Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.

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Proopiomelanocortin (POMC) is posttranslationally processed to several peptides including α-MSH, a primary regulator of energy balance that inhibits food intake and stimulates energy expenditure. However, another POMC-derived peptide, β-endorphin (β-EP), has been shown to stimulate food intake. In this study we examined the effects of intracerebroventricular (icv) β-EP on food intake and its ability to antagonize the negative effects of α-MSH on energy balance in male rats. A single icv injection of β-EP stimulated food intake over a 2- to 6-h period during both the light and dark cycles. This effect was, however, not sustained with chronic icv β-EP infusion. In the next study, a subthreshold dose of β-EP was injected together with Nle(4), d-Phe(7) (NDP)-MSH after a 16-h fast, and the negative effects of NDP-MSH on refeeding and body weight gain were partially reversed. Finally, peptide interactions were studied in a chronic icv infusion model. Weight gain and food intake were significantly suppressed in the NDP-MSH group during the entire study. A subthreshold dose of β-EP antagonized these suppressive effects on food intake and weight gain for the first 3 d. However on d 4-7, β-EP no longer blocked these effects. Of note, the stimulatory effect of β-EP on feeding and its ability to antagonize MSH were specific for β-EP(1-31) and were not observed with β-EP(1-27). This study highlights the importance of understanding how the balance between α-MSH and β-EP is maintained and the potential role of differential POMC processing in regulating energy balance.

Expanding upon previous observations of high POMC in EAS, this study specifically demonstrates elevated POMC levels can identify occult ectopic tumors. Elevations in AgRP also favor the diagnosis of EAS, suggesting AgRP should be further evaluated as a potential neuroendocrine tumor marker.

These data demonstrate that despite peripheral hyperleptinemia, positive energy balance is achieved during pregnancy by a relative decrease in central leptin concentrations and resistance to leptin's effects on target neuropeptides that regulate energy balance.