New metabolic aspects of Leptinmuscle cell activity and biological rhythms

Abstract

Understanding the molecular biological basis of the interaction between specific key nutrients and cells, such as muscle cells, is important since it would help pave the way for the development of new strategies to combat chronic and prevalent diseases, such as obesity and its associated metabolic disorders. Accordingly, in the present thesis two general objectives were considered: a) to study the role of specific free fatty acids (FFA) and Leptin in the regulation of energy metabolism capacity and the production of key myokines in muscle cells. This study was proposed considering previous results of the Laboratory where the thesis was performed; b) to investigate the presence of daily or biological rhythms of Leptin in mammalian milk, together with other related hormones (Ghrelin and Adiponectin), which may have potential future applications in formula milks. Altogether, the general objectives aimed to shed light onto new metabolic aspects of Leptin, in agreement with one of the main research lines of the Laboratory. Three time-sequential experimental models were used in this thesis: 1) a timereverse in vitro model of C2C12 muscle cells treated with FFA (an equimolar physiological mixture of oleic and linoleic acids) alone or in combination with exercise mimetics; 2) a time-forward in vitro model, again using C2C12 myocytes, to study the effects of Leptin treatment (at physiological concentrations) on key metabolic regulators of energy metabolism (both at the gene expression and post-translational modification levels); and 3) a time-sequential monitoring of pregnant rats (in vivo) at different days of lactation, analysing Leptin, Ghrelin and Adiponectin levels in milk and blood, together with morphometric parameters. The results of the first model are explained and discussed in Article 1, the results of the second model in Articles 2 and 3 and the results of the third model in Article 4. Briefly, part of the main results obtained showed that both exercise mimetics (adrenaline and AICAR) and FFA have profound effects inducing interleukin 6 (IL6) production in muscle cells, and that FFA potentiate the adenosine monophosphate activated protein kinase (AMPK) dependent induction of IL6 and are important factors in the muscleadipose tissue crosstalk. Furthermore, other significant results show that Leptin rapidly regulates the expression of key genes in the regulation of energy metabolism in muscle cells, such as those coding for peroxisome proliferator activated receptor gamma coactivator 1 α (PGC1α), Il6, carnitine palmitoyl transferase 1 (CPT1), uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4). These results are in line with the physiological adaptation to environmental conditions of nutrient partition and oxidation in muscle. Moreover, the early effect of Leptin treatment regarding PGC1α upregulation was outstanding and the mechanism involved was suggested to go beyond its previously known effect of AMPK activation. It was also described that Leptin probably plays a crucial role in muscle cells by modulating the post-translational modifications of PGC1α in order to adapt the cell to the nutritional environment, and that it is able to upregulate the expression of Mitofusin 2, important in mitochondrial function and dynamics. In the last part of the thesis, which further explores the physiological functions of Leptin, a biological fluctuation of its levels was described in rat milk during the early phase of lactation, which was also found for Ghrelin and Adiponectin, fitting with a circasemidian pattern. Importantly, a “milk Leptin surge” was discovered around midlactation and it is suggested that it may be related with the known Leptin surge in pups and with progeny development. In summary, the experiments performed and results obtained in the present thesis describe new aspects of Leptin in metabolic regulation, as well as of other important xviii signalling molecules/nutrients such as FFA, especially regarding the function of muscle cells and the possible roles of Leptin during lactation.