The hepatokine FGF21 and the adipokine chemerin have been implicated as metabolic regulators and mediators of inter-tissue crosstalk. While FGF21 is associated with beneficial metabolic effects and is currently being tested as an emerging therapeutic for obesity and diabetes, chemerin is linked to inflammation-mediated insulin resistance. However, dietary regulation of both organokines and their role in tissue interaction needs further investigation. The LEMBAS nutritional intervention study investigated the effects of two diets differing in their protein content in obese human subjects with non-alcoholic fatty liver disease (NAFLD). The study participants consumed hypocaloric diets containing either low (LP: 10 EN%, n = 10) or high (HP: 30 EN%, n = 9) dietary protein 3 weeks prior to bariatric surgery. Before and after the intervention the participants were anthropometrically assessed, blood samples were drawn, and hepatic fat content was determined by MRS. During bariatric surgery, paired subcutaneous and visceral adipose tissue biopsies as well as liver biopsies were collected. The aim of this thesis was to investigate circulating levels and tissue-specific regulation of (1) FGF21 and (2) chemerin in the LEMBAS cohort. The results were compared to data obtained in 92 metabolically healthy subjects with normal glucose tolerance and normal liver fat content. (1) Serum FGF21 concentrations were elevated in the obese subjects, and strongly associated with intrahepatic lipids (IHL). In accordance, FGF21 serum concentrations increased with severity of NAFLD as determined histologically in the liver biopsies. Though both diets were successful in reducing IHL, the effect was more pronounced in the HP group. FGF21 serum concentrations and mRNA expression were bi-directionally regulated by dietary protein, independent from metabolic improvements. In accordance, in the healthy study subjects, serum FGF21 concentrations dropped by more than 60% in response to the HP diet. A short-term HP intervention confirmed the acute downregulation of FGF21 within 24 hours. Lastly, experiments in HepG2 cell cultures and primary murine hepatocytes identified nitrogen metabolites (NH4Cl and glutamine) to dose-dependently suppress FGF21 expression. (2) Circulating chemerin concentrations were considerably elevated in the obese versus lean study participants and differently associated with markers of obesity and NAFLD in the two cohorts. The adipokine decreased in response to the hypocaloric interventions while an unhealthy high-fat diet induced a rise in chemerin serum levels. In the lean subjects, mRNA expression of RARRES2, encoding chemerin, was strongly and positively correlated with expression of several cytokines, including MCP1, TNFα, and IL6, as well as markers of macrophage infiltration in the subcutaneous fat depot. However, RARRES2 was not associated with any cytokine assessed in the obese subjects and the data indicated an involvement of chemerin not only in the onset but also resolution of inflammation. Analyses of the tissue biopsies and experiments in human primary adipocytes point towards a role of chemerin in adipogenesis while discrepancies between the in vivo and in vitro data were detected. Taken together, the results of this thesis demonstrate that circulating FGF21 and chemerin levels are considerably elevated in obesity and responsive to dietary interventions. FGF21 was acutely and bi-directionally regulated by dietary protein in a hepatocyte-autonomous manner. Given that both, a lack in essential amino acids and excessive nitrogen intake, exert metabolic stress, FGF21 may serve as an endocrine signal for dietary protein balance. Lastly, the data revealed that chemerin is derailed in obesity and associated with obesity-related inflammation. However, future studies on chemerin should consider functional and regulatory differences between secreted and tissue-specific isoforms.

The musculoskeletal system provides support and enables movement to the body, and its deterioration is a crucial aspect of age-related functional decline. Mesenchymal stromal cells (MSCs) play an important role in musculoskeletal homeostasis due to their broad differentiation potentials and their ability to support osteogenic and myogenic tissue maintenance and regeneration. In the bone, MSCs differentiate either into osteochondrogenic progenitors to form osteocytes and chondrocytes, or increasingly with age into adipogenic progenitors which give rise to bone-resident adipocytes. In skeletal muscle, during healthy regeneration MSCs provide regulatory signals that activate local, tissue-specific stem cells, known as satellite cells, which regenerate contractile myofibres. This process involves a significant cross-talk to immune cells stemming from both lymphoid and myeloid lineages. During ageing, muscle-resident MSCs undergo increased adipogenic lineage commitment, causing niche changes that contribute to fatty infiltration in muscles. These shifts in cell populations in bone lead to the loss of osteogenic cells and subsequently osteoporosis, or in muscle to impaired regeneration and to the development of sarcopenia. However, the signals that drive transition of MSCs into their respective cellular fates remain elusive. This thesis aims to elucidate the transcriptional shifts modulating cell states and cell types in musculoskeletal MSC fate determination. Single-cell RNA-sequencing (scRNA-seq) was used to characterise cell type-specific transcript regulation. State-of-the-art bioinformatics tools were combined with different analytical platforms that include both droplet-based scRNA-seq for large heterogeneous populations, and microfluidics-based scRNA-seq to assess small, rare subpopulations. For each platform, distinct computational pipelines were established including filtering steps to exclude low-quality cells, and data visualisation was performed by dimensionality reduction. Downstream analysis included clustering, cell type annotation, and differential gene expression to investigate transcriptional states in defined cell types during ageing and injury in the muscle and bone. Finally, a novel tool to assess publication activities in defined areas of research for the identified marker genes was developed. The results in the bone indicate that ageing MSCs increasingly commit towards an adipogenic fate at the expense of osteogenic specialisation. The data also suggests that significant cell population shifts of MSC-type fibro-adipogenic progenitors during muscle ageing underlie the pathologies observed in homeostatic and post-injury regenerative conditions. High-throughput visualisation of publication activity for candidate genes enabled more effective biological evaluation of scRNA-seq data. These results expose critical age-related changes in the stem cell niches of skeletal muscle and bone, highlight their respective sensitivity to nutrition and pathology, and elucidate novel factors that modulate stem cell-based regeneration. Targeting these processes might improve musculoskeletal health in the context of ageing and prevent the negative effects of pathological lineage determination.

Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis, thereby allowing mammals to maintain a constant body temperature in a cold environment. Thermogenic capacity of this tissue is due to a high mitochondrial density and expression of uncoupling protein 1 (UCP1), a unique brown adipocyte marker which dissipates the mitochondrial proton gradient to produce heat instead of ATP. BAT is actively involved in whole-body metabolic homeostasis and during aging there is a loss of classical brown adipose tissue with concomitantly reduced browning capacity of white adipose tissue. Therefore, an age-dependent decrease of BAT-related energy expenditure capacity may exacerbate the development of metabolic diseases, including obesity and type 2 diabetes mellitus. Given that direct effects of age-related changes of BAT-metabolic flux have yet to be unraveled, the aim of the current thesis is to investigate potential metabolic mechanisms involved in BAT-dysfunction during aging and to identify suitable metabolic candidates as functional biomarkers of BAT-aging. To this aim, integration of transcriptomic, metabolomic and proteomic data analyses of BAT from young and aged mice was performed, and a group of candidates with age-related changes was revealed. Metabolomic analysis showed age-dependent alterations of metabolic intermediates involved in energy, nucleotide and vitamin metabolism, with major alterations regarding the purine nucleotide pool. These data suggest a potential role of nucleotide intermediates in age-related BAT defects. In addition, the screening of transcriptomic and proteomic data sets from BAT of young and aged mice allowed identification of a 60-kDa lysophospholipase, also known as L-asparaginase (Aspg), whose expression declines during BAT-aging. Involvement of Aspg in brown adipocyte thermogenic function was subsequently analyzed at the molecular level using in vitro approaches and animal models. The findings revealed sensitivity of Aspg expression to β3-adrenergic activation via different metabolic cues, including cold exposure and treatment with β3-adrenergic agonist CL. To further examine ASPG function in BAT, an over-expression model of Aspg in a brown adipocyte cell line was established and showed that these cells were metabolically more active compared to controls, revealing increased expression of the main brown-adipocyte specific marker UCP1, as well as higher lipolysis rates. An in vitro loss-of-function model of Aspg was also functionally analyzed, revealing reduced brown adipogenic characteristics and an impaired lipolysis, thus confirming physiological relevance of Aspg in brown adipocyte function. Characterization of a transgenic mouse model with whole-body inactivation of the Aspg gene (Aspg-KO) allowed investigation of the role of ASPG under in vivo conditions, indicating a mild obesogenic phenotype, hypertrophic white adipocytes, impairment of the early thermogenic response upon cold-stimulation and dysfunctional insulin sensitivity. Taken together, these data show that ASPG may represent a new functional biomarker of BAT-aging that regulates thermogenesis and therefore a potential target for the treatment of age-related metabolic disease.