Preference for a high fat diet, but not hyperphagia following activation of mu opioid receptors is blocked in AgRP knockout mice

Abstract

Activation of mu opioid receptors (MOR) makes animals hyperphagic and selectively increases their preference for a high fat diet independent of their dietary preference. The orexigenic peptide Agouti Related Peptide (AgRP) also produces hyperphagia and increased the preference for a high fat diet. In this paper, we tested the hypothesis that the effect of MOR on feeding behavior will be attenuated in the absence of the orexigenic peptide AgRP. Immunohistochemical studies demonstrated that MOR are co-localized on AgRP neurons located in the arcuate nucleus. This finding is consistent with a role of MOR in mediating the release of AgRP. Our data also demonstrated that the wild-type (FVB) animals preferred a diet high in fat whereas the AgRP knockout (AgRP KO) mice did not. mRNA expression of MOR in the hypothalamus was not significantly different between AgRP KO mice and their wild-type control. In a dose-response experiment, the low dose (0.025 microg) of a MOR agonist, DAMGO, increased cumulative food intake in wild-type and AgRP KO mice. The low and middle (0.25 microg) dose of DAMGO significantly increased the amount of high fat diet eaten by the wild-type animals, but did not significantly change the amount of high fat diet eaten by the AgRP KO mice. The highest dose of DAMGO (2.5 microg) reduced food intake in the control and AgRP KO mice, probably due to somnolence. These data demonstrate that the increased preference for a high fat diet after stimulation of MOR is attenuated in the absence of AgRP, but the increase in food intake (i.e. hyperphagia) is not.

Figure 1A, 1B, 1C & 1D

Figure 1A is a representation of the area of the brain (medial arcuate nucleus; Bregma -3.30mm) that was used to identify the mu opioid receptors from FVB mice. Figure 1B, 1C and 1D are high power magnification (40×) of the arcuate nucleus of FVB mice on a chow diet. Magnification for the three micrographs is indicated by the 40μm scale in Panel D. Panel B is a neuron positively immunostained for mu opioid receptors. Panel C is a representative of a neurons that is immunostained for Agouti gene Related Peptide (AgRP). Panel D is an overlay of Panel B and Panel C which demonstrate that mu opioid receptors are located on AgRP neurons within the arcuate nucleus.

Figure 1A, 1B, 1C & 1D

Figure 1A is a representation of the area of the brain (medial arcuate nucleus; Bregma -3.30mm) that was used to identify the mu opioid receptors from FVB mice. Figure 1B, 1C and 1D are high power magnification (40×) of the arcuate nucleus of FVB mice on a chow diet. Magnification for the three micrographs is indicated by the 40μm scale in Panel D. Panel B is a neuron positively immunostained for mu opioid receptors. Panel C is a representative of a neurons that is immunostained for Agouti gene Related Peptide (AgRP). Panel D is an overlay of Panel B and Panel C which demonstrate that mu opioid receptors are located on AgRP neurons within the arcuate nucleus.

Figure 1A, 1B, 1C & 1D

Figure 1A is a representation of the area of the brain (medial arcuate nucleus; Bregma -3.30mm) that was used to identify the mu opioid receptors from FVB mice. Figure 1B, 1C and 1D are high power magnification (40×) of the arcuate nucleus of FVB mice on a chow diet. Magnification for the three micrographs is indicated by the 40μm scale in Panel D. Panel B is a neuron positively immunostained for mu opioid receptors. Panel C is a representative of a neurons that is immunostained for Agouti gene Related Peptide (AgRP). Panel D is an overlay of Panel B and Panel C which demonstrate that mu opioid receptors are located on AgRP neurons within the arcuate nucleus.

Figure 1A, 1B, 1C & 1D

Figure 1A is a representation of the area of the brain (medial arcuate nucleus; Bregma -3.30mm) that was used to identify the mu opioid receptors from FVB mice. Figure 1B, 1C and 1D are high power magnification (40×) of the arcuate nucleus of FVB mice on a chow diet. Magnification for the three micrographs is indicated by the 40μm scale in Panel D. Panel B is a neuron positively immunostained for mu opioid receptors. Panel C is a representative of a neurons that is immunostained for Agouti gene Related Peptide (AgRP). Panel D is an overlay of Panel B and Panel C which demonstrate that mu opioid receptors are located on AgRP neurons within the arcuate nucleus.

Figure 2

Hypothalamic mRNA expression of AgRP (Figure 2A) and MOR (Figure 2B). The mRNA expression of AgRP was not detectable in the knockout mice. The expression of MOR was similar between the wild-type control and AgRP knockout mice (p>0.05).

Figure 3

Dietary preference of wild-type control (3A) and AgRP knockout mice (3B). Preference is represented as a percentage of total food intake. Control animals preferred to eat significantly more of the high fat diet when given a choice between low fat and high fat diets simultaneously. In contrast, there was no preference for either diet with the AgRP knockout animals. Values are expressed as means ± SE; *p ≤ 0.05 vs. low fat.

Figure 4

Figure 4A. Cumulative food intake of wild-type control mice after icv injection of saline, 0.025, 0.25 or 2.5μg of DAMGO. Food intake is represented in grams. DAMGO dose of 0.025μg significantly increased cumulative food intake. The highest dose of DAMGO 2.5μg significantly decreased food intake, probably due to somnolence. Values are expressed as means ± SE; *p ≤ 0.05 vs saline. Figure 4B. The effect of saline, 0.025, 0.25, or 2.5μg of DAMGO on dietary selection (i.e. low fat or high fat diet) with FVB control mice. Food intake is presented in grams. The low (0.025μg) and high (2.5μg) dose of DAMGO significantly increased the grams of high fat diet consumed when compared to the low fat diet. The highest dose of DAMGO did not have any effect on dietary selection. Values are expressed as mean ± SEM. *p ≤ 0.05 vs low fat diet.

Figure 5

Figure 5A. Cumulative food intake of AgRP knockout mice after icv injection of saline, 0.025, 0.25 or 2.5μg of DAMGO. Food intake is represented in grams. DAMGO dose of 0.025μg significantly increased cumulative food intake. The highest dose of DAMGO 2.5μg significantly decreased food intake, probably due to somnolence. Values are expressed as means ± SE; *p < 0.05 vs saline. Figure 5B. The effect of saline, 0.025, 0.25, or 2.5μg of DAMGO on dietary selection (i.e. low fat or high fat diet) with AgRP knockout mice. Food intake is presented in grams. Neither treatment was able to significantly increase the preference for either diet. Values are expressed as mean ± SEM. *p > 0.05 vs low fat diet.