No description available.

The accumulation of excessive fat is a serious concern in both the livestock production and human health fields. Obesity is a condition of excessive energy storage in the form of body fat ( triacylglycerols ). The cellular basis for obesity is not yet understood but numerous factors have been suggested. Genetic factors and altered metabolism may be two cellular parameters that contribute to the excessive accumulation of fat. Adipocytes are responsive to extracellular signals, which have a dramatic effect on their metabolism implying that these metabolic responses may be the result of differences in the composition or responsiveness of adipocyte receptors. The purpose of this research was to identify adipocyte specific marker proteins and to determine if there are any differences in the expression of these proteins that may be associated with the conditions of genetic obesity or leanness. Identification of adipocyte-specific markers should allow for a better understanding of adipocyte growth and development and determination of the adipocytes role in energy metabolism. A hybridoma line was produced which secreted a monoclonal antibody (LA-1) directed against a novel 64-kD protein unique to porcine adipocyte plasma membranes, having an undetermined function in the unique physiology of the adipocyte. This protein was found to be expressed in genetically lean adipocytes but not adipocytes derived from genetically obese sources. In order to elucidate the role of this unique adipocyte-specific plasma membrane protein, a porcine adipocyte eDNA library was produced. This library was screened with LA-1 and a eDNA clone isolated. This eDNA clone was used to study the expression of the gene responsible for this unique protein at the nucleic acid level. Northern blot analysis revealed a 5000- and a 7000-base pair species of poly (A+) RNA present in total RNA isolated from contemporary porcine adipose tissue. Determination of the nucleic acid sequence of the eDNA clone should allow for the determination of the actual identity and possible function of this adipocyte-specific protein and the possible role it may serve in regulating adipocyte growth and development. / Graduation date: 1991

The primary goal was to study the influence of adipocyte number and volume, inflammation, insulin resistance, and genetic factors on indicators of liver injury, surrogate marker of non alcoholic fatty liver disease (NAFLD). The secondary goal was to explore the occurrence of NAFLD and its relationship with variations in liver function biomarkers. The first objective was to determine the association of plasma levels of monocyte chemoattractant protein-1 (MCP-1) with omental adipocyte number, insulin resistance and circulating concentrations of liver injury markers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in unrelated baboons. Significant associations of MCP-1 with other measured traits were established. The second objective was to examine if adiposity-related parameters are under genetic influence and to evaluate their genetic correlations with AST in pedigreed baboons. Adipocyte volume and number, body weight and plasma AST were heritable. Genetic correlations between adiposity-related phenotypes and AST were significant. A genome wide scan yielded a strong signal for adipocyte volume on chromosome 6. The third aim was to explore the genetic factors that influence variations in plasma levels of [gamma] glutamyl transferase (GGT) and albumin (ALB), and to evaluate their genetic correlations with cardiovascular risk factors in pedigreed baboons. Significant linkages for GGT and albumin were identified on chromosome 20_22 and chromosome 10, respectively. Genetic correlations between ALB and cardiovascular risk factors were significant. No statistically significant associations were found between GGT and cardiovascular-related phenotypes. The fourth objective was to investigate the prevalence of NAFLD and its association with altered liver protein levels in unrelated baboons. The influence of weight and insulin resistance on the occurrence of NAFLD was inconclusive. Significant relationships between the variations in plasma levels of liver injury biomarkers and severity of the disease could not be established. In conclusion, the first three studies provided observational and genetic evidence of a relationship between liver function markers and adiposity-related factors in baboons. However, the results of the fourth study do not provide conclusive evidence to suggest that body weight and insulin resistance play a significant role in the development of NAFLD in these baboons.

The current epidemic of obesity poses a substantial threat to public health worldwide. Obesity is associated with many deleterious health conditions, including type 2 diabetes, hypertension, dyslipidaemia, respiratory conditions, arthritis, and some forms of cancer. Moreover, the rising prevalence of obesity has been accompanied by a substantial increase in the cost of treating these conditions. Obesity results from a complex interaction between behavioural, environmental, and genetic factors. While the recent increase in the prevalence of obesity is largely due to behavioural factors (for example, physical inactivity); it has also been observed that genetic factors make a large contribution to individual susceptibility. In fact, studies indicate that as much as 50 - 80% of the variation in measures of obesity can be attributed to the effects of genes. Furthermore, closer examination of this genetic component using segregation analysis has indicated the presence of common genes for obesity, with large effects on the phenotype. However, these putative major genes for obesity have not yet been identified. The aim of this thesis was to investigate the role of three distinct genetic loci in obesity and related cardiovascular factors, including type 2 diabetes and dyslipidaemia. The aim of the first investigation was to test whether a common polymorphism (Pro12Ala) in the gene encoding peroxisome proliferator-activated receptor gamma 2 (PPAR-γ2) was associated with obesity and other cardiovascular risk factors in a large group of Caucasian subjects. PPAR-γ2 is an adipogenic transcription factor, which also regulates insulin sensitivity in adipose tissue. No association was observed between the Pro12Ala polymorphism and obesity in Caucasians, but obese subjects carrying the Ala allele displayed an altered blood lipid profile compared with obese Pro/Pro subjects. As the Pro12Ala polymorphism may exacerbate the risk of cardiovascular disease by modifying blood lipid profile in obesity, this relationship was examined further in a separate population. The aim of the second investigation was to determine whether the Pro12Ala polymorphism was associated with obesity, dyslipidaemia, diabetes and carotid intima-medial wall thickening in a population at high risk of developing cardiovascular disease. Australian Aboriginal people display high rates of mortality from cardiovascular disease, and it is possible that their increased susceptibility is due to genetic factors. However, the results from the Aboriginal population confirmed the results of the first study: there was no intrinsic association between the Pro12Ala variant and obesity. In addition, the Ala allele was not associated with deleterious changes in blood lipid profile, as it was in Caucasians. The aim of the third investigation was to confirm the presence of a quantitative trait locus (QTL) for obesity on chromosome 20q13. Highly polymorphic genetic markers in this region were tested for linkage and association with several measures of obesity in a Caucasian population. None of the measures of obesity were linked to or associated with markers spanning 20q13, suggesting that this chromosomal region does not contain a major locus for obesity in this Caucasian population. In the fourth investigation, the 5' sequence of Agouti Signalling Protein (ASIP) was identified. ASIP is a candidate gene for obesity, as it is expressed at high levels in adipocytes, and may participate in several obesity-related processes. Three new exons and two alternative promoters were identified for the ASIP gene. These results may lead to greater understanding of the role of ASIP in obesity and adipocyte metabolism; and may also be used to direct further research into genetic variation within this candidate gene. In conclusion, extensive study of two established candidate genetic loci revealed no association with measures of obesity. Therefore, it is likely that loci other than these make significant contributions to obesity in humans. Further investigation of novel candidate genes, such as ASIP, may allow the identification of novel genetic polymorphisms and new pathways important for the genetic basis of obesity.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

Single nucleotides in the first intron of FTO convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes (e.g. RPGRIP1L, IRX3) in the vicinity of FTO. This locus affects food intake, the browning of white adipose tissue and risk of type 2 diabetes (independent of its effects on body weight). FTO and RPGRIP1L expression are decreased in fibroblasts and iPSC-derived human neurons of individuals segregating for obesity risk alleles of FTO at rs8050136 and rs1421085. These alleles exhibit decreased binding of isoform p110 of the CUX1 transcription factor. This isoform activates transcription of both FTO and RPGRIP1L. The FTO locus conveys effects on adiposity via hyperphagia, in part, by regulating FTO and RPGRIP1L expression in the hypothalamus. We examined whether FTO and RPGRIP1L also modify adipogenesis and adipose tissue lipid storage. Such effects would influence systemic consequences of the hyperphagia driven by the actions of the genes in the hypothalamus. Given the role in energy homeostasis of genes encoding elements of the primary cilium, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. In confirmation, we find that Rpgrip1l+/− mice are hyperphagic and obese, and display diminished suppression of food intake in response to leptin administration. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO intronic locus. We describe effects of Rpgrip1l in adipocytes which may contribute to the adiposity phenotype observed in these animals, and possibly humans. Loss of Rpgrip1l in 3T3-L1 preadipocytes increased the number of cells capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes (using Adipoq-Cre) did not increase adiposity in mice fed chow or high fat diet. Neither did we observe any effects of Rpgrip1l knockdown in mature 3T3-L1 adipocytes in vitro. Thus, to the extent that Rpgrip1l affects cell-autonomous adipose tissue function, it appears to do so by effects conveyed in preadipocytes, a cell type in which the primary cilium – as a mediator of developmental signals – may have functional importance. We propose that decreased RPGRIP1L expression in preadipocytes in humans segregating for FTO-associated obesity risk alleles increases the potential storage capacity of adipose tissue. Such capacity would influence the metabolic consequences of positive energy balance due to the action of these alleles within the brain. Fto expression is upregulated during adipogenesis in murine and human cells in vitro, and is more highly expressed in isolated mouse adipocytes than in preadipocytes. Here we demonstrate that FTO is required for the maintenance of adipocyte lipid filling and endocrine function in murine 3T3-L1 cells and human adipose tissue-derived stromal cells. RNAseq analysis indicates that this effect on adipocyte programming is conveyed in part by modulation of C/ebpβ- and C/ebpδ-regulated transcription, consistent with reports that Fto acts a transcriptional coactivator. Fto-/- mice have normal fat mass in early life, but spontaneously lose adipose tissue as they age. We propose that Fto is required to maintain adipocyte viability, a function critical to the prevention of ectopic lipid accumulation in obese states. Such accumulation – both total and in specific anatomic regions – has adverse metabolic consequences. In addition to the developmental effects on adiposity mediated by RPGRIP1L, and the effects conveyed on adipocyte function related to FTO, the FTO locus could also impact systemic energy homeostasis by modifying production of humoral signals that are integrated centrally to regulate energy balance. We explored molecular modifiers of adipocyte production of leptin identified by GWAS that may modify obesity risk. The FTO locus was associated with circulating leptin concentration, but this association was abrogated when corrected for BMI, indicating that this locus does not contribute to adiposity by dysregulating leptin production. Our in vitro findings are consistent in this regard, as knockdown of Rpgrip1l and Fto in 3T3-L1 cells did not affect leptin production per adipocyte. These results, however, are not inconsistent with a role for FTO in maintenance of adipocyte viability.

No description available.

Obesity, or the presence of an excessive amount of body fat is a major public health problem in the United States and, increasingly, the rest of the world. The apparent drivers of the increased prevalence of obesity over the past several decades are environmental changes, e.g., dietary and lifestyle changes that interact with the individual’s genetic susceptibility for weight gain. In humans, obesity appears to be driven primarily by increases of energy intake relative to expenditure; that is, to uncompensated hyperphagia. The heritability of adiposity, i.e., the extent to which differences in adiposity among individuals living in the same environment can be attributed to genetic differences is estimated by twin and other studies to be about 50%. Large scale population-based association studies (e.g., GWAS) have suggested that genetic variants (e.g., SNPs) associated with susceptibility or resistance to obesity affect primarily the development and regulation of the central nervous system (CNS). In particular, SNPs in genes that play a role in brain cellular structures and molecular pathways known to regulate energy homeostasis, most notably, the leptin-melanocortin signaling pathway, are among the most highly associated with human obesity. For example, SNPs around the melanocortin receptor, MC4R, are associated with increased adiposity and mutations in MC4R represent the most prevalent genetic variations associated with monogenic obesity. Ultimately, however, relatively little is understood about the biological mechanisms by which an individual’s genetic sequence confers susceptibility or resistance to weight gain in a specific environment. Such understanding could open new avenues for the prevention and treatment of obesity and would advance our understating of genetic predisposition to other complex diseases. The goal of this research is to identify genomic regions contributing to susceptibility and resistance to hyperphagic obesity by analysis of whole genome sequence and hypothalamic gene expression data from two genetically related cohorts of Sprague-Dawley rats – the ‘Levin Rat’. Dr. Levin developed these animals by successive generations of selective breeding for differences in adiposity resulting from exposure to a calorically dense, highly palatable diet (described in detail in Chapter 2). These selectively bred diet-induced obese (DIO) and diet-resistant (DR) Levin rats have been the topic of a large body of physiological research (reviewed in Chapter 1) showing potentially important similarities to the physiology of human obesity. In particular, implication of diet-sensitive hyperphagia as the primary driver for the differential susceptibility of DIO (diet-induced obese) animals to gain weight in response to palatable diet; neuroanatomical and functional differences between DIO and DR in hypothalamic nuclei (e.g., ARH, PVH) and leptin signaling, prior to the development of obesity; and, neurophysiological differences between DIO and DR (diet-resistant) in ‘reward circuit’ nuclei (e.g., NAc) and their differential responses to pharmacological stimuli, e.g., cocaine, as well as palatable diet. These findings established the Levin rat as an interesting model for aspects of the biology of human obesity. Importantly, the genetic bases for these Levin rat phenotypes have remained unknown. Our efforts to elucidate the underlying genetics of this model system are, therefore, of potential relevance to human obesity. We obtained phenotypic, whole genome sequence (WGS) and hypothalamic gene expression (RNA-Seq) data from selected Levin rats and analyzed these data to identify several loci that are highly associated with the body weight phenotype in the Levin cohorts, as well as in a confirmation cohort of genetically related progeny being studied for phenotypes related to addictive behaviors. In Chapter 2, I describe our methods and approaches to collecting the relevant phenotypic and genetic data, and to selecting primary and confirmation cohorts for the WGS and RNA-Seq studies. In Chapter 3, I describe our bioanalytical and statistical approaches to the WGS data designed to detect genomic loci likely inherited Identical by Descent (IBD) from common ancestors of the DIO, or DR animals; such loci constitute candidate obesity susceptibility loci for the Levin rat. In Chapter 4, I present the phenotypic differences between the DIO and DR animals used for the study, the results of IBD analysis of the WGS, and indicate genes we found to be differentially expressed or spliced in their respective hypothalami. I also show, using confirmation groups (CG) of animals from the primary cohorts of Levin rats (not used for IBD mapping), that the identified susceptibility loci are highly associated with DIO/DR lineage. In Chapter 5 I show, using representative SNPs from the loci with the highest association to the obesity susceptibility phenotype, significant association between the ‘DIO’ genotype and increased body weight in an independent cohort of Levin rat progeny [designated Obesity Prone (OP) and Obesity Resistant (OR)] maintained by Dr. Ferrario (U. Michigan). Using gene set enrichment analyses (GSEA), I show that the candidate susceptibility loci are enriched in CNS genes and genes previously associated with obesity traits in human GWAS. I also analyzed the genes implicated by differential expression and/or splicing (RNA-Seq) in relation to the genetic susceptibility map (IBD analyses) and identified several genes whose hypothalamic differential regulation (e.g., expression, splicing) may be linked to genetic sequence variations between the DIO and DR animals. Interestingly, essentially, all of the identified genes have been previously implicated in body weight regulation in other species, including humans. Finally, I propose future studies to build upon this work in order to further refine the genetic susceptibility map, test the roles of putative candidate genes, and ultimately elucidate the genetic bases for the differences in body weight in this genetically complex mammalian model of diet-sensitive obesity: the Levin DIO/DR rats. In summary, the conclusions from these studies are that: - The 15 susceptibility loci we identified, span in total ~35 Mb, or 1.15% of the rat genome, likely contain a significant portion of the causal alleles underlying the Levin rat phenotype. - The majority of genetic variants in these susceptibility loci are ‘non-coding,’ e.g., intronic, in untranslated regions, or intergenic – similar to common obesity SNPs. - Our susceptibility map is enriched for genes governing CNS function and development, and its human syntenic genes/loci are enriched in obesity SNPs, identified by GWAS. Therefore, Levin rat obesity risk may have a genetic architecture similar to that in humans. - We identified several genes that are differentially expressed or spliced in the hypothalamus of DIO/DR animals, and are also implicated by our genetic map: Zfr, Slc24a2, Fhit, Adarb1, Lrp2. Interestingly, human orthologs of all of these genes have been implicated in obesity by GWAS or familial linkage studies, enhancing them as putative candidates for a role in the Levin rat obesity phenotypes. - Our studies further establish the Levin rat as a model system for polygenic human obesity and lay the foundation for further studies to elucidate the genetic basis of this interesting and important complex trait of hyperphagic diet-sensitive obesity.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Background: The obesity epidemic is the greatest public health problem of our time, and exerts an enormous health and economic burden by acting as a risk factor for multiple disorders and all-cause mortality. While environmental and social factors certainly contribute to the complex etiology of obesity, there is strong evidence of a substantial genetic component. The majority of obesity genes are involved the leptin-melanocortin receptor pathway governing energy homeostasis, but mutations affecting this circuit are often untreatable and rare, and an improved understanding of other genetic risk factors could aid in the development of novel therapies. In this thesis I study two obesity candidate genes with unclear direct relevance to disease: 1) rare structural variation at the 16p11.2 BP4-BP5 locus and 2) common variation in the Fat Mass and Obesity-Associated (FTO) gene. Methods: 1) I analyzed disinhibited eating measurements from families with 16p11.2 copy number variation (CNV) carriers, to test whether eating in the absence of hunger (EAH) and loss of control (LOC) eating behaviors mediate the dosage-dependent CNV-BMI relationship. 2) Using association data from a study of over 20,000 African Americans and 1,145 functional annotations from the Encyclopedia of Non-coding Elements (ENCODE) and Roadmap Epigenomics projects, I statistically fine-mapped the FTO locus to identify the SNP(s) and cellular contexts underlying the association between FTO and obesity. Results: 1) EAH due to external triggers mediates over 30% of the 16p11.2 deletion’s effect on obesity, while other EAH and LOC behaviors were not significant mediators. This result was independent of IQ deficits and autism related to the CNV, as well as parents’ feeding behaviors and practices. 2) Given 51 FTO SNPs’ association statistics, correlation, and overlap with functional annotations, rs9927317 and rs62033405 had the highest posterior probability of association with obesity. Obesity-associated SNPs may regulate expression of FTO and/or nearby genes through the activity of enhancers and 5’ ends of transcribed genes in the substantia nigra of the brain, bone chondrocytes, and white adipose. Conclusions: These results may help pinpoint the specific genes, regulatory elements, and cellular contexts through which the 16p11.2 and FTO loci exert their effects on obesity.