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The ability to repair cellular damage is reduced with aging, resulting in cellular senescence. Telomeres shorten as cells divide but the rate of telomere attrition is modulated by telomerase, an enzyme that adds nucleotides to the chromosome. Shelterin is a protein complex that acts as a negative regulator of telomerase. The aim of the present study was to investigate age-related differences in telomerase and shelterin responses to acute exercise. We hypothesized that acute exercise would stimulate an increased activity of telomerase (measured by telomerase reverse transcriptase, hTERT) without an increase in activity of shelterin (measured by telomeric repeat binding factor 2, TRF2) in both young and older individuals and that hTERT response would be attenuated in older individuals. Young (22 ± 2 y, n = 11) and older (60 ± 2 y, n = 8) men and women performed 30 min of cycling. Blood was collected pre-exercise and 30, 60, and 90-min post-exercise. The trial induced a significant hTERT response in the cohort as a whole (p < 0.05) with greater increases in the young as compared to the older group (time-by-group interaction p < 0.05). As expected, TRF2 did not change in response to the trial, however older individuals had a higher TRF2 response at 60 min (p < 0.05). There was an unexpected sex difference, regardless of age, where men had significantly greater hTERT and TRF2 responses to the acute exercise as compared to women (p < 0.05). These data support the hypothesis that aging is associated with attenuated telomerase activation in response to high-intensity exercise; however, this was only evident in men.A single session of high intensity interval cycling increased hTERT gene expression in young and older men but not in women.The hTERT response was attenuated in older men compared to young.TRF2 gene expression was increased in older men at 60 minute post-exercise.Sex differences in exercise-induced telomerase activation were a novel finding and need to be investigated in further detail.