ABSTRACT The differentiation process of human SW872 preadipocytes to mature adipocytes is accompanied by morphological changes associated with lipid droplets (LDs) formation and progressive modifications in differentiation markers' expression, as well as by enhanced mitochondrial biogenesis and membrane potential. Under the same circumstances, NADPH oxidase (NOX2)-derived reactive oxygen species (ROS) production was consistent at days 3 and 10 of differentiation and hardly appreciable at day 6. No mitochondrial ROS (mtROS) were detected until the 6th day, most probably owing to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response. On the contrary, mtROS formation was observed at day 10, along with large cytosolic LDs, oxidation of both cardiolipin and thioredoxin 2, and significant drop in mitochondrial glutathione. Therefore, these results show that the morphological and biochemical changes occurring during the SW872 adipogenic process were paralleled by the discontinuous release of NOX2-derived ROS. The mtROS formation was detected only in the late phase of adipogenesis and has been connected to mitochondrial dysfunction. The second generation antipsychotic drug Clozapine (CLZ) is considered a ‘gold standard’ for other treatment-resistant psychosis. However, it shows numerous disabling and serious side-effects which limits its clinical practice, such as an impairment of glucose, insulin, plasma lipid and body fat homeostasis, which can predispose CLZ-treated patients to Type 2 diabetes and cardiovascular disease and eventually to metabolic syndrome (MetS). Exposure of SW872 differentiating cells to CLZ slowed down adipogenesis and caused an early inhibition (day 3) of the expression of crucial transcription factors implicated in the differentiation process. In addition, CLZ dropped NOX2-derived ROS formation, by both NOX2 inhibition and ROS scavenging mechanisms. This effect had an impact on Nrf2-dependent downstream antioxidant responses, triggering an anticipated mitochondrial superoxide formation, associated with signs of mitochondrial dysfunction. Regarding this latter issue, the exposure of SW872 cells to CLZ negatively impacted on mitochondrial biogenesis, leading to decreased mitochondrial number and mass unexpectedly associated with an upregulation of the mitochondrial respiratory chain complexes. Under the same conditions CLZ reduced the cellular ATP levels and affected the compensatory glycolytic response, an effect particularly evident after exposure to complex I inhibitor Rotenone. Finally, the drug impaired the mitochondrial dynamics; indeed under the same conditions a significant downregulation of the main targets of fission and fusion (DRP1, FIS1, MFN1, MFN2 and OPA1) was detected in CLZ-treated cells. In conclusion, the results reported in this thesis may help to shed light on the molecular mechanisms explaining some of the adverse metabolic effects induced by CLZ.
REDOX MODULATION OF ADIPOGENESIS: Clozapine slows down the differentiation process and induces mitochondrial dysfunction by blunting early ROS formation
BLANDINO, GIULIA
2023
Abstract
ABSTRACT The differentiation process of human SW872 preadipocytes to mature adipocytes is accompanied by morphological changes associated with lipid droplets (LDs) formation and progressive modifications in differentiation markers' expression, as well as by enhanced mitochondrial biogenesis and membrane potential. Under the same circumstances, NADPH oxidase (NOX2)-derived reactive oxygen species (ROS) production was consistent at days 3 and 10 of differentiation and hardly appreciable at day 6. No mitochondrial ROS (mtROS) were detected until the 6th day, most probably owing to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response. On the contrary, mtROS formation was observed at day 10, along with large cytosolic LDs, oxidation of both cardiolipin and thioredoxin 2, and significant drop in mitochondrial glutathione. Therefore, these results show that the morphological and biochemical changes occurring during the SW872 adipogenic process were paralleled by the discontinuous release of NOX2-derived ROS. The mtROS formation was detected only in the late phase of adipogenesis and has been connected to mitochondrial dysfunction. The second generation antipsychotic drug Clozapine (CLZ) is considered a ‘gold standard’ for other treatment-resistant psychosis. However, it shows numerous disabling and serious side-effects which limits its clinical practice, such as an impairment of glucose, insulin, plasma lipid and body fat homeostasis, which can predispose CLZ-treated patients to Type 2 diabetes and cardiovascular disease and eventually to metabolic syndrome (MetS). Exposure of SW872 differentiating cells to CLZ slowed down adipogenesis and caused an early inhibition (day 3) of the expression of crucial transcription factors implicated in the differentiation process. In addition, CLZ dropped NOX2-derived ROS formation, by both NOX2 inhibition and ROS scavenging mechanisms. This effect had an impact on Nrf2-dependent downstream antioxidant responses, triggering an anticipated mitochondrial superoxide formation, associated with signs of mitochondrial dysfunction. Regarding this latter issue, the exposure of SW872 cells to CLZ negatively impacted on mitochondrial biogenesis, leading to decreased mitochondrial number and mass unexpectedly associated with an upregulation of the mitochondrial respiratory chain complexes. Under the same conditions CLZ reduced the cellular ATP levels and affected the compensatory glycolytic response, an effect particularly evident after exposure to complex I inhibitor Rotenone. Finally, the drug impaired the mitochondrial dynamics; indeed under the same conditions a significant downregulation of the main targets of fission and fusion (DRP1, FIS1, MFN1, MFN2 and OPA1) was detected in CLZ-treated cells. In conclusion, the results reported in this thesis may help to shed light on the molecular mechanisms explaining some of the adverse metabolic effects induced by CLZ.File | Dimensione | Formato | |
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