Evidence for possible period 2 gene mediation of the effects of alcohol exposure during the postnatal period on genes associated with maintaining metabolic signaling in the mouse hypothalamus.

Numerous studies have reported health and disease related problems in offspring with fetal alcohol spectrum disorders (FASD). Indeed, offspring exposed to alcohol during fetal development have problems ranging from stress disorders (Hellemans et al., 2010; Schneider et al., 2011), altered metabolic functioning (Chen et al., 2003), impairment in the immune response (Arjona et al, 2006), and disruptions in circadian rhythms (Chen et al., 2006; Handa et al., 2007). A critical component for regulation of stress, metabolic and immune functions is the proopiomelanocortin gene (POMC) (Boyadjieva et al., 2009; Sarkar et al., 2008), which has been shown to be a target of alcohol and clock genes (Agapito et al., 2010; Chen et al., 2006). Therefore, the resulting phenotypes of altered stress and metabolic responses due to developmental alcohol exposure may be in part due to effects on POMC producing neurons in the hypothalamus. Once transcribed, the POMC gene becomes a precursor for several bioactive peptides by posttranslational processing, including β-endorphin, adrenocorticotrophin (ACTH) and α-, β, and γ-melanocyte stimulating hormones (MSH), which are involved in regulation of food intake, metabolism, stress response (Millington, 2007) and immune regulation (Boyadjieva et al., 2006). POMC gene expression abnormalities are associated with obesity, hyperphagia, diabetes (Baker et al., 2005; Chen et al., 2005; Mizuno et al., 2003) and cancer (Sarkar et al., 2008). Thus POMC neurons are a key component regulating the metabolic signaling in the brain. Within the hypothalamus, several key genes, including signal transducer and activator of transcription 3 (Stat3), ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing 4 (Asb4), sirtuin 1 (Sirt1), and peroxisome proliferator-activated receptor gamma coactivator1α (Pgc1α), are associated with POMC function and play regulatory roles in metabolism. For example, Stat3 regulates POMC gene expression (Xu et al., 2007), and the mutation of this gene is associated with a severe obesity phenotype (Gao et al., 2004). Sirt1 is another key metabolic signaling gene that orchestrates adaptation to changing metabolic states in peripheral tissues (Cohen et al., 2004). Recently, Sirt1 has been found to localize in the arcuate nucleus of the hypothalamus and colocalizes with POMC producing neurons (Ramadori et al., 2008). In the liver both Sirt1 and Pgc1α peptides play a role to mediate the NAD+ mechanism. Interestingly, Pgc1α also colocalizes in POMC neurons, however the role that it plays in these neurons is unknown. Furthermore, gene expression levels of Sirt1 and Pgc1α are altered by alcohol exposure in the livers of adult rats (Lieber et al., 2008). Little is known about the function of Asb4, however, recent literature has indicated that Asb4 is expressed in the hypothalamic areas typically specific to regulating metabolic function. In POMC producing hypothalamic neurons, food intake regulates the gene expression level of Asb4 (Li et al., 2007), thus indicating its importance in metabolic sensing in the brain. Metabolic genes, in particular POMC and Sirt1, are expressed in a circadian manner, and are involved in core molecular clock function. (Chen et al., 2006; Grimaldi et al., 2009). Recently, clock genes have been connected to formation and progression of many diseases related to metabolic disorders (Ando et al., 2009; Bass et al., 2010). Clock genes, such as negative regulators, Period (Per1,2,3), and Cryptochrome (Cry1,2), and positive regulators, Clock and Bmal1, act in two tightly coupled transcriptional and translational feedback loops that are able to self-sustain a circadian rhythm (Ko and Takahashi, 2006). Interestingly, it appears that Per2 gene is a putative target of alcohol and may be linked to metabolic disease. Initial support for this notion stems from the evidence showing that alcohol exposure in adulthood or during fetal development alters the circadian expression of Per genes in the hypothalamus and peripheral tissues (Arjona et al, 2006; Chen et al., 2006). Additionally, Per2 gene mutant (Per2Brdm1) mice display enhanced alcohol consumption and preference (Spanagel et al., 2005), whereas Per1Brdm1 mutant mice do not show such an enhancement in alcohol drinking behavior (Zghoul et al., 2007). Evidence has shown, alcoholics with a specific set of polymorphisms in the Per2 gene consume less alcohol than alcoholics without the polymorphisms (Spanagel et al., 2005). Furthermore, under a metabolic disease state, the expression of Per genes is altered in peripheral tissues (Garaulet et al., 2010). However, there are no data available that connect Per genes in the mediation of ethanol’s programming of POMC-regulated metabolic functions. Therefore, we sought to determine whether postnatal alcohol exposure altered the expression levels of key metabolic genes in the hypothalamus of adult male and female mice.