Obesity-programmed mice are rescued by early genetic intervention

Abstract

Obesity is a chronic metabolic disorder affecting half a billion people worldwide. Major difficulties in managing obesity are the cessation of continued weight loss in patients after an initial period of responsiveness and rebound to pretreatment weight. It is conceivable that chronic weight gain unrelated to physiological needs induces an allostatic regulatory state that defends a supranormal adipose mass despite its maladaptive consequences. To challenge this hypothesis, we generated a reversible genetic mouse model of early-onset hyperphagia and severe obesity by selectively blocking the expression of the proopiomelanocortin gene (Pomc) in hypothalamic neurons. Eutopic reactivation of central POMC transmission at different stages of overweight progression normalized or greatly reduced food intake in these obesity-programmed mice. Hypothalamic Pomc rescue also attenuated comorbidities such as hyperglycemia, hyperinsulinemia, and hepatic steatosis and normalized locomotor activity. However, effectiveness of treatment to normalize body weight and adiposity declined progressively as the level of obesity at the time of Pomc induction increased. Thus, our study using a novel reversible monogenic obesity model reveals the critical importance of early intervention for the prevention of subsequent allostatic overload that auto-perpetuates obesity.

Figure 1. ArcPomc^–/– mice are obese and hyperphagic due to an impairment in hypothalamic Pomc expression.

(A) ArcPomc^– allele contains an insertion of a neomycin resistance cassette (neo), flanked by loxP sites, interrupting Pomc neuronal enhancer activity. Red oval, neuronal Pomc enhancer 1 (nPE1); asterisk, deletion of nPE2 (18); gray oval, Pomc promoter/pituitary enhancer; black rectangles, Pomc exons; arc and pit arrows, arcuate and pituitary Pomc transcription, respectively; X, inhibition of arcuate Pomc transcription. (B) Sections of arcPomc^+/– and arcPomc^–/– mice. Top two rows (POMC-EGFP): Mice transgenic for POMC-EGFP (19); upper panels show endogenous EGFP in POMC cells of coronal brain sections, and lower panels correspond to ACTH immunofluorescence (red, “POMC”) of the same slices. Arc, arcuate neurons. Third row (Pit): Pituitaries with ACTH immunofluorescent corticotrophs of the anterior lobe (AL) and melanotrophs of the intermediate lobe (IL). Fourth row (E14): Sagittal sections of E14 mice showing ACTH immunostaining. Pit, pituitary; 3v, third ventricle. Scale bars: 200 μm for lower- and 50 μm for higher-magnification photomicrographs. (C) Body weight curves of Pomc^+/+, arcPomc^+/–, and arcPomc^–/– mice. ArcPomc^–/– mice are obese (RMA, genotype × time effect: P < 0.001 for arcPomc^–/– vs. Pomc^+/+ and vs. arcPomc^+/– for both sexes). (D) Average daily food intake measured during 3 consecutive weeks at 5–6 months of age. (E) Hypothalamic Pomc mRNA expression normalized to 18S rRNA, relative to Pomc^+/+, in arbitrary units (AU). (D and E) n = 4–6 for each group. Error bars correspond to SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (OWA).

Figure 2. Arcuate Pomc rescue in the embryo normalizes food intake and body weight.

(A) The arcPomc^– line was crossed with an EIIa-Cre line to achieve germline deletion of the floxed neomycin (neo) cassette. After successive crosses, arcPomc^+/+ mice (homozygous for neo deletions but negative for the Cre transgene) were obtained. Red oval, neuronal Pomc enhancer 1 (nPE1); asterisk, deletion of nPE2 (18); gray oval, Pomc pituitary promoter/enhancer; black rectangles, Pomc exons; arc and pit arrows, arcuate and pituitary Pomc transcription, respectively; X, inhibition of arcuate Pomc transcription. (B) Body weight growth curves were completely normalized after neo removal in arcPomc^rcue/rcue mice (genotype effect: F[1,14] = 0.02, P = 0.88 for males and F[1,9] = 1.54, P = 0.25 for females with respect to arcPomc^rcue/rcue littermates; RMA). Error bars correspond to SEM. (C) Daily food intake was also normalized after neo removal in arcPomc^+/+ mice (genotype effect: F[1,14] = 0.02, P = 0.90 for males and F[1,9] = 0.52, P = 0.49 for females). Data correspond to averages of daily food intake measured during 3 consecutive weeks by the age of 5–6 months.

Figure 3. Response to Pomc rescue in mice with different initial body weights.

(A–C) Body weight curves of mice treated at different ages and magnitudes of obesity with either TAM (filled circles) or vehicle (VEH, open circles) as control. Sex and age of treatment are indicated. RMA between Pomc^+/+ and rescued arcPomc^–/–:Cre-ERT, from 12–24 weeks of age (P25 and P60) or from 31–34 weeks of age (P180), significant genotype-treatment (group) effects between Pomc^+/+ and rescued arcPomc^–/–:Cre-ERT at 12–24 weeks of age (P25 and P60) and 31–34 weeks of age (P180) are indicated. Gray bars denote the 5-day TAM or VEH treatment period. Circles represent averages of 3–8 mice. Error bars correspond to SEM. **P < 0.01, ***P < 0.001. (D) A representative P180-treated female mouse before (left) and 8 weeks after (right) treatment with TAM illustrating the marked loss of body mass. (E) Male arcPomc^–/– and arcPomc^–/–:Cre-ERT mice were weight matched to control lean mice by food restriction starting at weaning. The mice were then treated at P60 with TAM and provided chow ad libitum. Yellow circles show the combined average of 5 Pomc^+/+ and 2 Pomc^+/+:Cre-ERT mice.

Figure 4. The stage of Pomc rescue determines the magnitude of food intake improvement.

(A) Daily food intake of TAM-treated mice after body weight stabilization. (B) Changes in daily food intake before and after treatment of mice with TAM at P180. Only significant results for arcPomc^–/– and rescued arcPomc^–/–:Cre-ERT compared with Pomc^+/+ are indicated. (C) Food intake superimposed with body weight changes in a representative rescued female arcPomc^–/–:Cre-ERT mouse treated with TAM at P180. Gray bars denote the 5-day TAM treatment period. Circles and colored bars are averages of 3–8 mice. Error bars correspond to SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5. Serial measurements of metabolic parameters before and 4 weeks after TAM treatment in a cohort of female P60 mice.

(A) Body composition measured by NMR. ***P < 0.001, pairwise comparison of body fat by Bonferroni’s post hoc test. (B) Total locomotor activity in the horizontal plane measured by infrared beam breaks in the CLAMS chambers. The line graphs show average hourly activity counts over the final 24-hour period of a 72-hour continuous measurement. The dark period between 6 p.m. and 6 a.m. is indicated by the black bar below the x axis. The bar graph on the right shows the average hourly activity over the entire 12-hour dark period. ***P < 0.001, effect of TAM treatment in arcPomc^–/–:Cre-ERT mice. (C) VO₂ corrected to lean body mass (LBM) determined by indirect calorimetry in the CLAMS chambers. The line graphs show average hourly VO₂ over the final 24-hour period of a 72-hour continuous measurement. The dark period between 6 p.m. and 6 a.m. is indicated by the black bar under the x axis. The bar graph on the right shows the average hourly VO₂ over the entire 12-hour dark period. **P < 0.01, pairwise comparison by Bonferroni’s post hoc test. None of the 3 genotypes of mice showed a significant change in VO₂ after TAM treatment. All data shown are the mean ± SEM; n = 6–8 per genotype.

Figure 6. Pomc rescue restores compensatory hyperphagia induced by food deprivation.

Male and female mice treated with TAM at P180 were subjected to 24 hours of food deprivation at 33–35 weeks of age (7–9 weeks after TAM treatment). Food was then returned to the cages (refeeding period), and ad libitum intake was measured at 2, 8, 24, 48, and 72 hours after refeeding. Each bar represents average of food taken by mice of a genotype group (n = 5–7) during the indicated period, expressed as a percentage of their average individual pre-fasting daily food consumption. The corresponding raw data are shown in Supplemental Figure 4. Pre-fasting data represent average daily food intake of the same mice during 4 consecutive days before fasting. Error bars correspond to SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (OWA).