SummaryAlthough senescence has long been implicated in aging-associated pathologies, it is not clearly understood how senescent cells are linked to these diseases. To address this knowledge gap, we profiled cellular senescence phenotypes and mRNA expression patterns during replicative senescence in human diploid fibroblasts. We identified a sequential order of gain-ofsenescence phenotypes: low levels of reactive oxygen species, cell mass/size increases with delayed cell growth, high levels of reactive oxygen species with increases in senescence-associated b-galactosidase activity (SA-b-gal), and high levels of SA-b-gal activity. Gene expression profiling revealed four distinct modules in which genes were prominently expressed at certain stages of senescence, allowing us to divide the process into four stages: early, middle, advanced, and very advanced. Interestingly, the gene expression modules governing each stage supported the development of the associated senescence phenotypes. Senescence-associated secretory phenotype-related genes also displayed a stage-specific expression pattern with three unique features during senescence: differential expression of interleukin isoforms, differential expression of interleukins and their receptors, and differential expression of matrix metalloproteinases and their inhibitory proteins. We validated these phenomena at the protein level using human diploid fibroblasts and aging Sprague-Dawley rat skin tissues. Finally, disease-association analysis of the modular genes also revealed stage-specific patterns. Taken together, our results reflect a detailed process of cellular senescence and provide diverse genome-wide information of cellular backgrounds for senescence.

As Western diets continue to include an ever-increasing amount of sugar, there has been a rise in obesity and type 2 diabetes. To avoid metabolic diseases, the body must maintain proper metabolism, even on a high-sugar diet. In both humans and , excess sugar (glucose) is stored as glycogen. Here, we find that animals increased stored glycogen as they aged, whereas even young adult animals had increased stored glycogen on a high-sugar diet. Decreasing the amount of glycogen storage by modulating the glycogen synthase, , a key enzyme in glycogen synthesis, can extend lifespan, prolong healthspan, and limit the detrimental effects of a high-sugar diet. Importantly, limiting glycogen storage leads to a metabolic shift whereby glucose is now stored as trehalose. Two additional means to increase trehalose show similar longevity extension. Increased trehalose is entirely dependent on a functional FOXO transcription factor DAF-16 and autophagy to promote lifespan and healthspan extension. Our results reveal that when glucose is stored as glycogen, it is detrimental, whereas, when stored as trehalose, animals live a longer, healthier life if DAF-16 is functional. Taken together, these results demonstrate that trehalose modulation may be an avenue for combatting high-sugar-diet pathology.

SummaryGlycogen biogenesis and its response to physiological stimuli have often been implicated in age-related diseases. However, their direct relationships to cell senescence and aging have not been clearly elucidated. Here, we report the central involvement of enhanced glycogenesis in cellular senescence. Glycogen accumulation, glycogen synthase (GS) activation, and glycogen synthase kinase 3 (GSK3) inactivation commonly occurred in diverse cellular senescence models, including the liver tissues of aging F344 rats. Subcytotoxic concentrations of GSK3 inhibitors (SB415286 and LiCl) were sufficient to induce cellular senescence with increased glycogenesis. Interestingly, the SB415286-induced glycogenesis was irreversible, as were increased levels of reactive oxygen species and gain of senescence phenotypes. Blocking GSK3 activity using siRNA or dominant negative mutant (GSK3β β β β -K85A) also effectively induced senescence phenotypes, and GS knock-down significantly attenuated the stress-induced senescence phenotypes. Taken together, these results clearly demonstrate that augmented glycogenesis is not only common, but is also directly linked to cellular senescence and aging, suggesting GSK3 and GS as novel modulators of senescence, and providing new insight into the metabolic backgrounds of aging and aging-related pathogenesis.

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Mitochondrial complex II defect has recently been implicated in cellular senescence and in the ageing process of which a critical phenotype is retardation and arrest of cellular growth. However, the underlying mechanisms of how complex II defect affects cellular growth, remain unclear. In this study, we investigated the effect of complex II inhibition using a subcytotoxic dose (400 mM) of 2-thenoyltrifluoroacetone (TTFA), a conventional complex II inhibitor, on cell cycle progression. TTFA (400 mM) directly decreased KCN-sensitive cellular respiration rate to 67% of control and disrupted the mitochondrial membrane potential. In contrast to other respiratory inhibitors such as rotenone, antimycin A, and oligomycin, TTFA prolonged the duration of each phase of the cell cycle (G1, S, and G2/M) equally, thereby delaying overall cell cycle progression. This delay was accompanied by a biphasic increase of reactive oxygen species (ROS) and concurrent glutathione oxidation, in addition to a slight decrease in the cellular ATP level. Finally, the delay in cell cycle progression caused by TTFA was proved to be mainly due to ROS overproduction and subsequent oxidative stress, as evidenced by its reversal following pretreatment with antioxidants. Taken together, these results suggest that an overall delay in cell cycle progression due to complex II defects may contribute to ageing and degenerative diseases via inhibition of cellular growth and proliferation without arrest at any specific phase of the cell cycle.