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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.
Growth differentiation factor 15 (GDF15) is a stress-induced cytokine secreted into the circulation by a number of tissues under different pathological conditions such as cardiovascular disease, cancer or mitochondrial dysfunction, among others. While GDF15 signaling through its recently identified hindbrain-specific receptor GDNF family receptor alpha-like (GFRAL) has been proposed to be involved in the metabolic stress response, its endocrine role under chronic stress conditions is still poorly understood. Mitochondrial dysfunction is characterized by the impairment of oxidative phosphorylation (OXPHOS), leading to inefficient functioning of mitochondria and consequently, to mitochondrial stress. Importantly, mitochondrial dysfunction is among the pathologies to most robustly induce GDF15 as a cytokine in the circulation.
The overall aim of this thesis was to elucidate the role of the GDF15-GFRAL pathway under mitochondrial stress conditions. For this purpose, a mouse model of skeletal muscle-specific mitochondrial stress achieved by ectopic expression of uncoupling protein 1 (UCP1), the HSA-Ucp1-transgenic (TG) mouse, was employed. As a consequence of mitochondrial stress, TG mice display a metabolic remodeling consisting of a lean phenotype, an improved glucose metabolism, an increased metabolic flexibility and a metabolic activation of white adipose tissue.
Making use of TG mice crossed with whole body Gdf15-knockout (GdKO) and Gfral-knockout (GfKO) mouse models, this thesis demonstrates that skeletal muscle mitochondrial stress induces the integrated stress response (ISR) and GDF15 in skeletal muscle, which is released into the circulation as a myokine (muscle-induced cytokine) in a circadian manner. Further, this work identifies GDF15-GFRAL signaling to be responsible for the systemic metabolic remodeling elicited by mitochondrial stress in TG mice. Moreover, this study reveals a daytime-restricted anorexia induced by the GDF15-GFRAL axis under muscle mitochondrial stress, which is, mechanistically, mediated through the induction of hypothalamic corticotropin releasing hormone (CRH). Finally, this work elucidates a so far unknown physiological outcome of the GDF15-GFRAL pathway: the induction of anxiety-like behavior.
In conclusion, this study uncovers a muscle-brain crosstalk under skeletal muscle mitochondrial stress conditions through the induction of GDF15 as a myokine that signals through the hindbrain-specific GFRAL receptor to elicit a stress response leading to metabolic remodeling and modulation of ingestive- and anxiety-like behavior.