and a high-fat diet (HF). Agrp^−/− and wild-type mice exhibited comparable preferences and consumption of the high-fat diet during a 48-h exposure. (b) Both wild-type and Agrp^−/− male littermates gained more body weight after 10 weeks of maintenance on a high-fat diet (HF) than after 10 weeks of maintenance on chow (Chow) (HF groups: +/+, n = 10; −/−, n = 10) (Chow groups: +/+, n = 17; −/−, n = 20). ∗, P < 0.05 versus corresponding chow-fed groups at week 15.

FIG. 4.

Agrp^−/− mice exhibit normal responses to exogenous leptin and NPY. (a) Twice daily intraperitoneal dosing of 1.5 mg of leptin/kg on days 7 and 8 suppressed food consumption to similar extents in both male Agrp^−/− (n = 10) and wild-type (n = 12) littermate mice, relative to that observed with vehicle injections on days 1 to 6. On days 8 and 9 the daily food intake of both Agrp^−/− and wild-type mice was significantly lower than their corresponding average daily food intake from days 1 to 7. ∗, P < 0.005. (b) Injection of 0.25, 0.5, and 1.0 μg of NPY into the dorsal third cerebroventricle of male Agrp^−/− (n = 6 to 7) and wild-type (n = 9 to 12) littermate mice elicited similar dose-dependent increases in food intake. For both genotypes, all three doses of NPY significantly (P < 0.05) stimulated 2-h food intake relative to aCSF treatment.

FIG. 5.

Expression levels of NPY and MCH mRNAs in the hypothalamus of the wild-type and Agrp^−/− littermate mice (n = 5 per genotype). ∗, P < 0.05 versus wild-type mice. ARC, arcuate nucleus.

FIG. 6.

Growth curves of Agrp^−/−;Npy^−/− mice. (a) Growth curves of male and female wild-type (wtwt), Agrp^+/+;Npy^−/− (wtko), and Agrp^−/−;Npy^−/− (koko) mice (females, n = 9 to 11 per genotype; males, n = 10 per genotype). All mice were 3 months of age at the beginning of the study. (b) Cumulative food consumption of mice from panel a over the 7-week duration of the study.

FIG. 7.

Agrp^−/−;Npy^−/− mice display a normal hyperphagic response to fasting. Six- to 7-month-old female wild-type (wtwt), Agrp^+/+;Npy^−/− (wtko), and Agrp^−/−;Npy^−/− (koko) mice (n = 14 per genotype) were fasted for 48 h and then refed with regular chow. Cumulative food intake following refeeding and changes in body weight during the fasting and refeeding are shown.