Modulation of SIRT-1 as an anti-ageing target

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Embargoed until 2017-06-30
Copyright: Massudi, Hassina
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Abstract
Summary The NAD+-dependent deacetylase SIRT1 mediates metabolic and epigenetic adaptations to cellular stresses such as calorie restriction and regulates the pace of biological ageing in a variety of species. These effects become blunted with advancing age, for reasons that are not known. One possible cause is the age-associated decline in the co-substrate for SIRT1, NAD+. To test this, metabolic and health parameters were measured in old transgenic SIRT1 animals treated with vehicle alone or nicotinamide mononucleotide (NMN), a precursor for NAD+ synthesis to raise endogenous NAD+ levels. Our work suggests that over-expression of SIRT1 may be limited by declining NAD+ levels during old age. Furthermore, while whole body SIRT1 transgenic mice have improved glucose tolerance and insulin sensitivity, SIRT1 over-expression in skeletal muscle might not contribute to this effect. For example, muscle-specific SIRT1 overexpression in mice does not affect whole body insulin sensitivity, glucose disposal rates or insulin-stimulated glucose uptake. One possible interpretation of these findings is that SIRT1 might mediate its effects in the muscle not through muscle autonomous signalling, but through altered interactions with other cell types, which might signal to the muscle to alter mitochondrial biogenesis and function. In this study, mice overexpressing SIRT1 in the metabolic sensing, steriodogenic factor 1 (SF1) neurons improve expression of mitochondrial biogenesis transcription factors and mitochondrial function in muscle, distal to the site of SIRT1 over-expression. This points towards the previously unknown ability of SIRT1 to regulate mitochondrial function in distal tissues, via mechanisms that are not yet clear. Although SIRT1 has been more extensively characterized than other sirtuins, identification of proteins targeted for de-acylation by SIRT1 as well as other sirtuins remain an important challenge for the field due to the lack of a robust and high-throughput method to identify substrates. Current approaches to identify target substrates for sirtuins are primarily based on immuno-affinity techniques or testing specific deacetylases based on prior knowledge of a known acetylation site. The development of robust and high-throughput substrate screening methods would improve our knowledge of the repertoire of sirtuin de-acylase substrates. Here, an attempted strategy for identifying sirtuin substrates using a modified biotin switch method to capture de-acylated proteins is described, highlighting key points in the assay to reduce false positive protein identification, and also discusses the problems that were encountered in capturing de-acetylase substrates, and the non-specificity of the assay.
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Author(s)
Massudi, Hassina
Supervisor(s)
Sinclair, David
Wu, Lindsay
Morris, Margaret
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Publication Year
2016
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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