High circulating glucose and non-esterified free fatty acid (NEFA) levels can cause pancreatic β-cell failure. The molecular mechanisms of this β-cell glucolipotoxicity are yet to be established conclusively. In this thesis by exploring mitochondrial energy metabolism in INS-1E insulinoma cells and isolated pancreatic islets, a role of mitochondria in pancreatic β-cell glucolipotoxicity is uncovered. It is reported that prolonged palmitate exposure at high glucose attenuates glucose-stimulated mitochondrial respiration which is coupled to ADP phosphorylation. These mitochondrial defects coincide with an increased level of mitochondrial reactive oxygen species (ROS), impaired glucose-stimulated insulin secretion (GSIS) and decreased cell viability. Palmitoleate, on the other hand, does not affect mitochondrial ROS levels or cell viability and protects against the adverse effects of palmitate on these phenotypes. Interestingly, palmitoleate does not significantly protect against mitochondrial respiratory or insulin secretion defects and in pancreatic islets tends to limit these functions on its own. Furthermore, strong evidence suggests that glucolipotoxic-induced ROS are of a mitochondrial origin and these ROS are somehow linked with NEFA-induced loss in cell viability. To explore the mechanism of glucolipotxic-induced mitochondrial ROS and associated cell loss, uncoupling protein-2 (UCP2) protein levels and activity were probed in NEFA exposed INS-1E cells. It is concluded that UCP2 neither mediates palmitate-induced mitochondrial ROS production and the related cell loss, nor protects against these deleterious effects. Instead, UCP2 dampens palmitoleate protection against palmitate toxicity. Collectively, these data shed important new light on the area of glucolipotoxicity in pancreatic β-cells and provide novel insights into the pathogenesis of Type 2 diabetes.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

Uncoupling protein-2 (UCP2) may be linked to the attenuation of reactive oxygen species (ROS), but it is unclear whether this phenomenon pertains to the pancreatic beta cell. In this study, a UCP2-deficient mouse model was used to assess the importance of UCP2 to beta cell viability. We investigated the effect of UCP2 absence in response to a beta cell cytotoxic model of diabetes induction. In vivo treatment by the cytotoxic agent streptozotocin led to overall beta cell loss, but severity was not exacerbated by UCP2 deficiency. We also examined ROS production and cell viability in islet cells exposed to various stressors associated with oxidative stress. In vitro measurements of ROS and cell death in islet cells demonstrated that the response was not influenced by UCP2 expression. In contrast with UCP2 overexpression studies showing cytoprotection, this study reveals that beta cell survival is not compromised by the absence of UCP2.

Introdução: A apneia do sono causa hipóxia intermitente (HI), que tem sido implicada na resistência à insulina. A proteína desacopladora 2 (UCP2) no pâncreas é reguladora negativa da secreção de insulina. Objetivos do Estudo: Avaliar se a exposição animal ao modelo de apneia do sono influencia na produção de insulina via expressão do mRNA da UCP2. Desenho e intervenções: Camundongos C57BL machos foram expostos durante 35 dias à hipóxia intermitente (HI; n = 18) ou HI simulada (HIS; n = 18). Durante 8 horas diárias o grupo HI foi submetido a um total de 480 ciclos de 30 segundos de hipóxia progressiva a um nadir da fração inspirada de oxigênio de 8 ± 1%, seguido por 30 segundos de normóxia. Métodos e Resultados: A expressão do mRNA da UCP-2 no pâncreas, avaliada por PCR em tempo real, foi 18% mais baixa no grupo HI do que no grupo HIS (P = 0,14). Imunohistoquímica identificou maior número de células beta no grupo HI do que no HIS (P = 0,14). Os níveis de glicose no soro medidos por métodos colorimétricos enzimáticos foram menores no grupo HI do que no grupo HIS (P = 0,025). Níveis significativamente maiores de insulina no soro, quantificados por ELISA, e níveis significativamente mais baixos de glucagon, quantificados por EIA, foram observados no grupo HI, quando comparado ao grupo HIS. Os cálculos do modelo de avaliação da homeostase (HOMA) para a resistência à insulina sugerem maior sensibilidade em HI do que em camundongos HIS (P = 0,09). A função das células beta, avaliada por HOMA - β foi maior em camundongos HI do que nos HIS (P = 0,014). O peso corporal, ajustado ao consumo alimentar, foi relativamente estável durante o experimento. Não foram encontradas diferenças significativas no perfil lipídico. Conclusões: Estes resultados sugerem que a HI provoca alterações na função pancreática que não podem ser relacionadas com a expressão do mRNA da UCP2. / Background: Sleep apnea causes intermittent hypoxia (IH) that has been implicated in insulin resistance. Pancreatic uncoupling protein-2 (UCP2) is a negative regulator of insulin secretion. Study Objectives: To evaluate whether exposure to an animal model of sleep apnea influences insulin output via UCP2 expression. Design and interventions: Male C57BL mice were exposed during 35 days to intermittent hypoxia (IH; n=18) or to sham IH (SIH; n=18). During 8 hours daily the IH group underwent a total of 480 cycles of 30 seconds of progressive hypoxia to a nadir FIO2 of 8±1% followed by 30 seconds of normoxia. Measurements and Results: The expression of pancreatic UCP-2 mRNA assessed by real-time PCR was 18% lower (P=0.14) in the IH than in the SIH group. Immunohistochemistry identified higher number of beta cells in IH than in SIH group (P=0.14). Glucose levels measured in serum by enzymatic colorimetric methods was lower in IH than in SIH group (P=0.025). Significantly higher serum insulin quantified by ELISA and lower glucagon levels quantified by EIA were seen in the IH group when compared with SIH. Calculations of homeostasis model assessment (HOMA) for insulin resistance suggest higher sensitivity in IH than in SIH mice (P=0.09). HOMA for beta-cell function was higher in IH than in SIH mice (P=0.014). Body weight, adjusted for food intake, was relatively stable during the experiment. No significant differences in lipid profile were encountered. Conclusions: These results suggest that IH causes changes in pancreatic function that may be not related to expression of mRNA UCP2.

Introdução: A apneia do sono causa hipóxia intermitente (HI), que tem sido implicada na resistência à insulina. A proteína desacopladora 2 (UCP2) no pâncreas é reguladora negativa da secreção de insulina. Objetivos do Estudo: Avaliar se a exposição animal ao modelo de apneia do sono influencia na produção de insulina via expressão do mRNA da UCP2. Desenho e intervenções: Camundongos C57BL machos foram expostos durante 35 dias à hipóxia intermitente (HI; n = 18) ou HI simulada (HIS; n = 18). Durante 8 horas diárias o grupo HI foi submetido a um total de 480 ciclos de 30 segundos de hipóxia progressiva a um nadir da fração inspirada de oxigênio de 8 ± 1%, seguido por 30 segundos de normóxia. Métodos e Resultados: A expressão do mRNA da UCP-2 no pâncreas, avaliada por PCR em tempo real, foi 18% mais baixa no grupo HI do que no grupo HIS (P = 0,14). Imunohistoquímica identificou maior número de células beta no grupo HI do que no HIS (P = 0,14). Os níveis de glicose no soro medidos por métodos colorimétricos enzimáticos foram menores no grupo HI do que no grupo HIS (P = 0,025). Níveis significativamente maiores de insulina no soro, quantificados por ELISA, e níveis significativamente mais baixos de glucagon, quantificados por EIA, foram observados no grupo HI, quando comparado ao grupo HIS. Os cálculos do modelo de avaliação da homeostase (HOMA) para a resistência à insulina sugerem maior sensibilidade em HI do que em camundongos HIS (P = 0,09). A função das células beta, avaliada por HOMA - β foi maior em camundongos HI do que nos HIS (P = 0,014). O peso corporal, ajustado ao consumo alimentar, foi relativamente estável durante o experimento. Não foram encontradas diferenças significativas no perfil lipídico. Conclusões: Estes resultados sugerem que a HI provoca alterações na função pancreática que não podem ser relacionadas com a expressão do mRNA da UCP2. / Background: Sleep apnea causes intermittent hypoxia (IH) that has been implicated in insulin resistance. Pancreatic uncoupling protein-2 (UCP2) is a negative regulator of insulin secretion. Study Objectives: To evaluate whether exposure to an animal model of sleep apnea influences insulin output via UCP2 expression. Design and interventions: Male C57BL mice were exposed during 35 days to intermittent hypoxia (IH; n=18) or to sham IH (SIH; n=18). During 8 hours daily the IH group underwent a total of 480 cycles of 30 seconds of progressive hypoxia to a nadir FIO2 of 8±1% followed by 30 seconds of normoxia. Measurements and Results: The expression of pancreatic UCP-2 mRNA assessed by real-time PCR was 18% lower (P=0.14) in the IH than in the SIH group. Immunohistochemistry identified higher number of beta cells in IH than in SIH group (P=0.14). Glucose levels measured in serum by enzymatic colorimetric methods was lower in IH than in SIH group (P=0.025). Significantly higher serum insulin quantified by ELISA and lower glucagon levels quantified by EIA were seen in the IH group when compared with SIH. Calculations of homeostasis model assessment (HOMA) for insulin resistance suggest higher sensitivity in IH than in SIH mice (P=0.09). HOMA for beta-cell function was higher in IH than in SIH mice (P=0.014). Body weight, adjusted for food intake, was relatively stable during the experiment. No significant differences in lipid profile were encountered. Conclusions: These results suggest that IH causes changes in pancreatic function that may be not related to expression of mRNA UCP2.

Introdução: A apneia do sono causa hipóxia intermitente (HI), que tem sido implicada na resistência à insulina. A proteína desacopladora 2 (UCP2) no pâncreas é reguladora negativa da secreção de insulina. Objetivos do Estudo: Avaliar se a exposição animal ao modelo de apneia do sono influencia na produção de insulina via expressão do mRNA da UCP2. Desenho e intervenções: Camundongos C57BL machos foram expostos durante 35 dias à hipóxia intermitente (HI; n = 18) ou HI simulada (HIS; n = 18). Durante 8 horas diárias o grupo HI foi submetido a um total de 480 ciclos de 30 segundos de hipóxia progressiva a um nadir da fração inspirada de oxigênio de 8 ± 1%, seguido por 30 segundos de normóxia. Métodos e Resultados: A expressão do mRNA da UCP-2 no pâncreas, avaliada por PCR em tempo real, foi 18% mais baixa no grupo HI do que no grupo HIS (P = 0,14). Imunohistoquímica identificou maior número de células beta no grupo HI do que no HIS (P = 0,14). Os níveis de glicose no soro medidos por métodos colorimétricos enzimáticos foram menores no grupo HI do que no grupo HIS (P = 0,025). Níveis significativamente maiores de insulina no soro, quantificados por ELISA, e níveis significativamente mais baixos de glucagon, quantificados por EIA, foram observados no grupo HI, quando comparado ao grupo HIS. Os cálculos do modelo de avaliação da homeostase (HOMA) para a resistência à insulina sugerem maior sensibilidade em HI do que em camundongos HIS (P = 0,09). A função das células beta, avaliada por HOMA - β foi maior em camundongos HI do que nos HIS (P = 0,014). O peso corporal, ajustado ao consumo alimentar, foi relativamente estável durante o experimento. Não foram encontradas diferenças significativas no perfil lipídico. Conclusões: Estes resultados sugerem que a HI provoca alterações na função pancreática que não podem ser relacionadas com a expressão do mRNA da UCP2. / Background: Sleep apnea causes intermittent hypoxia (IH) that has been implicated in insulin resistance. Pancreatic uncoupling protein-2 (UCP2) is a negative regulator of insulin secretion. Study Objectives: To evaluate whether exposure to an animal model of sleep apnea influences insulin output via UCP2 expression. Design and interventions: Male C57BL mice were exposed during 35 days to intermittent hypoxia (IH; n=18) or to sham IH (SIH; n=18). During 8 hours daily the IH group underwent a total of 480 cycles of 30 seconds of progressive hypoxia to a nadir FIO2 of 8±1% followed by 30 seconds of normoxia. Measurements and Results: The expression of pancreatic UCP-2 mRNA assessed by real-time PCR was 18% lower (P=0.14) in the IH than in the SIH group. Immunohistochemistry identified higher number of beta cells in IH than in SIH group (P=0.14). Glucose levels measured in serum by enzymatic colorimetric methods was lower in IH than in SIH group (P=0.025). Significantly higher serum insulin quantified by ELISA and lower glucagon levels quantified by EIA were seen in the IH group when compared with SIH. Calculations of homeostasis model assessment (HOMA) for insulin resistance suggest higher sensitivity in IH than in SIH mice (P=0.09). HOMA for beta-cell function was higher in IH than in SIH mice (P=0.014). Body weight, adjusted for food intake, was relatively stable during the experiment. No significant differences in lipid profile were encountered. Conclusions: These results suggest that IH causes changes in pancreatic function that may be not related to expression of mRNA UCP2.

Diabetes is closely associated with increased oxidative stress, especially originating from the mitochondria. A mechanism to reduce increased mitochondria superoxide production is to reduce the mitochondria membrane potential by releasing protons across the mitochondria membrane. This phenomenon is referred to as mitochondria uncoupling since oxygen is consumed independently of ATP being produced and can be mediated by Uncoupling Proteins (UCPs). However, increased oxygen consumption is potentially detrimental for the kidney since it can cause tissue hypoxia. Therefore, this thesis aimed to investigate the role of mitochondria uncoupling for development of diabetic nephropathy.      UCP-2 was demonstrated to be the only isoform expressed in the kidney, and localized to tubular segments performing the majority of tubular electrolyte transport. Streptozotocin-induced diabetes in rats increased UCP-2 protein expression and correlated to increased non-transport dependent oxygen consumption in isolated proximal tubular cells. These effects were prevented by intense insulin treatment to the diabetic animals demonstrating a pivotal role of hyperglycemia. Importantly, elevated UCP-2 protein expression increased mitochondria uncoupling in mitochondria isolated from diabetic kidneys. Mitochondria uncoupling and altered morphology was also evident in kidneys from db/db-mice, a model of type-2 diabetes, together with proteinuria and glomerular hyperfiltration which are both clinical manifestations of diabetic nephropathy. Treatment with the antioxidant coenzyme Q10 prevented mitochondria uncoupling as well as morphological and functional alterations in these kidneys. Acute knockdown of UCP-2 paradoxically increased mitochondria uncoupling in a mechanism involving the adenosine nucleotide transporter. Increased uncoupling via adenosine nucleotide transporter decreased mitochondria membrane potential and kidney oxidative stress but did not affect glomerular filtration rate, renal blood flow, total kidney oxygen consumption or intrarenal tissue oxygen tension.      The role of increased mitochondria oxygen consumption was investigated by administering the chemical uncoupler dinitrophenol to healthy rats. Importantly, increased mitochondria oxygen consumption resulted in kidney tissue hypoxia, proteinuria and increased staining of the tubular injury marker vimentin, demonstrating a crucial role of increased oxygen consumption per se and the resulting kidney tissue hypoxia for the development of nephropathy.      Taken together, the data presented in this thesis establishes an important role of mitochondria uncoupling for the development of diabetic nephropathy.