Hypothalamic FTO is associated with the regulation of energy intake not feeding reward

Abstract

Background: Polymorphism in the FTO gene is strongly associated with obesity, but little is known about the molecular bases of this relationship. We investigated whether hypothalamic FTO is involved in energy-dependent overconsumption of food. We determined FTO mRNA levels in rodent models of short- and long-term intake of palatable fat or sugar, deprivation, diet-induced increase in body weight, baseline preference for fat versus sugar as well as in same-weight animals differing in the inherent propensity to eat calories especially upon availability of diverse diets, using quantitative PCR. FTO gene expression was also studied in organotypic hypothalamic cultures treated with anorexigenic amino acid, leucine. In situ hybridization (ISH) was utilized to study FTO signal in reward- and hunger-related sites, colocalization with anorexigenic oxytocin, and c-Fos immunoreactivity in FTO cells at initiation and termination of a meal.

Results: Deprivation upregulated FTO mRNA, while leucine downregulated it. Consumption of palatable diets or macronutrient preference did not affect FTO expression. However, the propensity to ingest more energy without an effect on body weight was associated with lower FTO mRNA levels. We found that 4-fold higher number of FTO cells displayed c-Fos at meal termination as compared to initiation in the paraventricular and arcuate nuclei of re-fed mice. Moreover, ISH showed that FTO is present mainly in hunger-related sites and it shows a high degree of colocalization with anorexigenic oxytocin.

Conclusion: We conclude that FTO mRNA is present mainly in sites related to hunger/satiation control; changes in hypothalamic FTO expression are associated with cues related to energy intake rather than feeding reward. In line with that, neurons involved in feeding termination express FTO. Interestingly, baseline FTO expression appears linked not only with energy intake but also energy metabolism.

Figure 1

Relative expression of FTO and feeding-related genes in the hypothalamus of mice. A. Mice were exposed for 48 h to palatable Intralipid or sucrose solutions in addition to chow (n = 8/group). B. Mice differed in body weight by ca. 2.6 g. Increased body weight was induced by 3-week exposure to the sucrose solution given in addition to chow (n = 8/group). C. Organotypic cultures of the hypothalamus were treated for 48 h with anorexigenic leucine versus untreated controls (n = 7/group). D. Mice differed in 5-day preference for fat (Intralipid) vs. sucrose (n = 7 per preference-based group). E. Mice differed in their propensity to ingest more calories during exposure to palatable diets ("big eaters", vs. "small eaters"; n = 11 and 10, respectively). A similar rat model was used as control of FTO expression in another species (FTO rat). F. Mice were deprived of food for 16 h preceding decapitation (n = 8/group). AGRP, Agouti-related protein; DYN, dynorphin; MC4R, melanocortin receptor-4; NPY, neuropeptide Y; ORX, orexin; POMC, proopiomelanocortin; KOR, kappa opioid receptor; MOR mu receptor; MCH, melanin concentrating hormone. * - P < 0.05; ** - P < 0.01.

Figure 2

ISH detection of FTO mRNA in sites involved in the regulation of energy- or reward-related feeding. Panels A-D show sites affecting mainly reward, whereas panels E and F depict energy-related areas. aca, anterior commisure; AcbC, nucleus accumbens core; AcbSh, Acb shell; ARC, arcuate nucleus; BST, bed nucleus of the stria terminalis; BSTMA, BST medial anterior; BSTMPL, BST medial posterolateral; DMH, dorsomedial nucleus; f, fornix; fr, fasciculus retroflexus; LC, locus corelueus; LH, lateral hypothalamus; LPO, lateral preoptic area; LV, lateral ventricle; ml, medial lemniscus; otr, optic tract; PBN, parabrachial nucleus; PVN, paraventricular nucleus; SHy, septohypothalamic nucleus; SCN, suprachiasmatic nucleus; scp, superior cerebellar peduncle; SON, supraoptic nucleus; VMH, ventromedial nucleus; VTA, ventral tegmental area; 3v, 3rd ventricle; 4v, 4th ventricle; 7n, facial nerve. Scale bar: 0.4 mm (A, B) 0.5 mm (C-F).

Figure 3

Colocalization of FTO and oxytocin. Oxytocin (brown cells) and FTO mRNA (blue cells) detected in the coronal section containing the PVN. Thin arrows, neurons expressing oxytocin and FTO; Open arrows, oxytocin cells devoid of FTO. Scale bar: 0.25 mm. Inserts: three-fold higher magnification of PVN neurons of interest.

Figure 4

Fos-IR FTO neurons in the PVN and ARC at initiation and termination of feeding. c-Fos was detected by IHC, whereas FTO mRNA by ISH. A. Percentage of Fos-positive FTO cells in the PVN and ARC. B and C. Coronal sections of mice perfused at the beginning and at the end of a meal. Thin arrows, Fos-positive nuclei, thick solid arrows, Fos-positive FTO cells, open arrows, FTO neurons devoid of Fos. Scale bar: 0.02 mm. D. Schematic representation of distribution of Fos-positive FTO cells (each blue point indicates one such cell) in the representative sections encompassing the PVN and ARC. dc, dorsal cap; lm, lateral magnocellular; mm, medial magnocellular; mp, medial parvocellular.