Calorie restriction does not elicit a robust extension of replicative lifespan inSaccharomyces cerevisiae

Abstract

Calorie restriction (CR) is often described as the most robust manner to extend lifespan in a large variety of organisms. Hence, considerable research effort is directed toward understanding the mechanisms underlying CR, especially in the yeast Saccharomyces cerevisiae. However, the effect of CR on lifespan has never been systematically reviewed in this organism. Here, we performed a meta-analysis of replicative lifespan (RLS) data published in more than 40 different papers. Our analysis revealed that there is significant variation in the reported RLS data, which appears to be mainly due to the low number of cells analyzed per experiment. Furthermore, we found that the RLS measured at 2% (wt/vol) glucose in CR experiments is partly biased toward shorter lifespans compared with identical lifespan measurements from other studies. Excluding the 2% (wt/vol) glucose experiments from CR experiments, we determined that the average RLS of the yeast strains BY4741 and BY4742 is 25.9 buds at 2% (wt/vol) glucose and 30.2 buds under CR conditions. RLS measurements with a microfluidic dissection platform produced identical RLS data at 2% (wt/vol) glucose. However, CR conditions did not induce lifespan extension. As we excluded obvious methodological differences, such as temperature and medium, as causes, we conclude that subtle methodspecific factors are crucial to induce lifespan extension under CR conditions in S. cerevisiae.I n the last decades, a nutritious diet low in calories, commonly referred to as calorie restriction (CR), has been reported to extend the lifespan of a broad range of organisms, such as yeast, worms, fruit flies, mice, rats, and monkeys (1-6). This seemingly evolutionary conserved effect of CR on aging has sparked intense investigation into its underlying molecular mechanisms, especially in the budding yeast Saccharomyces cerevisiae (7-10). Some studies suggest that nutrient-responsive pathways, such as target of rapamycin (TOR), protein kinase A (PKA), and Sch9 (11), mediate CR-induced lifespan extension, whereas others hypothesize that CR increases the activity of sirtuin deacetylases (e.g., SIR2) extending lifespan by suppressing ribosomal DNA recombination (9, 12, 13).In S. cerevisiae, lifespan can either be defined by the time cells remain viable in absence of nutrients, called chronological lifespan, or by the number of daughter cells a yeast cell produces during its life, which is referred to as replicative lifespan (RLS) (14). Although RLS can vary greatly between individual yeast cells, the average RLS (i.e., across a population of cells) is assumed to be a genotypic trait (15-19). However, besides CR, other environmental conditions, such as pH and carbon source, are known to have a strong impact on RLS (20, 21).Here, through a systematic analysis of RLS data of S. cerevisiae using data from more than 27,000 cells, we obtained indication that small sample sizes might be the cause for the often observed significant variation between RLS data. Further, we identified that partly biased data exis...

High-throughput analysis of yeast replicative aging using a microfluidic system

“…Recently, Huberts et al (33) reported that CR-induced lifespan extension could not be detected with their microfluidic dissection platform. In contrast, we observed a robust CR longevity effect with the HYAA-Chip that is consistent with results obtained from the conventional microdissection-based lifespan assay.…”

“…One possible explanation for this discrepancy is the very low retention rate of the cells in Huberts' device. As noted in their paper (33), 85% of all cells were lost before reaching the end of the lifespan, and in their analysis, experimental data obtained from cells that were washed out before death were incorporated into the lifespan curve as rightcensored data using the Kaplan-Meier analysis. Notably, due to the very low retention rate of their platform, the contribution of the washed out cells to the resultant data were significantly larger than that of the retained cells that were tracked throughout their entire lifespan.…”

The Natural Variation in Lifespans of Single Yeast Cells Is Related to Variation in Cell Size, Ribosomal Protein, and Division Time

“…2014 [21] was downloaded and lifespan data from BY4741 cells grown using the microfluidic platform in 2% glucose was used to evaluate lifespan curves generated with and without censored data.…”

“…The role of cell size is controversial. Cell size has both been suggested to be independent from replicative lifespan [13–17], and causal to it [18–20] and has also previously been studies in microfluidic devices [9–12,21–23]. Ribosomes, which are involved in cell size control [14], have been robustly shown to extend lifespan when knocked out in yeast and higher organisms [24–30], and have also been causally implicated in aging [31].…”

Microfluidic technologies for yeast replicative lifespan studies

“…In one case, Huberts et al reported that the longevity-promoting effect of caloric restriction is not seen in their microfluidic design. Their observations were accompanied by an interesting meta-analysis showing that studies showing the protective effects of dietary restriction in yeast generally reported control populations that were shorter-lived than the norm 52 . Later, Jo et al found that dietary restriction does increase longevity in cells aged in their device, noting that the conclusions of Huberts et al .…”