Metabólitos acumulados na acidemia propiônica comprometem a bioenergética mitocondrial e a homeostase do cálcio em coração e rins de ratos

A acidemia propiônica é um erro inato do metabolismo causado pela deficiência na atividade da enzima mitocondrial propionil-CoA-carboxilase (PCC). A doença é bioquimicamente caracterizada por predominante acúmulo de ácido propiônico (PA) no plasma e clinicamente por severa encefalopatia, além de cardiomiopatia e doença renal crônica. Alta excreção urinária dos ácidos 3-hidroxipropiônico (3OHPA), 2-metilcítrico (2MCA) e maleico (MA) também é reportada nos pacientes. Considerando que os mecanismos patogênicos das alterações cardíacas e renais são praticamente desconhecidos nessa doença, a presente tese investigou os efeitos do PA, 3OHPA, MA e 2MCA (0,05 – 5 mM) sobre críticos parâmetros da homeostase mitocondrial bioenergética e do Ca2+ em preparações mitocondriais e homogeneizados obtidos de coração e rim de ratos Wistar jovens (30 dias de vida), bem como em cultura de células cardíacas (H9c2) e renais (HEK-293). Foram determinados os parâmetros respiratórios estado 3 (estimulado por ADP), estado 4 (estimulado por oligomicina), estado desacoplado (estimulado por CCCP) e razão do controle respiratório (RCR), o potencial de membrana (ΔΨm), o conteúdo de NAD(P)H, a capacidade de retenção de Ca2+ e o inchamento mitocondrial. Além disso, foram avaliados a produção de ATP, a atividade de enzimas do ciclo do ácido cítrico e dos complexos da cadeia respiratória. A viabilidade celular também foi investigada após 24 horas de pré-incubação com o PA e MA. Observou-se que o MA inibiu os estados 3, 4 e desacoplado em preparações mitocondriais de coração e rim utilizando principalmente substratos precursores de NADH (glutamato mais malato, piruvato mais malato, glutamato ou α-cetoglutarato) em comparação ao FADH2 (succinato), que foram associados a uma diminuição na produção de ATP. Por outro lado, o PA e o 3OHPA provocaram efeitos inibitórios sobre a respiração mitocondrial mais moderados, enquanto o 2MCA não causou alterações. Os efeitos marcantes causados pelo MA podem estar em parte relacionados à inibição da α-cetoglutarato desidrogenase e glutamato desidrogenase, além de depleção de CoA pois a inibição da respiração mitocondrial causada pelo MA foi parcialmente revertida por suplementação dessa coenzima. Resultados similares foram obtidos em homogeneizados e cultura de células permeabilizadas desses tecidos, corroborando os achados obtidos em mitocôndrias isoladas. Somando-se a isso, o MA, e o PA em menor extensão, reduziram o ΔΨm, o conteúdo de NAD(P)H e a capacidade de retenção de Ca2+, bem como induziram inchamento em preparações mitocondriais de coração e rim na presença de Ca2+. Esses efeitos foram parcial ou totalmente prevenidos por ciclosporina A e ADP, sugerindo que a abertura do poro de transição de permeabilidade (PTP) mitocondrial possa estar envolvido. Finalmente, o MA reduziu a viabilidade e induziu morte em células renais. Analisando em conjunto, os resultados indicam que o MA, além do PA e 3OHPA de forma mais branda, atuam como inibidores metabólicos, bem como o MA e o PA induzem a abertura PTP, em coração e rins, comprometendo dessa forma a homeostase mitocondrial bioenergética e do Ca2+. Portanto, presume-se que uma disfunção mitocondrial causada pelos metabólitos acumulados na acidemia propiônica possa contribuir para a cardiomiopatia e insuficiência renal crônica que se manifestam nos pacientes acometidos por essa doença.Propionic acidemia is an inborn error of metabolism caused by a deficiency in the mitochondrial enzyme propionyl-CoA-carboxylase (PCC) activity. The disease is biochemically characterized by predominant propionic acid (PA) accumulation in plasma and clinically by severe encephalopathy, besides cardiomyopathy and chronic kidney disease. High urinary excretion of 3-hydroxypropionic (3OHPA), 2-methylcitric (2MCA), and maleic (MA) acids is also reported in patients. The pathogenic mechanisms of cardiac and renal alterations are practically unknown in this disease, the present thesis investigated the effects of PA, 3OHPA, MA, and 2MCA (0.05 – 5 mM) on critical parameters of mitochondrial bioenergetics and Ca2+ homeostasis in isolated mitochondria and homogenates obtained from heart and kidney of young Wistar rats (30 days old), as well as from cardiac (H9c2) and renal (HEK-293) cell cultures. The respiratory parameters state 3 (ADP-stimulated), state 4 (oligomycin- stimulated), uncoupled state (CCCP- stimulated) and respiratory control ratio (RCR), membrane potential (ΔΨm), NAD(P)H content, Ca2+ retention capacity, and mitochondrial swelling were determined. In addition, ATP production, citric acid cycle enzymes, and respiratory chain complexes activities were evaluated. Cellular viability was also investigated after 24 hours of pre-incubation with PA and MA. It was observed that MA inhibited states 3, 4 and uncoupled respiration in heart and kidney mitochondria using primarily NADH-linked substrates (glutamate plus malate, pyruvate plus malate, glutamate or α-ketoglutarate) as compared to FADH2 (succinate), which were associated with a decrease in ATP production. PA and 3OHPA caused milder inhibitory effects on mitochondrial respiration, and 2MCA did not cause any changes. The marked effects caused by MA may be related to the inhibition of α-ketoglutarate dehydrogenase and glutamate dehydrogenase, and CoA depletion since the inhibition of mitochondrial respiration caused by MA was partially reversed by supplementation of this coenzyme. Similar results were obtained in tissue homogenates and permeabilized cell cultures, corroborating the findings obtained in isolated mitochondria. In addition, MA, and PA, to a lesser extent, reduced the ΔΨm, NAD(P)H content, Ca2+ retention capacity, and induced swelling in Ca2+-loaded heart and kidney mitochondrial preparations. These effects were partial or totally prevented by cyclosporine A and ADP, suggesting mitochondrial permeability transition (MPT) pore opening may be involved. Finally, MA reduced viability and induced kidney cells death. Taken together, the data indicate that MA, and PA and 3OHPA, to a lesser degree, acts as metabolic inhibitors, as well as MA and PA induce MPT in the heart and kidney, thus compromising mitochondrial energy and Ca2+ homeostasis. Therefore, it is presumed that mitochondrial dysfunction caused by the metabolites accumulated in propionic acidemia may contribute to the cardiomyopathy and chronic renal failure that manifest in patients affected by this disease

Uncovering actions of type 3 deiodinase in the metabolic dysfunction-associated fatty liver disease (MAFLD)

Metabolic dysfunction-associated fatty liver disease (MAFLD) has gained worldwide attention as a public health problem. Nonetheless, lack of enough mechanistic knowledge restrains effective treatments. It is known that thyroid hormone triiodothyronine (T3) regulates hepatic lipid metabolism, and mitochondrial function. Liver dysfunction of type 3 deiodinase (D3) contributes to MAFLD, but its role is not fully understood. Objective: To evaluate the role of D3 in the progression of MAFLD in an animal model. Methodology: Male/adult Sprague Dawley rats (n = 20) were allocated to a control group (2.93 kcal/g) and high-fat diet group (4.3 kcal/g). Euthanasia took place on the 28th week. D3 activity and expression, Uncoupling Protein 2 (UCP2) and type 1 deiodinase (D1) expression, oxidative stress status, mitochondrial, Krebs cycle and endoplasmic reticulum homeostasis in liver tissue were measured. Results: We observed an increase in D3 activity/expression (p < 0.001) related to increased thiobarbituric acid reactive substances (TBARS) and carbonyls and diminished reduced glutathione (GSH) in the MAFLD group (p < 0.05). There was a D3-dependent decrease in UCP2 expression (p = 0.01), mitochondrial capacity, respiratory activity with increased endoplasmic reticulum stress in the MAFLD group (p < 0.001). Surprisingly, in an environment with lower T3 levels due to high D3 activity, we observed an augmented alpha-ketoglutarate dehydrogenase (KGDH) and glutamate dehydrogenase (GDH) enzymes activity (p < 0.05). Conclusion: Induced D3, triggered by changes in the REDOX state, decreases T3 availability and hepatic mitochondrial capacity. The Krebs cycle enzymes were altered as well as endoplasmic reticulum stress. Taken together, these results shed new light on the role of D3 metabolism in MAFLD

Early effects of LPS-induced neuroinflammation on the rat hippocampal glycolytic pathway

Neuroinflammation is a common feature during the development of neurological disorders and neurodegenerative diseases, where glial cells, such as microglia and astrocytes, play key roles in the activation and maintenance of inflammatory responses in the central nervous system. Neuroinflammation is now known to involve a neurometabolic shift, in addition to an increase in energy consumption. We used two approaches (in vivo and ex vivo) to evaluate the effects of lipopolysaccharide (LPS)-induced neuroinflammation on neurometabolic reprogramming, and on the modulation of the glycolytic pathway during the neuroinflammatory response. For this, we investigated inflammatory cytokines and receptors in the rat hippocampus, as well as markers of glial reactivity. Mitochondrial respirometry and the glycolytic pathway were evaluated by multiple parameters, including enzymatic activity, gene expression and regulation by protein kinases. Metabolic (e.g., metformin, 3PO, oxamic acid, fluorocitrate) and inflammatory (e.g., minocycline, MCC950, arundic acid) inhibitors were used in ex vivo hippocampal slices. The induction of early inflammatory changes by LPS (both in vivo and ex vivo) enhanced glycolytic parameters, such as glucose uptake, PFK1 activity and lactate release. This increased glucose consumption was independent of the energy expenditure for glutamate uptake, which was in fact diverted for the maintenance of the immune response. Accordingly, inhibitors of the glycolytic pathway and Krebs cycle reverted neuroinflammation (reducing IL-1β and S100B) and the changes in glycolytic parameters induced by LPS in acute hippocampal slices. Moreover, the inhibition of S100B, a protein predominantly synthesized and secreted by astrocytes, inhibition of microglia activation and abrogation of NLRP3 inflammasome assembly confirmed the role of neuroinflammation in the upregulation of glycolysis in the hippocampus. Our data indicate a neurometabolic glycolytic shift, induced by inflammatory activation, as well as a central and integrative role of astrocytes, and suggest that interference in the control of neurometabolism may be a promising strategy for downregulating neuroinflammation and consequently for diminishing negative neurological outcomes

Health-related quality of life in patients with type 1 diabetes mellitus in the different geographical regions of Brazil : data from the Brazilian Type 1 Diabetes Study Group

Background: In type 1 diabetes mellitus (T1DM) management, enhancing health-related quality of life (HRQoL) is as important as good metabolic control and prevention of secondary complications. This study aims to evaluate possible regional differences in HRQoL, demographic features and clinical characteristics of patients with T1DM in Brazil, a country of continental proportions, as well as investigate which variables could influence the HRQoL of these individuals and contribute to these regional disparities. Methods: This was a retrospective, cross-sectional, multicenter study performed by the Brazilian Type 1 Diabetes Study Group (BrazDiab1SG), by analyzing EuroQol scores from 3005 participants with T1DM, in 28 public clinics, among all geographical regions of Brazil. Data on demography, economic status, chronic complications, glycemic control and lipid profile were also collected. Results: We have found that the North-Northeast region presents a higher index in the assessment of the overall health status (EQ-VAS) compared to the Southeast (74.6 ± 30 and 70.4 ± 19, respectively; p < 0.05). In addition, North- Northeast presented a lower frequency of self-reported anxiety-depression compared to all regions of the country (North-Northeast: 1.53 ± 0.6; Southeast: 1.65 ± 0.7; South: 1.72 ± 0.7; Midwest: 1.67 ± 0.7; p < 0.05). These findings could not be entirely explained by the HbA1c levels or the other variables examined. Conclusions: Our study points to the existence of additional factors not yet evaluated that could be determinant in the HRQoL of people with T1DM and contribute to these regional disparities

Impairment of mitochondrial bioenergetics and permeability transition induction caused by major long-chain fatty acids accumulating in VLCAD deficiency in skeletal muscle as potential pathomechanisms of myopathy

cis-5-Tetradecenoic (cis-5) and myristic (Myr) acids predominantly accumulate in patients affected by very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. They commonly manifest myopathy with muscular pain and rhabdomyolysis, whose underlying mechanisms are poorly known. Thus, in the present study we investigated the effects of cis-5 and Myr on mitochondrial bioenergetics and Ca2+ homeostasis in rat skeletal muscle. cis-5 and Myr decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration, especially when mitochondria were supported by NADH-linked as compared to FADH(2)-linked substrates. In contrast, these fatty acids increased resting respiration (state 4). Similar effects were observed in skeletal muscle fibers therefore validating the data obtained with isolated mitochondria. Furthermore, cis-S and Myr markedly decreased mitochondrial membrane potential and Ca2+ retention capacity that were avoided by cyclosporin A plus ADP and ruthenium red, indicating that cis-5 and Myr induce mitochondrial permeability transition (MPT). Finally, docosanoic acid did not disturb mitochondria] homeostasis, indicating selective effects for Myr and cis-5. Taken together, our findings indicate that major long-chain fatty acids accumulating in VLCAD deficiency behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and MPT inducers. It is presumed that these pathomechanisms contribute to the muscular symptoms and rhabdomyolysis observed in patients affected by VLCAD deficiency62CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DO RIO GRANDE DO SUL - FAPERGS425914/2016-017/17728-82266-2551/14-

Análise da qualidade de prescrições de antimicrobianos comercializados em uma drogaria da Região Norte do Rio Grande do Sul

A resistência bacteriana tem sido descrita como preocupação mundial e está relacionada a diversos fatores como: uso abusivo de antimicrobianos, automedicação, falta de conhecimento e/ou descumprimento por parte dos profissionais envolvidos no tratamento. Frente a isso, a Agência Nacional de Vigilância Sanitária (ANVISA) criou a Resolução de Diretoria Colegiada (RDC) 20/2011 com a finalidade de facilitar a dispensação e controlar o consumo de antimicrobianos. O presente estudo tem a finalidade de analisar prescrições de antimicrobianos orais do ano de 2014 em uma drogaria no município de Erechim-RS, avaliando a sazonalidade climática, a prescrição pela Denominação Comum Brasileira (DCB) e a adequação das prescrições frente a RDC 20/2011. O total de prescrições analisadas foi de 2761. O antimicrobiano mais prescrito foi a Amoxacilina sendo prescrita 1069 vezes, destas 630 estavam em associação com Ácido Clavulânico. Apenas 36,28% das prescrições (1002) estavam com os antimicrobianos descritos pela DCB. Do total de prescrições, 97,57% das mesmas não estavam de acordo com a RDC 20/2011, apresentando a falta de vários itens como idade, sexo, DCB, nome completo do paciente, entre outros. É necessário elaborar mais estudos, campanhas de conscientização tanto para pacientes como para profissionais envolvidos no tratamento, e ter um maior controle tanto na prescrição como na dispensação destes medicamentos

Disturbance of bioenergetics and calcium homeostasis provoked by metabolites accumulating in propionic acidemia in heart mitochondria of developing rats

Propionic acidemia is caused by lack of propionyl-CoA carboxylase activity. It is biochemically characterized by accumulation of propionic (PA) and 3-hydroxypropionic (3OHPA) acids and clinically by severe encephalopathy and cardiomyopathy. High urinary excretion of maleic acid (MA) and 2-methylcitric acid (2MCA) is also found in the affected patients. Considering that the underlying mechanisms of cardiac disease in propionic acidemia are practically unknown, we investigated the effects of PA, 3OHPA, MA and 2MCA (0.05–5 mM) on important mitochondrial functions in isolated rat heart mitochondria, as well as in crude heart homogenates and cultured cardiomyocytes. MA markedly inhibited state 3 (ADP-stimulated), state 4 (non-phosphorylating) and uncoupled (CCCP-stimulated) respiration in mitochondria supported by pyruvate plus malate or α-ketoglutarate associated with reduced ATP production, whereas PA and 3OHPA provoked less intense inhibitory effects and 2MCA no alterations at all. MA-induced impaired respiration was attenuated by coenzyme A supplementation. In addition, MA significantly inhibited α-ketoglutarate dehydrogenase activity. Similar data were obtained in heart crude homogenates and permeabilized cardiomyocytes. MA, and PA to a lesser degree, also decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and Ca2+ retention capacity, and caused swelling in Ca2+-loaded mitochondria. Noteworthy, ΔΨm collapse and mitochondrial swelling were fully prevented or attenuated by cyclosporin A and ADP, indicating the involvement of mitochondrial permeability transition. It is therefore proposed that disturbance of mitochondrial energy and calcium homeostasis caused by MA, as well as by PA and 3OHPA to a lesser extent, may be involved in the cardiomyopathy commonly affecting propionic acidemic patients18665CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DO RIO GRANDE DO SUL - FAPERGS425914/2016-017/17728-817/2551-0000/800-

Experimental evidence that maleic acid markedly compromises glutamate oxidation through inhibition of glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase activities in kidney of developing rats

Maleic acid (MA), which has been reported to be highly excreted in propionic acidemia (PAcidemia), was demonstrated to cause nephropathy by bioenergetics impairment and oxidative stress, but the effects on kidney mitochondrial respiration has not yet been properly investigated. Therefore, the present study investigated the effects of MA (0.05-5mM), as well as of propionic (PA) and 3-hydroxypropionic (3OHPA) acids (5mM) that accumulate in PAcidemia, on mitochondrial respiration supported by glutamate, glutamate plus malate or succinate in mitochondrial fractions and homogenates from rat kidney, as well as in permeabilized kidney cells. MA markedly decreased oxygen consumption in state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respiration in glutamate and glutamate plus malate-respiring mitochondria, with less prominent effects when using succinate. We also found that PA significantly decreased state 3 and uncoupled respiration in glutamate- and glutamate plus malate-supported mitochondria, whereas 3OHPA provoked milder or no changes. Furthermore, glutamate dehydrogenase and alpha-ketoglutarate dehydrogenase activities necessary for glutamate oxidation were significantly inhibited by MA in a dose-dependent and competitive fashion. The MA-induced decrease of state 3 and uncoupled respiration found in mitochondrial fractions were also observed in homogenates and permeabilized renal cells that better mimic the in vivo cellular milieu. Taken together, our data indicate that MA, and PA to a lesser extent, disturb mitochondrial-oxidative metabolism in the kidney with the involvement of critical enzymes for glutamate oxidation. It is postulated that our present findings may be possibly involved in the chronic renal failure observed in patients with PAcidemia4581-299112CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPERGS - Fundação de Amparo à Pesquisa Do Estado Do Rio Grande Do SulFINEP - Financiadora de Estudos E Projetosnão tem404883/2013-317/2551-0000/800-601.06.0842-00573677/2008-5INCT - Instituto Nacional de Ciência e Tecnologia em Excitotoxicidade e Neuroproteçã

Uncovering Actions of Type 3 Deiodinase in the Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD)

Metabolic dysfunction-associated fatty liver disease (MAFLD) has gained worldwide attention as a public health problem. Nonetheless, lack of enough mechanistic knowledge restrains effective treatments. It is known that thyroid hormone triiodothyronine (T3) regulates hepatic lipid metabolism, and mitochondrial function. Liver dysfunction of type 3 deiodinase (D3) contributes to MAFLD, but its role is not fully understood. Objective: To evaluate the role of D3 in the progression of MAFLD in an animal model. Methodology: Male/adult Sprague Dawley rats (n = 20) were allocated to a control group (2.93 kcal/g) and high-fat diet group (4.3 kcal/g). Euthanasia took place on the 28th week. D3 activity and expression, Uncoupling Protein 2 (UCP2) and type 1 deiodinase (D1) expression, oxidative stress status, mitochondrial, Krebs cycle and endoplasmic reticulum homeostasis in liver tissue were measured. Results: We observed an increase in D3 activity/expression (p p p = 0.01), mitochondrial capacity, respiratory activity with increased endoplasmic reticulum stress in the MAFLD group (p p < 0.05). Conclusion: Induced D3, triggered by changes in the REDOX state, decreases T3 availability and hepatic mitochondrial capacity. The Krebs cycle enzymes were altered as well as endoplasmic reticulum stress. Taken together, these results shed new light on the role of D3 metabolism in MAFLD