Beta-cell failure is a key step in the progression from metabolic disorder to overt type 2 diabetes (T2D). This failure is characterised by both secretory defects and loss of beta-cell mass, the latter most likely through increases in the rate of apoptosis. Although the mechanisms underlying these beta-cell defects are unclear, evidence suggests that chronic exposure of beta-cells to elevated fatty acid (FA) plays a role in disease development in genetically susceptible individuals. Furthermore, it has been postulated that endoplasmic reticulum (ER) stress signalling pathways (the unfolded protein response; UPR) play a role in FA-induced beta-cell dysfunction. The broad aim of this thesis was to explore the nature of these relationships. Experiments detailed in this thesis demonstrate that MIN6 beta-cells mount a comprehensive ER stress response with exposure to elevated saturated fatty acid palmitate, but not the unsaturated fatty acid, oleate, within the low elevated physiological range. This response was time-dependent and involved both transcriptional and translational changes in UPR transducers and targets. The differential activation of ER stress in MIN6 beta-cells by saturated, but not unsaturated FA species may represent a mechanism of differential beta-cell death described in many studies with these FA. Furthermore, these experiments describe defects in ER to Golgi trafficking with chronic palmitate treatment, but not oleate or thapsigagin treatment, identifying this as a potential mechanism by which palmitate treatment induces ER stress. Moreover, these studies have shown the relevance to ER stress to a whole body model of T2D by demonstrating UPR activation in the islets of the db/db mouse. In conclusion, studies detailed in this thesis have demonstrated that ER stress occurs in in vitro and in vivo models of beta-cell lipotoxicity and apoptosis. In addition, these studies have identified defects in ER to Golgi trafficking as a mechanism by which palmitate treatment induces ER stress. These studies highlight the importance of ER stress in the development of T2D.

Chronic hyperglycemia, a hallmark of type 2 diabetes, can decrease β-cell function and mass (β-cell glucotoxicity); however, the mechanisms are incompletely understood. The objective was to examine the mechanisms of β-cell glucotoxicity using in vivo and ex vivo models. The hypothesis is that oxidative stress plays a causal role in high glucose-induced β-cell dysfunction in vivo via pathways that involve endoplasmic reticulum (ER) stress and JNK. The model of β-cell glucotoxicity was achieved by prolonged i.v. glucose infusion (to achieve hyperglycemia). In Study 1, 48h glucose infusion increased total and mitochondrial superoxide levels in islets, and impaired β-cell function in vivo and ex vivo. Co-infusion of the superoxide dismutase mimetic Tempol decreased total and mitochondrial superoxide, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. These results suggest that increased superoxide generation plays a role in β-cell glucotoxicity. In Study 2, 48h glucose infusion increased activation of the unfolded protein response (XBP-1 mRNA splicing and phospho-eIF2α levels). This was partially prevented by Tempol. Co-infusion of the chemical chaperone 4-phenylbutyrate with glucose decreased spliced XBP-1 levels, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. Co-infusion of 4-phenylbutyrate also decreased total and mitochondrial superoxide induced by high glucose. These results suggest that 1) ER stress plays a causal role in high glucose-induced β-cell dysfunction, and 2) there is a link between oxidative stress and ER stress in high glucose-induced β-cell dysfunction in vivo. In Study 3, JNK inhibition using the inhibitor SP600125 in rats or JNK-1 null mice prevented high glucose-induced β-cell dysfunction ex vivo and in vivo. SP600125 prevented high-glucose-induced β-cell dysfunction without decreasing total and mitochondrial superoxide levels. Both Tempol and 4-phenylbutyrate prevented JNK activation induced by high glucose. These results suggest a role of JNK activation in high glucose-induced β-cell dysfunction downstream of increased superoxide generation and ER stress in vivo. Together, the results suggest that 1) oxidative stress, ER stress and JNK activation are causally involved in β-cell glucotoxicity, and 2) High glucose-induced oxidative stress and ER stress are linked, and both impair β-cell dysfunction via JNK activation in vivo.

Chronic hyperglycemia, a hallmark of type 2 diabetes, can decrease β-cell function and mass (β-cell glucotoxicity); however, the mechanisms are incompletely understood. The objective was to examine the mechanisms of β-cell glucotoxicity using in vivo and ex vivo models. The hypothesis is that oxidative stress plays a causal role in high glucose-induced β-cell dysfunction in vivo via pathways that involve endoplasmic reticulum (ER) stress and JNK. The model of β-cell glucotoxicity was achieved by prolonged i.v. glucose infusion (to achieve hyperglycemia). In Study 1, 48h glucose infusion increased total and mitochondrial superoxide levels in islets, and impaired β-cell function in vivo and ex vivo. Co-infusion of the superoxide dismutase mimetic Tempol decreased total and mitochondrial superoxide, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. These results suggest that increased superoxide generation plays a role in β-cell glucotoxicity. In Study 2, 48h glucose infusion increased activation of the unfolded protein response (XBP-1 mRNA splicing and phospho-eIF2α levels). This was partially prevented by Tempol. Co-infusion of the chemical chaperone 4-phenylbutyrate with glucose decreased spliced XBP-1 levels, and prevented high glucose-induced β-cell dysfunction in vivo and ex vivo. Co-infusion of 4-phenylbutyrate also decreased total and mitochondrial superoxide induced by high glucose. These results suggest that 1) ER stress plays a causal role in high glucose-induced β-cell dysfunction, and 2) there is a link between oxidative stress and ER stress in high glucose-induced β-cell dysfunction in vivo. In Study 3, JNK inhibition using the inhibitor SP600125 in rats or JNK-1 null mice prevented high glucose-induced β-cell dysfunction ex vivo and in vivo. SP600125 prevented high-glucose-induced β-cell dysfunction without decreasing total and mitochondrial superoxide levels. Both Tempol and 4-phenylbutyrate prevented JNK activation induced by high glucose. These results suggest a role of JNK activation in high glucose-induced β-cell dysfunction downstream of increased superoxide generation and ER stress in vivo. Together, the results suggest that 1) oxidative stress, ER stress and JNK activation are causally involved in β-cell glucotoxicity, and 2) High glucose-induced oxidative stress and ER stress are linked, and both impair β-cell dysfunction via JNK activation in vivo.

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity-related inflammation and insulin resistance in adipose tissue. However, the mechanisms responsible for induction of ER stress are presently unclear. Proper ER redox state is crucial for oxidative protein folding and secretion and impaired protein folding in ER leads to induction of unfolded protein response and ER stress. However, while ER redox state is more oxidizing compared to the rest of the cell, its regulation is poorly understood. In order to determine the effects of ER redox state on development of ER stress and insulin resistance, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. In this study we attempted to develop and characterize a sensitive tool to study the ER redox state in adipocytes in real-time by targeting a new generation of redox-sensitive green fluorescent protein (roGFP) to ER. The roGFP1-iL sensor targeted to the ER is termed ‘eroGFP1-iL’ by convention. The ER-targeting eroGFP1-iL construct contains the signal peptide from adiponectin and the ER retention motif KDEL and has a midpoint reduction potential of -229 mV in vitro in oxidized and reduced lipoic acid. Despite having a midpoint reduction potential that is 50 mV higher than the previously determined midpoint reduction potential of the ER, eroGFP1-iL was found capable of detecting both oxidizing and reducing changes in the ER. In an attempt to determine the mechanisms by which roGFP1-iL detects oxidizing changes, we found that, first, glutathione mediated the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitated dimerization of roGFP1-iL, which in effect shifted the equilibrium from oxidized monomers to dimers, thereby increasing the molecule’s reduction potential compared with a dithiol redox buffer like lipoic acid. From this study, we concluded that the glutathione redox couple in ER significantly raised the reduction potential of roGFP1-iL in vivo by facilitating its dimerization while preserving its ratiometric nature, which makes it suitable for monitoring oxidizing and reducing changes in ER with high reliability in real-time. The ability of roGFP1-iL to detect both oxidizing and reducing changes in ER and its dynamic response in glutathione redox buffer between approximately -190 and -130 mV in vitro suggest a range of ER redox potential consistent with those determined by earlier approaches that did not involve fluorescent sensors. Our primary aim in developing eroGFP1-iL as a redox-sensing tool was to be able to assess whether redox changes represent an early initiator of ER stress in obesity-induced reduction in high molecular weight (HMW) adiponectin in circulation. Hypoxia is a known mediator of redox changes. We found that oligomerization of HMW adiponectin was impaired in the hypoxic conditions observed in differentiated fat cells. The redox-active antioxidant ascorbate was found capable of reversing hypoxia-induced ER stress. Lastly, we demonstrated that changes in ER redox condition is associated with ER stress response and is implicated in the mechanism of action of the insulin-sensitizing agent troglitazone and desensitizing agent palmitate. Using the redox sensing property of eroGFP1-iL, palmitate was found to be an effective modulator of redox changes in the ER and troglitazone was found to cause oxidizing changes in the ER. The action of palmitate in causing aberrant ER redox conditions was associated with aberrant HMW adiponectin multimerization. Palmitate-induced ER stress was ameliorated by troglitazone. Taken together, the data suggest a potential role of ER redox changes in ER stress and impaired protein folding in adipocytes.

No description available.

Beta-cell failure is a key step in the progression from metabolic disorder to overt type 2 diabetes (T2D). This failure is characterised by both secretory defects and loss of beta-cell mass, the latter most likely through increases in the rate of apoptosis. Although the mechanisms underlying these beta-cell defects are unclear, evidence suggests that chronic exposure of beta-cells to elevated fatty acid (FA) plays a role in disease development in genetically susceptible individuals. Furthermore, it has been postulated that endoplasmic reticulum (ER) stress signalling pathways (the unfolded protein response; UPR) play a role in FA-induced beta-cell dysfunction. The broad aim of this thesis was to explore the nature of these relationships. Experiments detailed in this thesis demonstrate that MIN6 beta-cells mount a comprehensive ER stress response with exposure to elevated saturated fatty acid palmitate, but not the unsaturated fatty acid, oleate, within the low elevated physiological range. This response was time-dependent and involved both transcriptional and translational changes in UPR transducers and targets. The differential activation of ER stress in MIN6 beta-cells by saturated, but not unsaturated FA species may represent a mechanism of differential beta-cell death described in many studies with these FA. Furthermore, these experiments describe defects in ER to Golgi trafficking with chronic palmitate treatment, but not oleate or thapsigagin treatment, identifying this as a potential mechanism by which palmitate treatment induces ER stress. Moreover, these studies have shown the relevance to ER stress to a whole body model of T2D by demonstrating UPR activation in the islets of the db/db mouse. In conclusion, studies detailed in this thesis have demonstrated that ER stress occurs in in vitro and in vivo models of beta-cell lipotoxicity and apoptosis. In addition, these studies have identified defects in ER to Golgi trafficking as a mechanism by which palmitate treatment induces ER stress. These studies highlight the importance of ER stress in the development of T2D.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER protein folding capacity to restore protein folding homeostasis. Unfolded proteins activate UPR signalling across the ER membrane to the nucleus by promoting oligomerisation of IRE1, a conserved transmembrane ER stress receptor. Despite significant research, the mechanism of coupling ER stress to IRE1 oligomerisation and activation has remained contested. There are two proposed mechanisms by which IRE1 may sense accumulating unfolded proteins. In the direct binding mechanism, unfolded proteins are able to bind directly to IRE1 to drive its oligomerisation. In the chaperone inhibition mechanism, unfolded proteins compete for the repressive BiP bound to IRE1 leaving IRE1 free to oligomerise. Currently, these two mechanisms respectively lack compelling in vivo and in vitro evidence required to assess their validity. The work presented here first describes in vivo experiments that identify a role of the ER co-chaperone ERdj4 as an IRE1 repressor that promotes a complex between the luminal Hsp70 BiP and the luminal stress-sensing domain of IRE1α (IRE1LD). This is then built on by a series of in vitro experiments showing that ERdj4 catalyses formation of a repressive BiP-IRE1LD complex and that this complex can be disrupted by the presence of competing unfolded protein substrates to restore IRE1LD to its default, dimeric, and active state. The identification of ERdj4 and the in vitro reconstitution of chaperone inhibition establish BiP and its J-domain co-chaperones as key regulators of the UPR. This thesis also utilises the power of Cas9-CRISPR technology to introduce specific mutations into the endogenous IRE1α locus and to screen for derepressing IRE1α mutations. Via this methodology, two predicted unstructured regions of IRE1 are found to be important for IRE1 repression. Finally, this thesis challenges recent in vitro findings concerning the direct binding mechanism.

A neurodegeneração é o resultado da destruição progressiva e irreversível dos neurônios no sistema nervoso central, apresentando causas desconhecidas e mecanismos patológicos não totalmente elucidados. Fatores como a idade, o aumento da formação de radicais livres e/ou estresse oxidativo, defeito no metabolismo energético, a inflamação e acúmulo de elementos neurotóxicos e de proteínas malformadas no lúmen do retículo endoplasmático (RE) contribuem para o desenvolvimento dos processos neurodegenerativos. O sistema canabinoide tem sido proposto como neuroprotetor em diversos modelos de neurodegeneração como hipóxia aguda e epilepsia, isquemia cerebral, lesão cerebral e modelos de estresse oxidativo. Assim, este trabalho teve como objetivo investigar o papel do sistema canabinoide em uma linhagem de neuroblastoma (Neuro 2a) submetida a condições de estresse oxidativo (H2O2), inflamação (LPS) e estresse do RE (tunicamicina), avaliando parâmetros de viabilidade celular e vias de sinalização envolvidas. Nossos resultados mostram que o agonista canabinoide ACEA foi capaz de proteger as células da morte celular causada pela inflamação e pelo estresse de retículo endoplasmático, mas não pelo estresse oxidativo. Esse efeito neuroprotetor exercido pelo ACEA parece pelo menos em parte ocorrer via receptor CB1 no modelo de inflamação e ser independente deste receptor no modelo de estresse de RE. Os efeitos neuroprotetores observados envolveram a modulação dos níveis de proteínas pré-apoptóticas, CHOP e Caspase 12, e da proteína relacionada à sobrevivência celular ERK 1/2. Nossos dados sugerem um papel neuroprotetor do sistema canabinoide em mecanismos relacionados aos processos neurodegenerativos e propõem a manipulação desse sistema como possível alvo terapêutico. / Neurodegeneration is the result of progressive and irreversible destruction of neurons in the central nervous system, with unknown causes and pathological mechanisms not fully elucidated. Factors such as age, increased formation of free radicals and/or oxidative stress, defects in energetic metabolism, inflammation and accumulation of neurotoxic factors and misfolded proteins in the lumen of the endoplasmic reticulum (ER) contribute to the development of neurodegenerative processes. The cannabinoid system has been proposed as neuroprotector in several models of neurodegeneration such as acute hypoxia and epilepsy, cerebral ischaemia, brain injury and oxidative stress models. This work aimed to investigate the role of the cannabinoid system in a neuroblastoma line (Neuro 2a) submitted to oxidative stress (H2O2), inflammation (LPS) and ER stress (tunicamycin) conditions, assessing cell viability parameters and signaling pathways involved. Our results show that the ACEA cannabinoid agonist was able to protect cells from cell death caused by inflammation and ER stress, but not from oxidative stress. This neuroprotective effect exerted by ACEA appears to occur at least in part via the CB1 receptor in inflammation model and it seems to be independent of this receptor in the ER stress model. The neuroprotective effects observed involved the modulation of the levels of pre-apoptotic proteins CHOP and Caspase 12 and the cell survival related protein ERK 1/2. Our data suggest a neuroprotective role of the cannabinoid system in mechanisms related to neurodegenerative processes and propose it as possible therapeutic target.

Recentemente, verificou-se que diferentes protocolos de overtraining (OT) com mesma carga externa, mas realizados em declive (OTR/down), aclive (OTR/up) e sem inclinação (OTR) levaram ao acúmulo de gordura hepática. Sabe-se que perturbando a homeostase do retículo endoplasmático (ER) ocorre o estresse do RE que também está associada com presença de gordura hepática em modelos animais. Com isso, verificamos os efeitos desses modelos de OT nas proteinas relacionadas ao estresse do RE (BiP, IRE1, PERK, eIF2alpha, ATF6beta, and GRP94), apoptose (CHOP, Caspase-3, 4 and 12, Bax and TRAF2) e inflamação (SAPKJNK and IKK) no fígado de camundongos C57BL/6. Uma vez que o treinamento aeróbio pode diminuir o estresse do RE cardíaco e aumentar a capacidade do exercício, também verificou se a queda de desempenho induzida pelos protocolos de OT estaria relacionada com o estresse do RE e apoptose no coração dos animais. Os animais foram divididos em naive (N, animais sedentários), controle (CT, animais sedentários submetidos as avaliações de desempenho), treinado (TR), OTR/down, OTR/up and OTR. Os testes de rotarod, incremental, exaustivo e força de preensão foram usados para avaliar o desempenho. Trinta e seis horas após o teste de força de preensão, os fígados e corações (ventrículo esquerdo) foram removidos e usados para técnica de immunoblotting. Todos os protocolos de OT levaram a respostas similares em relação aos parâmetros de desempenho e mostraram valores significativamente menores de ATF6beta hepática quando comparados com o grupo N. O grupo OTR/down exibiu valores inferiores de caspase-3 clivada no fígado quando comparado com o grupo CT. As proteínas cardíacas relacionadas ao estresse do RE, apoptose e inflamação não foram moduladas nos grupos experimentais. / Newly, we verified that different running overtraining (OT) protocols with same external load but performed in downhill (OTR/down), uphill (OTR/up) and without inclination (OTR), directed to hepatic fat accumulation. Knowing the disruption of endoplasmic reticulum (ER) homeostasis is linked to animal models of fatty liver, we explored the effects of these OT models on the proteins related to ER stress (i.e., BiP, IRE1, PERK, eIF2alpha, ATF6beta, and GRP94), apoptosis (CHOP, Caspase-3, 4 and 12, Bax and TRAF2) and inflammation (SAPKJNK and IKK) in livers of C57BL/6 mice. Because aerobic training can diminish cardiac ER stress and increase exercise capacity, we also verified whether the performance decrease induced by our OT protocols is linked to ER stress and apoptosis in mouse hearts. Rodents were divided into naive (N. sedentary mice), control (CT, sedentary mice submitted to the performance evaluations), trained (TR), OTR/down, OTR/up and OTR groups. Rotarod, incremental load, exhaustive and grip force tests were used to estimate performance. Thirteen six hours after the grip force test, the livers and cardiac muscles (i.e., left ventricle) were removed and used for immunoblotting. All OT protocols led to similar responses of the performance parameters and showed significantly lower values of hepatic ATF6beta compared to the N group. The OTR/down group exhibited inferior values of liver cleaved caspase-3 compared to the CT group. The cardiac proteins related to ER stress and apoptosis were not modulated in the experimental groups.

Orientador: Maria Marluce dos Santos Vilela / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T17:02:47Z (GMT). No. of bitstreams: 1 Ramalho_VanessaDomingues_D.pdf: 1441004 bytes, checksum: a45d3f4f8825288e2959115ef25fc13a (MD5) Previous issue date: 2014 / Resumo: A agamaglobulinemia ligada ao X (XLA; OMIM#300755) é caracterizada por um bloqueio na diferenciação dos linfócitos B na medula óssea, levando à profunda hipogamaglobulinemia e reduzido número ou ausência de linfócitos B periféricos. Os pacientes são susceptíveis a infecções recorrentes por bactérias encapsuladas e enterovírus. XLA é causada por mutações no gene da tirosina quinase de Bruton (BTK). Contudo, não há estudos de relação entre expressão protéica e o tipo de mutação, nem sobre as conseqüências da retenção intracelular do excesso de proteínas mal formadas. Os objetivos deste trabalho foram avaliar a expressão de BTK e sua relação com o tipo de mutação em pacientes com XLA, assim como verificar suas conseqüências nos sensores de estresse do retículo endoplasmático. O diagnóstico de XLA foi baseado em infecções recorrentes, níveis significativamente reduzidos de IgM, IgG e IgA, linfócitos B circulantes <2% e mutação identificada no gene BTK. A expressão dos transcritos de BTK foi avaliada por PCR quantitativo em tempo real em oito pacientes XLA e oito controles. Pela mesma técnica, foi avaliada a expressão de 10 genes do estresse do retículo endoplasmático em seis pacientes e seis controles. Foram caracterizadas quatro mutações missense, uma mutação nonsense, dois frameshifts e um defeito em sítio de splicing. As mutações do tipo nonsense, frameshift e defeito em sítio de splicing levaram à formação de stop codon prematuro. Foi detectado um perfil de expressão de BTK diferenciado nos pacientes com mutações com stop codon prematuro em comparação aos pacientes com mutações missense e controles saudáveis. Especificamente, os pacientes com mutações com stop codon prematuro apresentaram redução da expressão de BTK (P = 0,004). No entanto, verificamos que as mutações missense não afetaram a expressão de BTK. Por meio de imunocitoquímica, encontramos que as mutações com stop codon prematuro levaram à deficiência da expressão da proteína BTK e as do tipo missense resultaram na localização anormal da proteína no citoplasma celular, o que evidencia a síntese de proteína não funcional. Os pacientes com XLA apresentaram expressão aumentada do marcador de estresse do retículo endoplasmático XBP1 (P = 0,002). Em conclusão, a quantificação da expressão de mRNA para BTK é uma ferramenta para diferenciar as conseqüências mutacionais em pacientes com XLA. Ela também pode contribuir para o estudo de transcritos em outras doenças genéticas com diferentes tipos de mutação. Este é o primeiro relato de estresse do retículo endoplasmático na agamaglobulinemia ligada ao X / Abstract: X-linked agammaglobulinemia (XLA, OMIM # 300755) is characterized by a block in differentiation of B lymphocytes in the bone marrow, leading to profound hypogammaglobulinemia and few or no peripheral B lymphocytes. Patients are susceptible to recurrent infections by encapsulated bacteria and enteroviruses. XLA is caused by mutations in the Bruton tyrosine kinase gene (BTK). However, there have been no studies on the relationship between protein expression and the type of mutation, nor on the consequences of the disruption of protein folding that results in intracellular retention. The objectives of this study were to evaluate BTK expression and its mutation type in patients with XLA, as well as to verify their consequences on the endoplasmic reticulum stress sensors. The XLA diagnosis was based on recurrent infections, significantly reduced levels of IgM, IgG and IgA, circulating B lymphocytes <2% and BTK gene mutation identified. The expression of BTK transcripts was assessed by quantitative real-time PCR in eight XLA patients and eight control subjects. By the same technique, the expression of 10 endoplasmic reticulum stress genes was measured in six patients and six controls. Four missense mutations, one nonsense mutation, two frameshifts and a splice site defect were characterized. Mutations of the nonsense type, frameshift and splice site defect led to a premature stop codon formation. A differential profile of expression of BTK was detected in patients with mutations that led to a premature stop codon compared to patients with missense mutations and healthy controls. Specifically, patients with mutations resulting in a premature stop codon exhibited reduced expression of BTK gene (P = 0.004). However, it was found that missense mutations did not affect BTK expression. By immunocytochemistry, we found that mutations with a premature stop codon impaired expression of BTK protein and that missense mutations led to an abnormal localization of the protein in the cell cytoplasm, showing the synthesis of a non-functional protein. Patients with XLA showed increased expression of the endoplasmic reticulum stress marker XBP1 (P = 0.002). In conclusion, the quantification of mRNA expression for BTK is a tool to differentiate mutational consequences in patients with XLA. It can also contribute to the study of transcripts in other genetic diseases with different types of mutation. This is the first report on endoplasmic reticulum stress in X-linked agammaglobulinemia / Doutorado / Saude da Criança e do Adolescente / Doutora em Ciências