GRB14/COBLL1 locus has been shown to be associated with body fat distribution (FD), but neither the causal gene nor its role in metabolic diseases has been elucidated. We hypothesize that GRB14/COBLL1 may act as the causal genes for FD-related SNPs (rs10195252 and rs6738627), and that they may be regulated by SNP to effect obesity-related metabolic traits. We genotyped rs10195252 and rs6738627 in 2860 subjects with metabolic phenotypes. In a subgroup of 560 subjects, we analyzed GRB14/COBLL1 gene expression in paired visceral and subcutaneous adipose tissue (AT) samples. Mediation analyses were used to determine the causal relationship between SNPs, AT GRB14/COBLL1 mRNA expression, and obesity-related traits. In vitro gene knockdown of Grb14/Cobll1 was used to test their role in adipogenesis. Both gene expressions in AT are correlated with waist circumference. Visceral GRB14 mRNA expression is associated with FPG and HbA1c. Both SNPs are associated with triglycerides, FPG, and leptin levels. Rs10195252 is associated with HbA1c and seems to be mediated by visceral AT GRB14 mRNA expression. Our data support the role of the GRB14/COBLL1 gene expression in body FD and its locus in metabolic sequelae: in particular, lipid metabolism and glucose homeostasis, which is likely mediated by AT GRB14 transcript levels.

Rainbow trout (Oncorhynchus mykiss) is an important freshwater fish whose glucose intolerance, white muscle lactate retention and high lipolytic inertia, have interested comparative physiologists for decades. Recent advancements in mammalian G-protein coupled receptor deorphanization research have identified many endogenous metabolites as regulators of energy metabolism, including lactate and long-chain fatty acids. In addition to being essential metabolic fuels, lactate and long-chain fatty acids regulate lipolysis and lipogenesis by binding to hydroxycarboxylic acid receptor 1 (HCAR1) and the free fatty acid receptors (FFAR1 and 4), respectively. Therefore, the goal of this thesis was to quantify the effects of exogenous lactate and lipids on glucose and fatty acid mobilization in rainbow trout and identify potential signaling mechanisms by monitoring the expression and activity of key glycolytic, gluconeogenic, lipolytic, lipogenic and β-oxidation targets in the liver, muscle and adipose tissue. In Chapter 2, in vivo measurements of metabolic fuel kinetics show that lactate (i) strongly reduced hepatic glucose production by substituting glucose for lactate and (ii) exhibited no lipolytic suppression suggesting HCAR1 signaling is weak in trout. In Chapter 3, in vivo measurements of energy mobilization show that Intralipid strongly induced lipolysis by saturating circulating lipases while transcriptional induction of gluconeogenesis compensates for the acute reduction in hepatic glucose production. Intralipid infusion increased total fatty acid concentration and altered fatty acid composition while suppressing lipid metabolism of trout liver and adipose tissue. In Chapter 4, I identify the presence (hcar1 and ffar1) and absence (ffar4) of these G-protein coupled receptor genes in the rainbow trout genome and describe their evolutionary origins, using in silico approaches of microsynteny, amino acid sequence similarity and critical residue conservation. However, their importance in fish physiology remains relatively unknown, thus future studies are warranted to further investigate such metabolic signals.

There is concern over the impact that petroleum related compounds (PRCs) associated with mining activity in the Athabasca Oil Sands Region (AOSR) are having on local wildlife. With the increase in oil sands mining activity in the AOSR there has been a corresponding decline in the fertility of indicator species in the AOSR. One of the primary sources of PRCs in the environment is oil sands process affected water (OSPW), which is stored in tailings ponds. Several PRCs, including naphthenic acid fraction components (NAFC), have endocrine disrupting effects, which may, in part, explain reduced fertility in indicator species. For example, male North American river otters (Lontra canadensis) living in areas impacted by mining activity have lower baculum strength those unaffected by mining activity. Weaker baculums are associated with increases in fracture rates and reduced fertility. Baculum strength is maintained throughout life by bone remodeling, a process that requires the differentiation of osteoblasts. NAFCs can impact several pathways integral to the development and path selection of mesenchymal stem cells into osteoblasts or adipocytes. Therefore, the objective of this thesis was to test the hypothesis that NAFCs inhibit osteoblast differentiation and induce adipocyte differentiation from progenitor cells. We exposed osteoblast progenitor cells and adipocyte progenitor cells to NAFCs. We demonstrated that NAFCs inhibit osteoblast differentiation and activate the glucocorticoid receptor pathway. We also found that NAFCs do not induce adipogenesis in adipocyte precursor cells. Lastly, we showed that NAFCs are PPARγ ligands that inhibit the expression of PPARγ associated genes. These insights into the effects of NAFCs on osteoblast and adipocyte progenitor cells suggest NAFCs may contribute to lower baculum strength and impaired adipose tissue function of animals living in the AOSR. These effects my reduce the fertility and population of wildlife in the AOSR. / Thesis / Master of Science (MSc) / There is concern that chemicals from oil sands mining in the Athabasca oil sands region are hurting the reproductive health of animals in the wild. Some of these animals, including bears, wolves, and river otters, need a bone in their penis called a baculum to reproduce. Studies have shown that some chemicals, including those from mining activity can make the baculum bone weaker. For bone to stay strong, bone cells always need to be developing to fix the bone tissue. The goal of our study was to find how chemicals from mining activity can affect the development of bone cells. We found that a group of chemicals that come from oil sands mining called naphthenic acid fraction components (NAFCs) stop bone cells from developing and making new bone. We also know that having more fat cells in bone is associated with weaker bones. We also looked at whether NAFCs could increase the development of fat cells. However, NAFCs did not increase the development of fat cells. Together, this research shows that NAFCs can make bones like the baculum weaker by slowing the development of new bone, but not by increasing fat cells. Our research suggests that exposure to NAFCs may make baculums weaker which may be bad for the reproductive health of animals living near oil sands mining activity.

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Chronic venous insufficiency (CVI) is characterized by valve dysfunction and venous hypertension, leading to erythrocytes’ extravasation into the tissue over time. CVI patients present dermal manifestations as; hyperpigmentation of the leg due to iron accumulation, histological changes regarding the dermal layer and fat (lipodermatosclerosis), and a high risk of developing a leg ulcer. For decades, researchers have studied CVI, and chronic venous ulcer (CVU) and iron have been considered critical pathological factors in this context, especially concerning oxidative stress and ROS formation. However, a clear understanding of the pathogenic effects of iron on the tissue network and the cross-talk of resident immune and skin tissue cells in the course of CVI is still missing. Therefore, in this project, we aim to investigate the pathological effect of iron overload on the cross-talk of resident immune and tissue cells in the skin. In the current thesis work, biopsies from CVI patients were analysed. Immunohistochemistry (IHC) and spectrometric assay confirmed a massive iron accumulation in their dermis and hypodermis. To dissect the different skin cells’ responses to iron overload, in vitro techniques were exploited. Mimic the erythrocyte overload in a macrophage (M2-like) culture revealed a shift in the gene signature towards inflammatory activation states of these cells. Hence, cytokine analysis confirmed an evident pro-inflammatory activation of macrophages (Ma). A mouse model with skin iron overload was generated to investigate the effect of iron overload in a more complex picture. Here, it was confirmed that iron induces an expansion of immune cells and a pro-inflammatory activation in the skin with a shift in resident macrophage subtypes, which was coupled to the adipose layer. Indeed, the dWAT of these mice shirked and showed clear signs of lipolysis. Moreover, IHC and IF staining of iron-mice skin showed increased cellularity of the lower dermis, which was linked to an expansion of the fibroblast population (dermis and stromal vascular fraction of the dWAT). Consistent with in vitro data, ECM genes were downregulated in the dermis, which may explain changes in the skin architecture of these mice. In this thesis, the newly established mouse model made it possible to understand how iron may affect skin cells in CVI patients and can be extremely useful for future research to develop a new therapeutic approach. This work wants to highlight the significance of iron overload in the skin, which affects cellular cross-talk, altering skin homeostasis and possibly leading to an ulcer.

Since overweight and obesity reached pandemic proportions, the understanding of underlying causes became a complex research area. Within this context, the white adipose tissue acts as an endocrine organ, producing adipokines. Investigations of adipokines, their molecular structure, physiological impact and pro- or anti-inflammatory functions are in focus of attention to identify their metabolic role in mediating obesity induced metabolic modifications. Therefore, this work analyzed whether 14 different adipokines correlate with diverse eating behavior types and individual BMI values and how this associations are possibly mediated by these eating behavior phenotypes. Therefore, 557 participants of the Sorbs and 3101 participants of LIFE Leipzig Adult completed the German version of the Three-Factor-Eating Questionnaire to assess the eating behavior types “restraint”, “disinhibition” and “hunger”. Serum levels of 14 adipokines, including adiponectin, adipocyte fatty acid binding protein (AFABP), angiopoietin-related growth factor (AGF), chemerin, fibroblast growth factor (FGF)-19, FGF-21, FGF-23, insulin-like growth factor (IGF)-1, interleukin (IL) 10, irisin, progranulin, vaspin, pro-neurotensin (pro-NT) and pro-enkephalin (PENK) were measured. Based on significant correlations between the following adipokines: PENK, pro-NT, adiponectin, FGF-19, FGF-21, IGF-1, chemerin, progranulin, AGF and AFABP with different eating behavior items and BMI, mediation analyses were constructed, including the eating behavior item as mediation variable. Here, positive associations between chemerin, AFABP or leptin and BMI in Sorbian women was mediated by higher restraint or disinhibited eating, respectively. Additionally, in Sorbian women, the negative relation between IGF-1 and BMI was mediated by higher disinhibition and the positive link between AGF and BMI by lower disinhibition. In Sorbian men, the negative relationship between PENK and BMI was mediated by lower disinhibition and hunger, whereas the negative relation between IGF-1 and BMI was mediated by higher hunger. In the LIFE-Adult women ́s cohort, associations between chemerin and BMI were mediated by decreased hunger or disinhibition, respectively, whereas relations between PENK and BMI were fully mediated by decreased disinhibition. In summary, this work provides evidence, that the adipokines PENK, IGF-1, chemerin, AGF, AFABP and leptin may impact the development of obesity by directly modifying individual eating behavior.