Neuronal Pomc expression and body weight are synergistically regulated by distal upstream enhancers nPE1 and nPE2

LAM D; OTERO-CORCHON V; DE SOUZA FS; MEECE K; WARDLAW S; RUBINSTEIN M; LOW MJ

Boston

Congreso; 93rd Annual Meeting of the Endocrine Society (ENDO 2011); 2011

The Endocrine Society

Products of the proopiomelanocortin (Pomc) gene play an important role in regulating body weight, and disruption of Pomc function results in severe obesity. Previously, we identified distal upstream enhancers of the Pomc gene, called neuronal Pomc enhancers 1 and 2 (nPE1 and nPE2). These enhancers appear to specifically control Pomc transcription in the arcuate nucleus of the hypothalamus (ARC), since each enhancer drives transgene expression to Pomc cells in the ARC but not the pituitary. Moreover, deletion of both enhancers disrupts ARC but not pituitary transgene expression. To probe the functional importance of the enhancers, we generated and characterized mice bearing specific homozygous deletions of nPE1 (delta1 mice), nPE2 (delta2 mice), or both (delta1-delta2 mice). The nPE-deficient mice were generated by Cre-mediated excision of a LoxP-flanked neomycin selection cassette (Fneo) at the respective enhancer site. Prior to excision of the neomycin cassette, Fneo delta1-delta2 mice had drastic reductions in Pomc transcription (<0.4% of wild-type ARC Pomc mRNA levels) and accordingly developed severe obesity. Deletion of nPE2 did not substantially affect ARC Pomc transcription as measured by qRT-PCR of dissected hypothalamus. In contrast, delta1 mice retained only 20-25% of wild-type Pomc mRNA levels and 25-30%of wild-type levels of Pomc-derived peptide alpha-melanocyte stimulating hormone (alpha-MSH) measured by RIA. delta1-delta2 mice retained just 2-3% of wild-type ARC Pomc mRNA levels and 10-15% of wild-type alpha-MSH levels, indicating that the two enhancers may interact synergistically. Anterior pituitary Pomc mRNA levels were more than two-fold higher in delta1-delta2 mice than wild-types, possibly due to lack of indirect inhibitory effects of ARC Pomc products on pituitary Pomc expression. The phenotype of nPE-deficient mice closely reflected the extent of Pomc disruption. delta2 mice showed no discernable altered phenotype, delta1 mice showed moderate obesity, and delta1-delta2 mice showed severe obesity approximating that of Fneo delta1-delta 2 mice. Both male and female delta1-delta2 and Fneo delta1-delta2 mice displayed fasting hyperglycemia, but only females were glucose intolerant. delta1 mice exhibited normal glucose homeostasis. In summary, our data show that two distal enhancers play a synergistic role in controlling transcription of Pomc in ARC neurons but not in pituitary cells. Disruption of the enhancers causes a severe obesity phenotype, indicating that these enhancers are critical for the normal regulation of energy balance.