Investigação da participação da AMPK em aspectos do catabolismo lipídico induzido pela privação alimentar
Data
2023-05-25
Autores
Silva, Sofia Beatriz e 
Orientadores
Cunha, Fernanda Marques da
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: Investigar a participação da Proteína Quinase Ativada por Adenosina
Monofosfato (AMPK) em aspectos do catabolismo de lipídios induzido pela privação
alimentar. Métodos: A contribuição da β-oxidação para a longevidade de animais
selvagens e animais com ganho ou perda de função da AMPK foi analisada em
condições ad libitum ou de privação de alimento na ausência ou presença perhexilina,
inibidor farmacológico da entrada de ácidos graxos de cadeia longa na matriz
mitocondrial. A contribuição da AMPK na manutenção da saúde foi analisada em
animais adultos com perda ou ganho de função para AMPK e sua movimentação foi
medida em líquido nos dias 1, 5 e 10 de vida adulta. Para definir o impacto da alteração
da atividade da AMPK e da privação alimentar sobre a abundância de lipídios neutros,
animais adultos foram ou não submetidos a 6 horas de privação alimentar, e
posteriormente corados com Oil-Red O. A participação da AMPK sobre o controle da
expressão de proteínas envolvidas no catabolismo de lipídios foi investigada por meio
da quantificação de RNA mensageiro, realizada em animais controle ou privados de
alimento por 6 horas. O perfil temporal de ativação da AMPK em resposta à falta de
alimento, foi determinado pela quantificação da fosforilação da treonina 172 da enzima
por western blotting em animais selvagens submetidos à privação alimentar.
Resultados: A privação alimentar aumenta o tempo de vida de animais selvagens,
sendo que esse aumento depende da β-oxidação. A perda de função para AMPK
diminui o tempo de vida em condições ad libitum e previne totalmente o aumento do
tempo de vida induzido pela privação alimentar. O ganho de função para AMPK tem
o efeito oposto, aumentando o tempo de vida em condições ad libitum assim como a
resposta à privação alimentar. Em animais com AMPK constitutivamente ativa, há
menor perda de movimentação durante o envelhecimento quando comparado à
linhagem selvagem ou com perda de função para AMPK. O ganho ou perda de função
da AMPK não parece afetar o consumo de lipídios de reserva e a expressão de
enzimas do catabolismo lipídico após 6 horas de privação de alimentos em
comparação ao grupo selvagem Por fim, a ativação da AMPK parece ser tardia, porém
duradoura. Conclusões: A AMPK tem ativação tardia, em resposta à privação
alimentar. A ativação dessa enzima é necessária para o aumento no tempo de vida
em resposta à privação alimentar, assim como para a manutenção da saúde do animal
durante o envelhecimento em dieta ad libitum. Seis horas de privação alimentar levam
ao consumo de lipídios corporais, assim como à alteraçaõ da expressão de enzimas
envolvidas no catabolsimo de lipídios, efeitos que não parecem depender da AMPK.
Objective: To investigate the participation of AMP-Activated Protein Kinase (AMPK) in aspects of lipid catabolism induced by bacterial deprivation. Methods: The contribution of β-oxidation to the longevity of wild-type worms and worms with AMPK gain- or loss-of-function was analyzed under ad libitum or bacterial deprivation conditions in the absence or presence of perhexiline, a pharmacological inhibitor that prevents the entry of long-chain fatty acids into the mitochondrial matrix. The contribution of AMPK to healthspan was analyzed in animals with AMPK loss- or gain- of-function through the thrashing assay on days 1, 5 and 10 of adulthood. To define the impact of alteration in AMPK activity and bacterial deprivation on the abundance of neutral lipids, adult animals were or not starved for 6 hours, and subsequently stained with Oil-Red O. The impact of AMPK on the control of expression of proteins involved in lipid catabolism was investigated by quantification of messenger RNA. The time course of AMPK activation in response to bacterial deprivation was determined by quantification of phosphorylation of the threonine 172 residue by western immunoblotting in wild-type animals. Results: Bacterial deprivation increased the lifespan of wild-type animals in a β-oxidation-dependent manner. Loss of function for AMPK shortend the lifespan under ad libitum conditions and completely prevented bacterial deprivation-induced lifespan increase. The gain of function for AMPK had the opposite effect, increasing the lifespan under ad libitum conditions as well as the response to bacterial deprivation. Animals with constitutively active AMPK, presented a minor loss of movement during aging when compared to the wild type or the lossof- function groups. Worms with AMPK gain- or loss-of-function for consumed similar amounts of neutral lipid and had similar modulation of expression of enzymes involved in lipid catabolism after 6 hours of bacterial deprivation when compared to wild-type animals. Finally, AMPK activation in response to bacterial deprivation was apparent only after 24 hours of diet and lasted at least 48 hours. Conclusions: AMPK seems to be activated at a late stage of bacterial deprivation. Activation of this enzyme is necessary for increasing lifespan in response to bacterial deprivation, as well as for maintaining animal health during aging in control conditions. Six hours of bacterial deprivation leads to the consumption of body lipids, as well as alterations in the expression of enzymes involved in lipid catabolism, parameters that are not altered by AMPK gain- or loss-of function, suggesting that these effects do not seem to be dependent on AMPK signaling, at least in the first 6 hours of starvation.
Objective: To investigate the participation of AMP-Activated Protein Kinase (AMPK) in aspects of lipid catabolism induced by bacterial deprivation. Methods: The contribution of β-oxidation to the longevity of wild-type worms and worms with AMPK gain- or loss-of-function was analyzed under ad libitum or bacterial deprivation conditions in the absence or presence of perhexiline, a pharmacological inhibitor that prevents the entry of long-chain fatty acids into the mitochondrial matrix. The contribution of AMPK to healthspan was analyzed in animals with AMPK loss- or gain- of-function through the thrashing assay on days 1, 5 and 10 of adulthood. To define the impact of alteration in AMPK activity and bacterial deprivation on the abundance of neutral lipids, adult animals were or not starved for 6 hours, and subsequently stained with Oil-Red O. The impact of AMPK on the control of expression of proteins involved in lipid catabolism was investigated by quantification of messenger RNA. The time course of AMPK activation in response to bacterial deprivation was determined by quantification of phosphorylation of the threonine 172 residue by western immunoblotting in wild-type animals. Results: Bacterial deprivation increased the lifespan of wild-type animals in a β-oxidation-dependent manner. Loss of function for AMPK shortend the lifespan under ad libitum conditions and completely prevented bacterial deprivation-induced lifespan increase. The gain of function for AMPK had the opposite effect, increasing the lifespan under ad libitum conditions as well as the response to bacterial deprivation. Animals with constitutively active AMPK, presented a minor loss of movement during aging when compared to the wild type or the lossof- function groups. Worms with AMPK gain- or loss-of-function for consumed similar amounts of neutral lipid and had similar modulation of expression of enzymes involved in lipid catabolism after 6 hours of bacterial deprivation when compared to wild-type animals. Finally, AMPK activation in response to bacterial deprivation was apparent only after 24 hours of diet and lasted at least 48 hours. Conclusions: AMPK seems to be activated at a late stage of bacterial deprivation. Activation of this enzyme is necessary for increasing lifespan in response to bacterial deprivation, as well as for maintaining animal health during aging in control conditions. Six hours of bacterial deprivation leads to the consumption of body lipids, as well as alterations in the expression of enzymes involved in lipid catabolism, parameters that are not altered by AMPK gain- or loss-of function, suggesting that these effects do not seem to be dependent on AMPK signaling, at least in the first 6 hours of starvation.