Energy balance includes energy intake and energy expenditure. Either excessive food intake or insufficient physical activity will increase the body mass and cause obesity, a worldwide health problem. In the US, more than two-thirds of people are obesity or overweight. Conversely, it is well accepted that reducing energy intake can increase the life span and the resistance to age-related diseases. MicroRNAs are highly conserved non-coding RNA molecules with a length of 21-23 nucleotides. Recent studies show that numerous microRNAs are associated with the regulation of oxidative stress, inflammation, insulin signaling, apoptosis, and angiogenesis that relate to obesity. However, the role of microRNAs in the regulation of energy balance in central nervous system remains unknown, especially within the hypothalamus, a primary site of energy balance control. In this project, microRNA, and mRNA were profiled using microarray technology. Furthermore, quantitative proteomics were used to identify differential protein levels during fasting, and in a genetically obese mouse model, Mice were given either a 24-hour fast, or ad libitum access to food. Hypothalamic RNA and microRNA samples were analyzed by microarray, using both the Affymetrix and Toray 3D mRNA and microRNA platforms. No microRNAs were found to be differentially expressed between two treatments, whereas numerous mRNAs were significantly regulated by fasting, including 7 cell cycle related genes. Hypothalamic protein samples from WT and N2KO mice treated either to ad lib feeding or 24-hour fasting were analyzed by MSE quantitative proteomics. Over 650 proteins were identified with some proteins showing significantly different abundances between or among the four groups. Between ad lib fed WT and N2KO mice, 53 proteins were differentially expressed, with some of these linked to neurodegeneration, NAD synthesis, and the citrate acid cycle (TCA). Overall, the results of this study suggest that while microRNA-mediated mechanisms are not significant modulators of hypothalamic gene expression upon a 24 hour fast, cell cycle gene expression changes represent a major contributor to the fasting response. Moreover, Nlhl2 might play an important role in the neurodegeneration and mitochondrial metabolism. / Ph. D.

Prader-Willi Syndrome (PWS) is a genetic disease that results in abnormal hormone levels, developmental delay, intellectual disability, hypogonadism, and excessive appetite. The disease is caused by a de novo genetic deletion in chromosome 15. While many of the deleted genes have been identified, there is little known about their molecular function. There is evidence that a cluster of non-coding RNA genes in the deleted region known as the SNORD116 genes may be the most critical genes deleted in Prader-Willi Syndrome. It is unknown what the SNORD116 genes do at the molecular level, but recent evidence suggests they regulate the expression of other genes involved in the neuroendocrine system. Specifically, the SNORD116 gene is implicated in regulation of NHLH2, a transcription factor gene which plays a key role in development, hormonal regulation, and body weight. In this study we identify phylogenetically conserved regions of SNORD116 and predict interactions with its potential downstream RNA targets. We show that mouse Snord116 post-transcriptionally increases Nhlh2 RNA levels dependent on its 3'UTR and protects it from degradation within 45 minutes of its transcription. Additionally, a single nucleotide variant within Nhlh2 at the predicted Snord116 interaction site may disrupt Snord116's protective effect. This is the first observation of a molecular mechanism for Snord116, identifying its role in RNA stability, and leads us closer to understanding Prader-Willi Syndrome and finding a possible treatment. However, Snord116 in vitro knockdown or paternally inherited in vivo deletion fail to detect differential expression of Nhlh2, likely due to missing the key timepoint of Snord116 regulatory effects on Nhlh2 RNA soon after its transcriptional stimulation, and dependent on leptin signals. Furthermore, the hypothalamic mRNA expression profile of PWS mouse models fed a nutraceutical dietary supplement of conjugated linoleic acid reveals minimal overall changes, while the effect of diet may be stronger than genotype and potentially changes gene expression of metabolic molecular pathways. / Doctor of Philosophy / Prader-Willi Syndrome is a genetic disease that results in abnormal hormone levels, slow development, intellectual disability, gonad deficiency, and excessive appetite. The disease is caused by a genetic deletion in chromosome 15 that is almost always a spontaneous mutation not inherited from the parents. While many of the deleted genes have been identified, there is little known about what their molecular function is. There is evidence that a cluster of genes in the deleted region known as the SNORD116 genes may be the most critical genes deleted in Prader-Willi Syndrome. It is unknown what the SNORD116 genes do at the molecular level, but recent evidence suggests that it regulates other genes involved in the hormone system. Specifically, the SNORD116 gene is implicated to regulate the levels of NHLH2, a gene which plays a key role in development, hormonal regulation, and body weight. In this study we identify key regions of SNORD116 and predict interactions with its potential downstream targets. We show that SNORD116 increases NHLH2 levels and slows its degradation at the RNA transcript level. This is the first observation of a molecular mechanism for SNORD116 and leads us closer to understanding Prader-Willi Syndrome and finding a possible treatment. However, other mouse models of Snord116 deletion fail to find differences in Nhlh2. This is likely due to missing a brief key timepoint and hormonal signal when Nhlh2 is most subject to Snord116's effects. Furthermore, PWS mouse models fed a supplement intended for weight loss leads to mild overall gene expression changes in the hypothalamus, a brain region that regulates many hormonal signals including appetite and energy balance. The effect of diet may be stronger than genotype in this brain region, with diet potentially changing the activity of metabolic molecular pathways.

Mutations in the melanocortin 4 receptor (MC4R) are the most frequent cause of monogenetic forms of human obesity. Despite its importance, the MC4R signaling pathways and transcriptional regulation that underly the melanocortin pathway are far from being fully understood. The transcription factor Nescient Helix Loop Helix 2 (Nhlh2), is known to influence the melanocortin pathway. It regulates the transcription of genes by binding to the E-Box binding sites present in the promoter region. Here in this dissertation, Nhlh2's role as a transcriptional regulator of Mc4r and the effects of deletion of Nhlh2 on peripheral energy expenditure, glucose homeostasis and fatty acid oxidation are reported. To investigate the transcriptional mechanisms of Mc4r and the involvement of Nhlh2, gene expression analysis, DNA-protein binding, transactivation assays, and SiRNA induction were used. We show that Nhlh2 regulates the transcription of Mc4r by binding to the three E-Boxes present on the promoter at -553, -361 and +47. Further, SiRNA knockdown of Nhlh2 in the N29/2 cell line depresses Mc4r expression which suggests the requirement of Nhlh2 for Mc4r transcription. Development of adult onset obesity in the absence of evident hyperphagia questions the ability of mice which lacks Nhlh2 (N2KO) to utilize energy substrate efficiently. To test the effect of deletion of Nhlh2 in N2KO, body composition analysis, tissue specific characterization, fatty acid oxidation and glucose and insulin homeostasis were assessed. N2KO mice have a higher fat content than WT at the age of 12 weeks. There are architectural differences in adipose tissue of N2KO. White adipose tissue (WAT) shows infiltration of macrophages, and increased mRNA and serum levels of interleukin 6 which suggests the presence of a systemic inflammatory state in the N2KO mice. Sympathetic nervous system tone is reduced in both brown adipose tissue (BAT) and WAT, as evidenced by gene expression analysis, and this may be because of overall reduced melanocortinergic tone in N2KO mice. N2KO mice have an impaired glucose tolerance on the basis of their late glucose clearance on glucose (non-significant) and insulin (significant) challenges. Fatty acid oxidation (FAO) is higher in red fibers of skeletal muscle, and the respiratory exchange ratio (RER) is lower in N2KO, which is indicative of using fat as a preferential energy source. Increased expression of genes involved in the lipid metabolism in skeletal muscle and liver supports the RER and FAO, and are indicative of high turnover of lipids in N2KO. Findings from these studies implicate Nhlh2 as a transcriptional regulator of Mc4r which has a direct relevance to the ever increasing epidemic of obesity. Characterization of N2KO mice sheds light on the adult onset obesity phenotype. Knowledge gained from these findings will help us understand the monogenetic form of obesity more completely and could lead to the design of improved pharmacological therapies that target Nhlh2 or Mc4r or modify physical activity behavior. / Ph. D.

Nescient Helix Loop Helix 2 (Nhlh2) is a member of the basic helix-loop-helix transcription factor family. Mice with a targeted deletion of Nhlh2, called N2KO mice, show adult onset obesity in both males and females. Nhlh2 regulates other genes by binding to the E-box in the promoter region of these genes. This transcription factor regulates many other transcription factors including MC4R and PC1/3 which are associated with human obesity. The Nhlh2 promoter has been analyzed for putative transcription factors binding sites. These putative binding sites have been tested to be the regulators of Nhlh2 by transactivation assays with mutant promoters, Electrophoretic Shift Assay (EMSA), and Chromatin Immunoprecipitation Assay (ChIP) as methods to investigate the DNA-protein binding. The results of these experiments showed that the Nhlh2 promoter has five Signal Transducer and Activator of Transcription 3 (Stat3) binding site motifs at -47, -65, -80, -281, -294 and two Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB) binding site motifs at -67 and -135. While NFκB acts as a negative regulator of Nhlh2, this research showed that Stat3 acts as a regulator for the Nhlh2 basal expression and leptin stimulation. The ChIP assay using chromatin from mouse hypothalamus and antibodies against Stat3 and the NFκB subunits P50, P65, and c-Rel demonstrated that all of these antibodies were able to pull down the part of the Nhlh2 promoter containing the binding sites of Stat3 and NFκB. The EMSA results not only demonstrated that NFκB and Stat3 binding site motifs are real binding sites, but also exists the possibility of a relationship between these transcription factors to regulate Nhlh2 expression with leptin stimulation. An effort in analyzing the human NHLH2 3'UTR showed that one of the SNPs located at position 1568 in the NHLH2 mRNA (NHLH2A^1568G) which converts adenosine to guanine might have the potential to decrease the mRNA stability. For more investigation about this SNP, the mouse Nhlh2 tail was cloned into 2 different vectors and these vectors were subjected to site directed mutagenesis to create the 3'UTR SNP that convert A to G. One of these vectors used luciferase as a reporter gene for expression while the other one was used to measure Nhlh2 mRNA stability. These vectors were transfected into hypothalamic cell line N29/2 to test the effect of this SNP on Nhlh2 expression. This study demonstrated that this SNP down regulated luciferase expression and also decreased Nhlh2 mRNA stability. Taken together, this study demonstrated that Nhlh2 could be regulated transcriptionally by both NFκB and Stat3 transcription factors and post-transcripitionally by the 3'UTR SNP that converts adenosine to guanine. / Ph. D.