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Improving human mesenchymal stem cell-derived hepatic cell energy metabolism by manipulating glucose homeostasis and glucocorticoid signaling

Publication . Rodrigues, Joana S.; Faria-Pereira, Andreia; Camões, Sérgio P.; Serras, Ana S.; Morais, Vanessa A.; Ruas, Jorge Lira; Miranda, Joana P

Introduction: The development of reliable hepatic in vitro models may provide insights into disease mechanisms, linking hepatocyte dysmetabolism and related pathologies. However, several of the existing models depend on using high concentrations of hepatocyte differentiation-promoting compounds, namely glucose, insulin, and dexamethasone, which is among the reasons that have hampered their use for modeling metabolism-related diseases. This work focused on modulating glucose homeostasis and glucocorticoid concentration to improve the suitability of a mesenchymal stem-cell (MSC)-derived hepatocyte-like cell (HLC) human model for studying hepatic insulin action and disease modeling. Methods: We have investigated the role of insulin, glucose and dexamethasone on mitochondrial function, insulin signaling and carbohydrate metabolism, namely AKT phosphorylation, glycogen storage ability, glycolysis and gluconeogenesis, as well as fatty acid oxidation and bile acid metabolism gene expression in HLCs. In addition, we evaluated cell morphological features, albumin and urea production, the presence of hepatic-specific markers, biotransformation ability and mitochondrial function. Results: Using glucose, insulin and dexamethasone levels close to physiological concentrations improved insulin responsiveness in HLCs, as demonstrated by AKT phosphorylation, upregulation of glycolysis and downregulation of Irs2 and gluconeogenesis and fatty acid oxidation pathways. Ammonia detoxification, EROD and UGT activities and sensitivity to paracetamol cytotoxicity were also enhanced under more physiologically relevant conditions. Conclusion: HLCs kept under reduced concentrations of glucose, insulin and dexamethasone presented an improved hepatic phenotype and insulin sensitivity demonstrating superior potential as an in vitro platform for modeling energy metabolism-related disorders, namely for the investigation of the insulin signaling pathway.

2023Journal article

Open access

Unraveling the specific role of synaptic mitochondria

Publication . Faria-Pereira, Andreia; Epifânio, Vanessa Alexandra dos Santos Morais

The brain is one of the organs with the highest energy demands in the body and the majority of these energy requirements are fulfilled by mitochondria. Most of the energy used in the brain is required for synaptic transmission, either in propagation of action potentials or for neurotransmission (Harris, Jolivet and Attwell, 2012). Being synapses one of the highest energy-consuming brain structures, as well as one of the most molecularly dynamic sites in the brain able to change within milliseconds from a resting to a stimulated state, it is expected that mitochondria population at synapses – synaptic mitochondria - are adapted to the synaptic environment and present different properties from other brain mitochondria - non-synaptic mitochondria. Indeed, morphological (Chavan et al., 2015a), proteomic (Kelly L. Stauch, Purnell and Fox, 2014; Völgyi et al., 2015), lipidomic (Kiebish et al., 2008), activity of OxPhos complexes (Almeida et al., 1995; Villa, Gorini and Hoyer, 2006) and pathological (Du et al., 2010) differences have been found between synaptic and non-synaptic mitochondria. Unexpectedly, not much is known on the functional consequences of these differences and more specifically whether synaptic mitochondria have specialized bioenergetic properties adapted to the synaptic environment. Therefore, in the first part of this thesis, we aim at dissecting the functional bioenergetic fingerprint of synaptic mitochondria in comparison with non-synaptic mitochondria, by providing an extensive bioenergetic characterization of synaptic mitochondria. We observed that synaptic mitochondria have a higher energetic flexibility to respond to different respiratory stimuli. They demonstrate higher activity of Complex IV and more drastically of Complex V, whose organization in oligomers is favourable in this mitochondrial population. On the other hand, a lower enzymatic Complex I activity was found in synaptic mitochondria, which is accompanied by a lower FMN content, NAD+ content and lower N-module core subunits expression pointing towards a lower N-module function. Despite overall Complex I activity being lower in synaptic mitochondria, the combined enzymatic activity of Complex I and III was significantly increased in this population and when dissecting Complex I expression and activity, it was demonstrated that, in synaptic mitochondria, Complex I is preferentially integrated into supercomplexes in comparison with non-synaptic mitochondria. We hypothesised that differential arrangement of Complex I is a functional bioenergetic signature of synaptic mitochondria and may constitute a protective mechanism so that synaptic mitochondria in resting conditions are still able to provide ATP for basal synaptic activity but keeping low ROS damage derived from individual Complex I activity. Our results seem to indicate that under non-stimulating conditions mitochondria at synapses have already a prepared “energetic machinery” on-hold to help responding to the higher energetic demands upon synaptic stimulation. Additionally, to study mitochondria in synapses and neurons, in vitro primary neuronal cultures are widely used and, hence constant protocol optimizations and new culture media are formulated, as is the case of BrainPhys medium (Bardy et al., 2015). BrainPhys medium was formulated to mimic physiological brain environment (Bardy et al., 2015). Therefore, in the second part of this thesis we aimed to assess the effects of BrainPhys medium on mouse neuronal maturation and to characterize how this physiologically-formulated medium impacted on neuronal bioenergetics in comparison with widely-used Neurobasal medium. Our findings demonstrate that neurons maintained in BrainPhys medium formed dense neuronal networks, abundantly stained for neuronal and synaptic markers and are functionally active. Additionally, mouse neurons in BrainPhys medium have significantly higher ATP levels, as well as, increased mitochondrial activity and Complex IV subunit expression, especially at latter maturation stages. Overall, our results corroborated previous data (Bardy et al., 2015; Jackson et al., 2018; Satir et al., 2020) showing the adequacy of BP medium to generate robust neuronal networks in mouse primary neurons and elucidated that neurons maintained in BP medium are more reliant on mitochondrial activity, as occurs in physiological conditions.

2022-07Doctoral thesis

Open access