PHD PROGRAM IN HEALTH AND PHYSICAL ACTIVITY CYCLE XXVII
“The role of sirtuins in modulating hormonal, biochemical, and cellular responses in aging”
Elisa Grazioli
Supervisor: Prof. Daniela Caporossi
Abstract:
During my doctoral program, the main focus was on the role of sirtuins in modulating hormonal, biochemical, and cellular responses, and also on the possible link between these modulation and physical activity status. We evaluated the genetic and epigenetic contribution to the interindividual variability of this response through the variance components analysis in a twin model. I was also involved in several different projects that will be discussed in the final part of my thesis, were I contributed with my competences.
The first part of my PhD was devoted to acquire the basic knowledge on Sirtuins that, as NAD+ dependent deacetylase, play a key role in the regulation of many processes related to homeostasis, such as the regulation of metabolism, apoptosis, DNA repair and inflammatory response. Studies on humans reported a relation between the alterations of their expression and the incidence of various diseases such as metabolic, cardiovascular, cancer and neurodegenerative diseases. Accordingly to literature, the maintenance of their optimal level of activity is considered important to ensure a low risk of pathologies related with the aging process. Indeed the well-known and studied SIRT1 has been correlated with the redox homeostasis maintenance and with levels of oxidative damage.
To study the role of sirtuins on some biomarkers of the ageing phenotype, we enrolled young (20-40 years old) and old (65-85 years old) female monozygotic and dizygotic twin couples analysing the expression of SIRT1 and SIRT2, the global lysine acetylation, the Histone H4 acetylation and the protein carbonylation in PBMCs, as well as the plasma level of oxidized and reduced glutathione and of malondialdehyde.
The twin model allowed us to focus on the intra-pair correlation of all the markers reported above. Our analysis reported a decreased SIRT1 levels in the old group that should lead to a reduced response to redox and inflammatory homeostasis and to the stress response. We also evidenced an interesting relation between the global proteins acetylation and level and aging. The Western Blot analysis showed higher expression of this global acetylation in the Monozygotic Old Twin than in the Young, which could support the new link between lysine acetylation, metabolic enzyme and aging.
Global Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression, it targets large macromolecular
complexes involved in diverse cellular processes, such as chromatin remodelling, cell cycle, splicing, nuclear transport etc. Recently many studies focused the attention on the hypothesis that this acetylation may play a key role in the coordination of different metabolic pathways in response to extracellular conditions. However, the increase of global protein acetylation during aging remains still unclear. Another important finding of this study is the strong correlation revealed between SIRT1 level and protein carbonylation in monozygotic old twin that confirm the association between this protein and the oxidative system. This data is also supported by our findings on serum GSH, GSSG and MDA level in old and young monozygotic twins.
Interestingly, we reported that the genetic component seems to be influenced by aging in lymphocytes, especially regarding the antioxidant system (MnSOD and GSH). Life span and other senescent processes are very sensitive to genetic background, despite how genetics can influence oxidative stress is still difficult to understand; further researches on twin model are needed in this direction, in order to understand this important link and the possible interaction between genetic and epigenetic effects in the aging process.
The monozygotic twin sample was also analysed for the metabolic and hormonal profile in relation with aging and fitness level. Again, thanks to this very interesting model, we evaluated the intra-pair correlation of all analysis reported below. In the specific, regarding the metabolic and hormonal profile we studied the different expressions of IGF-1, IGF2, Leptin, c-peptide, Ghrelin, gip, gip-1, Glucagon, Insulin, pai-1, Resistin and Visfatin, also with respect the subjects’ level of daily physical activity (Baecke questionnaire) and fitness (Vo2max, 1RM strength test for upper and lower limb, balance tests, flexibility tests). According to literature, all these markers have a strong link with the aging process, and most of them (such as Leptin, Insulin, Visfatin, etc) have a high impact on several metabolic diseases, which occur during the last part of the life. Studies reported that physical activity could counteract this decline, helping in the prevention and treatment of these modulations. The results of this second study reported a decreased trend in the expression of several metabolic hormones with age, such as Ghrelin, which is a peptide hormone secreted by gastric mucosal cells that stimulates release of human growth hormone at the hypothalamic and pituitary levels. Despite the cellular and molecular mechanisms of impaired angiogenesis in aging are not well defined yet, it seems that this important hormone
could induces angiogenesis via ERK2 pathway and, for this reason, may be linked with the aging process. Also Leptin activity seems to decline with aging, despite in our study we evidenced not significant results. This hormone is related to the stimulation of lipid metabolism and regulation of energy homeostasis and it is important in the limitation of excessive storage of adipose tissue in humans. Studies reported that the stimulation of this specific hormone could be related to longevity, due to its important role against obesity and diabetes. We evidenced interesting but not significant increase on the Insulin and Resistin level in Old Monozygotic Twin, this second hormone seems to be linked with obesity and insulin resistance, which are very common processes of aging. Several evidence reported that exercise can counteract the mechanisms of these hormone despite the specific pathways are still unclear. But our finding on physical activity level support the thesis reported in literature, whereby we reported that the general level of fitness is very low in the older group as well as the level of fat mass are higher in this group than in the younger one. An interesting discover was evidenced in the intra-pair correlation of functional parameters, especially in the monozygotic old group compared to the younger; in fact we experienced that Vo2 peak, FC max, power (5-time chair test), strength (Hangrip test) and flexibility of some specific joints (shoulder and trunk) seems to be influenced by genetic. Despite some cases are reported in literature, more studies will be necessary in order to evaluate how genetic can influence these physical parameters during aging.
During the last part of my program I collaborated with Ivan Dimauro on a project that extended the data from a previous experimental protocol to the effects of 12-week of strength training on the SIRT1 and SIRT2 expression in the elderly and on the possible correlation with telomere length and epigenetic modifications. We took advantage from blood samples collected before and after explosive-type resistance training (EMRT) in elderly subjects. In particular, given the extensive evidence that aging can impact the genome modifying its structure-function relationship through epigenetic changing (i.e. DNA methylation) and altered telomere dynamics, we tested the hypothesis that EMRT may attenuate the age-associated genomic changes. In particular, length telomere leukocyte (LTL), global DNA methylation, SIRT1, SIRT2 and global protein acetylation, were evaluated in PBMCs. We believe that this study would enlighten some important details on the capacity of a specific protocol of
physical exercise to maintain DNA integrity and preventing age-related shortening of telomeres. Indeed, our results showed an increase of the telomere length in the training group, after the 12weeks of exercise and also a significant decreases in the global DNA methylation compared with the sedentary group. Although we found a tendency to increase of SIRT 1 protein expression, any significant changing was reached following the training program, neither for both sirtuins (1 and 2) nor for the global acetylation level. However, these results confirm our preliminary hypothesis, and give us new ideas to implement the knowledge of this interesting new topic.