Differential expression of feeding-related genes in <i>Nbea+/−</i> and WT mice under different feeding paradigms.

<p><i>Ad libitum</i>-fed, sated <i>Nbea</i>+/− and WT mice showed a differential expression of the dynorphin (DYN) mRNA in the hypothalamus <i>(A)</i> and of the melanocortin (MC) receptor-3 mRNA in the brainstem <i>(B)</i>. 16-h food deprivation differently affected expression of mRNAs encoding DYN, proopiomelanocortin (POMC), opioid-like receptor-1 (ORL1) and corticotropin releasing hormone (CRH) in the hypothalamus <i>(A)</i>, whereas no differences between the genotypes were triggered by food deprivation in the brainstem <i>(B)</i>. Exposure to the palatable HFHS diet did not affect gene expression differently in the <i>Nbea</i>+/− and WT animals. Only DYN mRNA was increased in the standard chow-fed controls <i>(C)</i>. * P<0.05; error bars, ± SEM. The following other genes were also analyzed, but their expression levels did not differ between the genotypes: AGRP, Agouti-related protein; AVP, vasopressin; CCK, cholecystokinin; CRHR, CRH receptor; DOR, delta opioid receptor; ENK, enkephalin; GHSR, growth hormone secretagogue receptor; GLP1R1, glucagon-like peptide 1 receptor 1; KOR, kappa opioid receptor; MCH, melanin concentrating hormone; MOR, mu opioid receptor; NPY, neuropeptide Y; ORX, orexin.</p

Association with BMI and body weight as continuous traits in adults and children for <i>NBEA</i> rs17775456 and rs7990537.

<p>Beta indicates transformed beta-values. P indicates p-values adjusted for significant covariates.</p

Differential feeding behavior of <i>Nbea+/−</i> and WT mice.

<p>Male mice were tested at the age of 8–10 weeks, prior to the manifestation of a significant body weight difference between the genotypes. Compared to WT mice, <i>Nbea</i>+/− mice <i>(A)</i> ate more <i>ad libitum</i> of the energy-dense and palatable high-fat high-sugar (HFHS) solid diet; <i>(B)</i> ate more standard chow upon 2-h refeeding after overnight food deprivation; <i>(C)</i> consumed more of the caloric and palatable 4.1% Intralipid, 10% sucrose, 10% glucose and 10% fructose solutions; but <i>(D)</i> they consumed the same amounts of tastants which do not contain calories, i.e. saline, 0.1% saccharin or 0.05% sucralose, despite their palatability. <i>Nbea</i>+/− mice <i>(E)</i> were not resistant to leptin, but they were <i>(F)</i> more sensitive to the anorexigenic naltrexone (NTX) than their WT counterparts. *, P<0.05; **, P<0.01; ***, P<0.001; error bars, ± SEM.</p

Odds ratio for overweight and obesity depending on <i>NBEA</i> genotypes in adults and children.

<p>Data are number of subjects in each group and number of subjects for each genotype (G) (% in each group). Allele frequency (A) for each group is given in percentage. GRR indicate genotype relative risk with a 95% confidence interval (CI). Odds ratio (OR) with a 95% confidence interval (CI) was calculated assuming an additive model. Association with overweight in the adult cohort was determined comparing subjects with normal weight (BMI<25 kg/m²) and overweight (BMI≥25 kg/m²). P indicates p-values adjusted for age and gender.</p

First-line phenotyping in the German Mouse Clinic: Body composition (DXA) and energy assimilation parameters of 18–20 week-old WT and <i>Nbea</i>+/− mice, fed with standard chow <i>ad libitum</i> over 5 days (n = 7/group for energy metabolism, n = 15/group for DXA; means±SD).

*<p>Fat and lean mass, food intake and daily metabolized energy were analyzed using a linear model including body mass as covariate.</p>**<p>Feces production was analyzed using a linear model including food intake as covariate to adjust for differences in overall food consumption.</p

Nbea-haploinsufficient mice develop higher body weight due to higher adipose tissue mass.

<p>(A) Western blot analysis of whole brain demonstrates that Nbea protein expression in <i>Nbea</i>+/− mice is half of that in WT mice. The same blot was sequentially developed with anti-Nbea, anti-Lrba and anti-Cadherin. 1, ½ and ¼ indicate a dilution series of loaded protein. (B) Male and female <i>Nbea</i>+/− mice develop higher body mass than WT controls. From week 14 on we continuously detected a significant genotype effect on body mass in males and females combined (week 14–15 p<0.05, week 16–19 p<0.01, afterwards p<0.001). (C,D) qNMR scans of mice aged 6–22 weeks shows that increased body mass of <i>Nbea</i>+/− mice is caused by increased fat tissue mass (significant genotype effect in females between week 6–21, in males in week 13–16, linear model with body mass as covariate). (E) The in-out-difference between daily metabolizable energy and daily energy expenditure was significantly increased in <i>Nbea</i>+/− mice at the age of 8 weeks. (F) Plasma insulin and <i>(G)</i> leptin at 22 weeks of age were significantly increased in <i>Nbea</i>+/− mice. (H,I) High-fat feeding from age 14 weeks accelerates weight gain, more pronouncedly in <i>Nbea</i>+/− than in WT mice. * within males, ⁺within females, P<0.05; ** within males, ⁺⁺within females, P<0.01; *** within males, ⁺⁺⁺within females, P<0.001; error bars, ± SEM. In part <i>H</i>, all genotype differences were significant for males with at least P<0.05 (except weeks −2 and 7, n.s.) and for females with at least P<0.001 (except week −2, p<0.01).</p