Modulação do fluxo autofágico pelo decanoato de nandrolona em hipotálamo de camundongos envelhecidos

INTRODUÇÃO: O envelhecimento recente da população mundial têm apresentado à ciência e medicina moderna novos desafios e questionamentos quanto aos mecanismos neurobiológicos do envelhecimento. O envelhecimento fisiológico é responsável por um grupo de características clínicas marcantes, tais como declínio do metabolismo energético, perda da proteostase gerando acúmulo protéico, bem como, um significativo desbalanço na regulação hormonal androgênica. Atribui-se ao desbalanço da homeostase protéica disfunções no sistema autofágico. Neste contexto destaca-se a importância do tecido hipotalâmico como importante alvo dos hormônios gonadais, bem como regulador do metabolismo energético global. Baseado nestas premissas, a administração de hormônios androgênicos esteroides exógenos têm sido proposta desde o século passado, visando reverter sintomas clínicos característicos do envelhecimento. Entretanto, a modulação da maquinaria autofágica pelos hormônios esteroides e o seu impacto no balanço energético ainda não foram definidos. MÉTODOS: Neste trabalho administramos decanoato de nandrolona (ND, 15 mg/Kg, s.c.) por 15 dias em camundongos CF1 machos de 3 meses 18 meses. Após o sacrifício, foram analisadas proteínas da maquinaria autofágica no hipotálamo. RESULTADOS: Os animais envelhecidos apresentam um acúmulo de autofagossomos evidenciado pela redução na razão LC3II/LC3I e o tratamento com ND reverteu este perfil. Observamos um aumento no imunoconteúdo de Beclin-1 nos animais envelhecidos independentemente de ND. Finalmente, o tratamento com ND aumentou a ativação da via AMPK. CONCLUSÃO: Este estudo demonstra um acúmulo de autofagossomos associado ao envelhecimento e o tratamento com ND ativa o fluxo autofágico de maneira AMPK-dependente.INTRODUCTION: Populational ageing has presented modern science and medicine new challenges and questions about the neurobiological mechanisms of aging. Physiological aging is responsible for a group of marked clinical features, such as a decline in energy metabolism, loss of proteostase, generating protein accumulation, as well as a significant imbalance in androgen hormonal regulation. It is attributed a dysfunction in the autophagic system the imbalance of protein homeostasis. In this context, the importance of hypothalamic tissue as an important target of gonadal hormones as well as regulator of global energy metabolism is highlighted. Based on these premises, the administration of exogenous steroid androgenic hormones has been proposed since the last century, aiming to revert clinical symptoms characteristic of aging. However, the modulation of autophagic machinery by steroid hormones and its impact on energy balance have not yet been defined. METHODS: In this work we administered nandrolone decanoate (ND, 15 mg / kg, s.c.) for 15 days in male CF1 mice aged 18 months and 3 months. After the euthanasia, proteins from the autophagic machinery were analyzed in the hypothalamus. RESULTS: Aged animals presented an accumulation of autophagosomes evidenced by the reduction in LC3II / LC3I ratio, and treatment with ND reversed this profile. We observed an increase in the immunocontent of Beclin-1 in aged animals independently of ND. Finally, treatment with ND increased the activation of the AMPK pathway. CONCLUSION: This study demonstrates an accumulation of autophagosomes associated with aging and treatment with ND activates autophagic flow in an AMPK-dependent manne

Decanoato de nandrolona e AMPK modulam a macroautofagia e bioenergética cerebral no envelhecimento

Sabe-se que alterações na homeostase energética e proteica ao longo do envelhecimento, estão associadas com doenças neurodegenerativas. Fisiologicamente, a mitocôndria funciona como uma organela integradora do metabolismo energético oxidativo influenciando o sistema de depuração de proteínas desenoveladas e organelas disfuncionais, autofagia. O envelhecimento pode levar à ruptura funcional destes sistemas resultando em prejuízos neuroenergéticos e acúmulo de proteínas tóxicas. Neste sentido, o declínio nos níveis de testosterona, um hormônio anabólico, tem sido proposto como um potencializador do declínio das funções fisiológicas no envelhecimento. O objetivo deste estudo foi investigar se a suplementação com decanoato de nandrolona (ND), um derivado sintético da testosterona, em animais envelhecidos melhora o metabolismo energético mitocondrial e mecanismos autofágicos. Camundongos machos albino CF1 de 3 e 18 meses foram submetidos à análise basal de captação de fluorodesoxiglicose (18FDG) através de tomografia por emissão de pósitrons (microPET-FDG). Posteriormente eles foram alocados em 4 grupos que receberam uma injeção diária subcutânea de ND (15mg/kg; Grupos 3 m/ND e 18 m/ND) ou veículo oleoso (VEH) (Grupos 3 m/VEH; 18 m/VEH), durante 15 dias. Os animais foram resubmetidos à análise de microPET-FDG, e logo após a eutanásia foi realizada a respirometria de alta resolução, para avaliação funcional dos estados de respiração mitocondrial em preparações de sinaptossomas. Para tanto, utilizamos um protocolo com substratos energéticos, um desacoplador e inibidores do sistema de transporte de elétrons. Ainda, os imunoconteúdos hipotalâmicos de AMPK e pAMPKT172, bem como de LC3 foram avaliados como biomarcadores de homeostase energética e de fluxo autofágico, respectivamente. Os resultados demonstram que os camundongos envelhecidos não apresentaram alterações significativas na captação cerebral de 18FDG e nos estados de respiração mitocondrial. Também, os animais envelhecidos apresentaram menor razão pAMPKT172/ AMPK, Beclin-1, BCL-2 e LC3II/LC3I em relação aos animais jovens controle. A suplementação de ND nos animais jovens aumentou a captação de 18FDG e o consumo de oxigênio no estado routine sem influenciar os outros estados mitocondriais. A ND nos animais envelhecidos não aumentou a captação de 18FDG, e nem o consumo de oxigênio nos estados mitocondriais. Nos animais envelhecidos a ND aumentou a razão pAMPKT172/ AMPK e LC3II/LC3I, bem como a eficiência da fosforilação oxidativa mitocondrial. Observamos que este mecanismo não se dá por modulação de Beclin-1. Nossos resultados sugerem que envelhecimento não culmina necessariamente em modificações funcionais da capacidade energética cerebral, embora os biomarcadores moleculares de estado energético e fluxo autofágico estejam diminuídos. A ND melhorou a eficiência bioenergética mitocondrial e o fluxo autofágico sem aumentar a captação de 18FDG. Estes benefícios são provavelmente mediados pela reprogramação da sinalização via AMPK.Physiologically, the mitochondria integrates oxidative metabolism and cellular mechanisms responsible for turnover of dysfunctional organelles and proteins namely autophagy. Conversely, aging disrupt this functional interplay, leading to neuroenergetic deficits coupled with protein aggregation. In this sense, the decay in testosterone levels, an anabolic hormone, has been proposed as a trigger to accelerate functional decline during ageing. Thus, in this study we investigated whether nandrolone decanoate (ND) supplementation, a synthetic analog of testosterone, in aged animals influences mitochondrial respiratory complex activity along with autophagic flux. Albino CF1 mice of 3 (3m) and 18 (18m) months of age where submitted to baseline fluorodeoxyglucose (18FDG) uptake analysis, through positron emission tomography scans (microPETFDG). They were separated in 4 groups that received daily subcutaneous injections for 15 days of either ND (15mg/kg) (Groups 3m/ND; 18m/ND), or equivalent volume of vehicle (Groups 3m/VEH; 18m/VEH). Mice were resubmitted to microPET-FDG analysis and euthanised. High resolution respirometry was performed to assess mitochondrial respiratory states function and respiratory control ratio (RCR) in synaptossomes of entire left hemisphere. For such, a protocol of substratres, uncouplers and inhibitors of electron transport system were sequentially titrated. Also, hypothalamic immunocontent of AMPK e pAMPKT172, as well as LC3, were assessed as biomarkers, proxy of homeostatic energy balance and autophagic flux, respectively. Results demonstrate that aged animals do not display significant alterations nor in brain 18FDG uptake, neither in mitochondrial respiratory states Routine, Leak, Oxphos and Uncoupled. Also, aged animals displayed reduced pAMPKT172/ AMPK ratio, and increased LC3-II compared to adult controls. ND supplementation in adults increased cerebral 18FDG uptake, as well as increased Routine mitochondrial oxygen consumption rate (OCR). Curiously, ND supplementation in aged animals did neither lead to 18FDG uptake, nor alterations in mitochondrial states OCR. In aged animals ND treatment caused an increased pAMPKT172/ AMPK ratio, LC3-II turnover, as well as increased mitochondrial oxidative phosphorylation efficiency, as evidenced by increased RCR. ND supplementation does not incur in significant alterations of Beclin- 1/BCL-2 immunocontents. Our results suggest that aging does not culminate necessarily in functional alterations in mitochondrial capacity to produce ATP, although molecular biomarkers of energetic status and autophagic flux are reduced. ND improved mitochondrial ATP synthesis and LC3-II turnover in aged mice without increasing 18FDG uptake. These benefits are probably mediated by reprogramming of AMPK signalling

Adaptações metabólicas como potenciais componentes da terapia e biomarcadores prognósticos no TCE

O trauma cranioencefálico é definido como uma lesão mecânica ao tecido encefálico, que está amplamente associada à mortalidade, e consequências crônicas aos pacientes que sobrevivem; particularmente neurodegeneração e prejuízos cognitivos. Apesar de ser fundalmentamente uma lesão mecânica as consequências clínicas do TCE têm como substrato principal alterações metabólicas que são desencadeadas imediatamente após à lesão, e que se perpetuam e difundem pelo tecido cerebral. Este dano secundário ao trauma, altera funções celulares, impede a recuperação e conduz as células para um caminho de neurodegeneração apoptótica. Portanto, entender e desvendar os componentes destas abnormalidades pode proporcionar indicadores clínicos de prognóstico e respostas a terapias, assim como alvos terapêuticos contra os mecanismos neurotóxicos associados ao TCE. O principal dilema atual relacionado ao TCE grave é sua limitação em relação à disponibilidade de arsenais terapêuticos efetivos e biomarcadores prognósticos específicos. Considerando o papel das alterações metabólicas na patofisiologia do trauma e suas consequências a longo prazo, realizamos dois estudos: (1) Um estudo pré-clínico para explorar o potencial neuroprotetor da trealose, considerado um indutor de autofagia AMPK dependente, cujo impacto na bioenergética após o TCE grave ainda não foi explorado; (2) Um estudo clínico, para desvendar as alterações no catabolismo purinérgico e identificar assinaturas purinômicas de mortalidade e de desfecho neurofuncional a longo prazo em pacientes com TCE grave. As principais descobertas desta tese revelam que a suplementação com trealose levou à melhora da atividade do complexo mitocondrial I e II nos estágios agudo e subagudo de recuperação do TCE, concomitante à atenuação de marcadores de neurodegeneração e redução do prejuízo cognitivo. Também, demonstramos no líquido cefalorraquidiano de pacientes com TCE, assinaturas do trauma sobre as taxas de interconversão de purinas, e que os níveis extracelulares de GTP e Guanosina no líquor possuem respectivamente, valor preditor de mortalidade a curto prazo e de desfechos clínicos funcionais desfavoráveis em longo prazo.Traumatic brain injury is defined as a mechanical injury to brain tissue, largely associated with mortality, and chronic consequences for patients who survive, particularly neurodegeneration and cognitive impairment. The main current dilemma related to severe TBI is its limitation regarding the availability of effective therapeutic arsenals and specific prognostic biomarkers. Despite being fundamentally a mechanical injury, the clinical consequences of TBI have as their main substrate metabolic changes that are perpetuated and spread throughout the brain tissue. This secondary damage alters cellular functions, impedes recovery, and drives cells down a path of neurodegeneration. Therefore, understanding and unraveling the components of these abnormalities can provide clinical indicators of prognosis and responses to therapies as well as therapeutic targets against the neurotoxic mechanisms associated with TBI. Considering the role of metabolic alterations in the pathophysiology of trauma and its long-term consequences, we carried out two studies: (1) A preclinical study to explore the neuroprotective potential of trehalose, considered an inducer of AMPK-dependent autophagy, whose impact on bioenergetics after severe TBI had not yet been explored; and (2) A clinical study, to unravel changes in purinergic catabolism and identify purinomic signatures of mortality and long-term neurofunctional outcome in patients with severe TBI. The main findings of this thesis reveal that trehalose supplementation leads to improved mitochondrial complex I and II activity in the acute and subacute stages of TBI recovery, concomitant with attenuation of neurodegeneration markers and reduction of cognitive impairment. Also, we demonstrated in the cerebrospinal fluid of patients with TBI, a signature interconversion rate and dynamic of purines, and that the CSF levels of GTP and Guanosine respectively, display predictive value for short-term mortality and for unfavorable long-term functional clinical outcomes

Mortality of septic shock patients is associated with impaired mitochondrial oxidative coupling efficiency in lymphocytes : a prospective cohort study

Background: Septic shock is a life-threatening condition that challenges immune cells to reprogram their mitochondrial metabolism towards to increase ATP synthesis for building an appropriate immunity. This could print metabolic signatures in mitochondria whose association with disease progression and clinical outcomes remain elusive. Method: This is a single-center prospective cohort study performed in the ICU of one tertiary referral hospital in Brazil. Between November 2017 and July 2018, 90 consecutive patients, aged 18 years or older, admitted to the ICU with septic shock were enrolled. Seventy-five patients had Simplified Acute Physiology Score (SAPS 3) assessed at admission, and Sequential Organ Failure Assessment (SOFA) assessed on the first (D1) and third (D3) days after admission. Mitochondrial respiration linked to complexes I, II, V, and biochemical coupling efficiency (BCE) were assessed at D1 and D3 and Δ (D3–D1) in isolated lymphocytes. Clinical and mitochondrial endpoints were used to dichotomize the survival and death outcomes. Our primary outcome was 6-month mortality, and secondary outcomes were ICU and hospital ward mortality. Results: The mean SAPS 3 and SOFA scores at septic shock diagnosis were 75.8 (± 12.9) and 8 (± 3) points, respectively. The cumulative ICU, hospital ward, and 6-month mortality were 32 (45%), 43 (57%), and 50 (66%), respectively. At the ICU, non-surviving patients presented elevated arterial lactate (2.8 mmol/L, IQR, 2–4), C-reactive protein (220 mg/L, IQR, 119–284), and capillary refill time (5.5 s, IQR, 3–8). Respiratory rates linked to CII at D1 and D3, and ΔCII were decreased in non-surviving patients. Also, the BCE at D1 and D3 and the ΔBCE discriminated patients who would evolve to death in the ICU, hospital ward, and 6 months after admission. After adjusting for possible confounders, the ΔBCE value but not SOFA scores was independently associated with 6-month mortality (RR 0.38, CI 95% 0.18–0.78; P = 0.009). At a cut-off of − 0.002, ΔBCE displayed 100% sensitivity and 73% specificity for predicting 6-month mortality. Conclusions: The ΔBCE signature in lymphocytes provided an earlier recognition of septic shock patients in the ICU at risk of long-term deterioration of health status

Elevated glutamate and lactate predict brain death after severe head trauma

Objective: Clinical neurological assessment is challenging for severe traumatic brain injury (TBI) patients in the acute setting. Waves of neurochemical abnormalities that follow TBI may serve as fluid biomarkers of neurological status. We assessed the cerebrospinal fluid (CSF) levels of glutamate, lactate, BDNF, and GDNF, to identify potential prognostic biomarkers of neurological outcome. Methods: This cross-sectional study was carried out in a total of 20 consecutive patients (mean [SD] age, 29 [13] years; M/F, 9:1) with severe TBI Glasgow Coma Scale ≤ 8 and abnormal computed tomography scan on admission. Patients were submitted to ventricular drainage and had CSF collected between 2 and 4 h after hospital admission. Patients were then stratified according to two clinical outcomes: deterioration to brain death (nonsurvival, n = 6) or survival (survival, n = 14), within 3 days after hospital admission. CSF levels of brain-derived substances were compared between nonsurvival and survival groups. Clinical and neurological parameters were also assessed. Results: Glutamate and lactate are significantly increased in nonsurvival relative to survival patients. We tested the accuracy of both biomarkers to discriminate patient outcome. Setting a cutoff of >57.75, glutamate provides 80.0% of sensitivity and 84.62% of specificity (AUC: 0.8214, 95% CL: 54.55–98.08%; and a cutoff of >4.65, lactate has 100% of sensitivity and 85.71% of specificity (AUC: 0.8810, 95% CL: 54.55–98.08%). BDNF and GDNF did not discriminate poor outcome. Interpretation: This early study suggests that glutamate and lactate concentrations at hospital admission accurately predict death within 3 days after severe TBI

Modulação do fluxo autofágico pelo decanoato de nandrolona em hipotálamo de camundongos envelhecidos

INTRODUÇÃO: O envelhecimento recente da população mundial têm apresentado à ciência e medicina moderna novos desafios e questionamentos quanto aos mecanismos neurobiológicos do envelhecimento. O envelhecimento fisiológico é responsável por um grupo de características clínicas marcantes, tais como declínio do metabolismo energético, perda da proteostase gerando acúmulo protéico, bem como, um significativo desbalanço na regulação hormonal androgênica. Atribui-se ao desbalanço da homeostase protéica disfunções no sistema autofágico. Neste contexto destaca-se a importância do tecido hipotalâmico como importante alvo dos hormônios gonadais, bem como regulador do metabolismo energético global. Baseado nestas premissas, a administração de hormônios androgênicos esteroides exógenos têm sido proposta desde o século passado, visando reverter sintomas clínicos característicos do envelhecimento. Entretanto, a modulação da maquinaria autofágica pelos hormônios esteroides e o seu impacto no balanço energético ainda não foram definidos. MÉTODOS: Neste trabalho administramos decanoato de nandrolona (ND, 15 mg/Kg, s.c.) por 15 dias em camundongos CF1 machos de 3 meses 18 meses. Após o sacrifício, foram analisadas proteínas da maquinaria autofágica no hipotálamo. RESULTADOS: Os animais envelhecidos apresentam um acúmulo de autofagossomos evidenciado pela redução na razão LC3II/LC3I e o tratamento com ND reverteu este perfil. Observamos um aumento no imunoconteúdo de Beclin-1 nos animais envelhecidos independentemente de ND. Finalmente, o tratamento com ND aumentou a ativação da via AMPK. CONCLUSÃO: Este estudo demonstra um acúmulo de autofagossomos associado ao envelhecimento e o tratamento com ND ativa o fluxo autofágico de maneira AMPK-dependente.INTRODUCTION: Populational ageing has presented modern science and medicine new challenges and questions about the neurobiological mechanisms of aging. Physiological aging is responsible for a group of marked clinical features, such as a decline in energy metabolism, loss of proteostase, generating protein accumulation, as well as a significant imbalance in androgen hormonal regulation. It is attributed a dysfunction in the autophagic system the imbalance of protein homeostasis. In this context, the importance of hypothalamic tissue as an important target of gonadal hormones as well as regulator of global energy metabolism is highlighted. Based on these premises, the administration of exogenous steroid androgenic hormones has been proposed since the last century, aiming to revert clinical symptoms characteristic of aging. However, the modulation of autophagic machinery by steroid hormones and its impact on energy balance have not yet been defined. METHODS: In this work we administered nandrolone decanoate (ND, 15 mg / kg, s.c.) for 15 days in male CF1 mice aged 18 months and 3 months. After the euthanasia, proteins from the autophagic machinery were analyzed in the hypothalamus. RESULTS: Aged animals presented an accumulation of autophagosomes evidenced by the reduction in LC3II / LC3I ratio, and treatment with ND reversed this profile. We observed an increase in the immunocontent of Beclin-1 in aged animals independently of ND. Finally, treatment with ND increased the activation of the AMPK pathway. CONCLUSION: This study demonstrates an accumulation of autophagosomes associated with aging and treatment with ND activates autophagic flow in an AMPK-dependent manne