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Repeated temperature fluctuation extends the life span of Caenorhabditis elegans in a daf-16-dependent fashion
Mechanisms of Ageing and Development 129 (2008) 507–514
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Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Repeated temperature fluctuation extends the life span of Caenorhabditis elegans in a daf-16-dependent fashion Thushara Galbadage, Phil S. Hartman * Texas Christian University, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2007 Received in revised form 21 March 2008 Accepted 12 April 2008 Available online 2 May 2008
Thermocyclers were utilized to regularly shift nematodes between 12 8C and 25 8C throughout their life spans. When wild-type worms (N2) were ‘‘thermocycled’’ between 12 8C and 25 8C at 10-min intervals they lived almost as long as those that were incubated constantly at 12 8C. Shifting at 1-min or 1-h intervals lessened this effect. Similar results were observed for the long-lived mutants daf-2, eat-2 and clk-1, each of which prolongs life span through different mechanisms. In contrast, the life span of a daf-16 mutant was not prolonged by thermocycling worms, indicating that the effect is mediated by an insulinlike signaling pathway. To elucidate the molecular basis for the life span extension, two transgenic strains were employed in which heat shock proteins (HSPs) drove expression of the green fluorescent protein (GFP) gene. As expected, both HSPs were expressed at significantly higher levels in animals grown at 25 8C. Moreover, HSP expression in the thermocycled worms approximated that of animals grown at 25 8C more so than animals grown at 12 8C. This suggests that incubation at the higher temperatures for short time intervals induced stress-responsive gene expression that led to significant life span extension. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Temperature Life span Daf-16 HSP Caenorhabditis elegans Aging PCR
1. Introduction Caenorhabditis elegans, Drosophila and fish have been used extensively as model systems to study the temperature dependence of life span. The life spans of most cold-blooded organisms (poikilotherms) are strongly temperature-dependant. For example, C. elegans live much longer at lower temperatures as opposed to higher ones, with intermediate life spans at intermediate temperatures (e.g., Klass, 1977; Hosono et al., 1982). Drosophila shows similar variations in life spans (Vermeulen and Bijlsma, 2004). Several hypotheses have been advanced to explain these life span relationships. The most prominent of these is the ‘‘rate-ofliving’’ theory, first advanced by Pearl in 1928 and subsequently modified by Shaw and Bercaw (1962). This theory posits that life spans decrease as a function of increased temperature because of overall increases in the rate of metabolism. However, it has been documented that lower body temperature does not always depress metabolism (reviewed by Liu and Walford, 1972). In addition, it has been suggested that metabolism may be fundamentally
* Corresponding author at: Texas Christian University, Biology Department TCU, Box 298930, Fort Worth, TX 76129, United States. Tel.: +1 817 257 6176; fax: +1 817 257 6177. E-mail address: [email protected] (P.S. Hartman). 0047-6374/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.04.012
different at various temperatures, such that a ‘‘metabolic reorganization’’ might occur as organisms are passed from one temperature to another (Fry and Hochachka, 1970; Somero, 1972; Yen et al., 2004). Considerable insights have been derived from shifting animals from one temperature to another and assaying life span. For example, while fish live longer at low temperatures than at high temperatures, they live even longer when transferred from a higher temperature to a lower one midway through their lifetime (Liu and Walford, 1972, 1975). This follows the classic experiments of Maynard Smith (1962), who showed that Drosophila alternated between 30 8C (for 1 day) and 20 8C (for 3 days) lived almost as long as those kept continuously at 20 8C. He postulated that the adult fruit fly’s life span can be divided into two phases (‘aging or induction’ and ‘dying or development’). A similar phenomenon was observed with C. elegans. Specifically, animals reared at high temperatures lived much longer when shifted to lower temperature after they had developed into adults, but reversing this sequence did not prolong life span (Hosono et al., 1982). A slightly different experimental regimen has been employed to elucidate the molecular basis of hormesis. Hormesis can be defined as life span extensions that result from transient exposure to stressful conditions (Johnson et al., 2002; Cypser and Johnson, 2003). Exposure presumably induces a battery of genes that better protect against the normal ‘‘wear-and-tear’’ of aging. A variety of
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stressors including heat can promote hormesis in C. elegans (Lithgow et al., 1995; Butov et al., 2001; Cypser and Johnson, 2001, 2002, 2003; Yashin et al., 2001, 2002; Olsen et al., 2006). Olsen et al. (2006) have recently demonstrated that repeated thermal stress throughout the worm’s life span enabled animals to live longer than a single treatment. They also showed that levels of two heat shock proteins correlated well with the life span data; that is, both HSP-4 and HSP-16 were dramatically induced by the mild thermal treatment offered animals. We were interested in exploring the effects of more regular heat fluctuation on C. elegans life span. To do so, we employed thermocyclers in a novel fashion. Specifically, rather than amplifying DNA, we developed conditions enabling worms to be grown and maintained in microfuge tubes in a thermocycler. This allowed us to systemically shift temperatures between 12 8C and 25 8C, the upper and lower bounds for C. elegans growth. We discovered that shifting animals at 10-min increments resulted in a life span extension that was dependent upon a functional daf-16 gene. We also observed the induction of HSP-4 and HSP-16 in the ‘‘thermocycled’’ worms. Collectively, these data suggest that incubation at the higher temperature induced a daf-16-dependent increase in expression of stress-responsive genes, which in turn led to significant life span extension. 2. Materials and methods 2.1. Life span analyses 2.1.1. Strains The following strains were employed in this study: N2 (wild type), CB1370 [daf2(e1370)], CB1375 [daf-18(e1375)]CB4876 [clk-1(e2519)], DA465 [eat-2(ad465)], DR27 [daf-16(m27)], GR1307 [daf-16(mgDf50)], CL2070 [dvIs70 (hsp-16.2::GFP; rol6(su1006)] and SJ4005 [zcIs4(hsp-4::GFP)]. All were obtained from the Caenorhabditis Genetics Center and were maintained on NGM agar plates (Brenner, 1974) seeded with Escherichia coli OP50. 2.1.2. Synchronization of worms Synchronous populations of embryos were obtained by lysing gravid hermaphrodites in alkaline bleach as described by Emmons et al. (1979). Once washed free of the alkaline bleach by centrifugation, the embryos were inoculated on to NGM agar plates (Brenner, 1974) seeded with E. coli OP50. The plates were then incubated at 16 8C for 3 days before being transferred to liquid culture and subjected to a specific temperature regimen. This ensured that all worms, including the temperaturesensitive mutant daf-2, developed into adults rather than arresting as dauer larvae. 2.1.3. Life span determinations So that animals could be incubated in thermocyclers, microfuge tubes were employed for all experimental protocols. After incubating the synchronous populations at 16 8C for 3 days, larvae were counted and transferred into S medium (Lewis and Fleming, 1995) in these microfuge tubes. Different environmental conditions were tested in order to establish optimal growth conditions. These included comparisons of: (i) microfuge tubes of 500 ml and 600 ml volumes; (ii) inoculations of five worms versus ten worms per microfuge tube; (iii) different volumes of S medium, varying from 50 ml to 120 ml; (iv) different amounts of E. coli OP50 as a food source, from 1 ml to 10 ml at concentrations of between 2 109 and 1010 bacteria per milliliter (with bacteria first grown in LB broth and then resuspended after centrifugation in S buffer); and (v) either two or four holes drilled into the microfuge tubes to maintain a steady gas exchange. After a number of trials, 600 ml microfuge tubes with two holes having five worms per microfuge tube, in 100 ml of S-medium and 5 ml of E. coli (2 109 bacteria/ml) proved to be optimal in that life spans were both consistent from experiment to experiment and in keeping with reports in the literature. Therefore this set of conditions was employed for the remainder of the study. Worms were transferred into autoclaved, sterile microfuge tubes either every other day or once every 3 days, dependent upon the strain and stage of life of the worms used. Two-day transfers occurred during the fecund period to minimize contamination with progeny and fungi. Worms were monitored, counted and fed on a daily basis. In addition, 10–30 ml of S medium was added to compensate for loss owing to evaporation. These transactions typically took about 10 min and occurred at ambient temperature, which presumably had little effect on life span. Five microliters of E. coli was added daily. In reporting the life spans of the nematodes the events of ‘‘bagging’’ were not censored; on the contrary all the deaths of a cohort of worms were considered to be natural. Bagging occurs when larvae hatch internally and eventually kill a gravid
adult, creating a ‘‘bag’’ of larvae constrained by the remnant cuticle of the adult. Bagging was not censored because the overall rates observed were either less than or in the range of 10–15% for a cohort, with the exception of daf-16 worms grown at 18.5 8C, which had bagging rates of 20–25%. Thus, it was judged that premature deaths owing to bagging did not significantly alter the mean life spans in these experiments. The first day each worm was observed dead was considered to be its maximum life span. Death was assumed when no movement was observed, either of a spontaneous nature or in response to mechanical stimulation. 2.1.4. Thermocylers Two thermocyclers (PCR Sprint – SPRT001 and GeneMate – FPR0G05G) were employed for shifting the temperature at constant time intervals between 12 8C and 25 8C. The thermocyclers were programmed in such a way that they fluctuated indefinitely between these two temperatures. The ramp-times from 12 8C to 25 8C and vice versa were about 35–50 s, and were dependent on the thermocycler used. Each thermocycler could accommodate up to 20 microfuge tubes, thus allowing incubation of up to 100 worms at a given time. All temperatures were measured within 0.3 8C using a wire probe attached to a calibrated thermometer. The probe was placed in a microfuge tube containing S medium, which was positioned in the thermocyclers or incubators. Experimental worms were incubated in thermocyclers for their entire lifetimes, save the first 3 days, when they were incubated at 16 8C. Controls from the same population of synchronized worms were maintained at constant temperatures of 12 8C, 18.5 8C or 25 8C to match to the number of experimental worms. 2.2. HSP expression analyses Transgenic animals were synchronized and grown as described above, with thermocyclers utilized as described previously. One cohort of each strain was temperature shifted every 10 min between 12 8C and 25 8C. Each strain was also maintained at constant temperatures of 12 8C and 25 8C. As for the life span experiments, these worms were also transferred to fresh S medium once every 3 days. During the transfer process, three worms from each treatment were removed and observed under a fluorescent microscope (Zeiss Axioskop equipped with Nomarski optics and epifluorescence). Still images were taken using a digital camera (Sony DSC S-75). To reduce the movement of the worms a ceramic plate precooled to 20 8C was placed below the glass slide for a few seconds before obtaining the images. The process of imaging the transgenic worms was completed in less than 5 min to reduce any external stresses. Worms that were observed under the fluorescent microscope were then discarded. Fluorescent images of three worms from each of the three temperature treatments (i.e., 10-min shifts in a thermocycler, constant incubation at 12 8C, constant incubation at 25 8C) were obtained and quantified using the NIH Image (also known as ImageJ) software. In reporting the observed intensities, the worms were quantified, and the background ‘‘noise’’ was subtracted from the absolute intensities, yielding the relative GFP intensities. This procedure was carried out throughout the lifetimes of these worms and images were taken, first at 3-day intervals and then at 6-day intervals. This experimental procedure was repeated twice and the combined results are presented.
3. Results 3.1. Life span analyses 3.1.1. Wild-type (N2) worms As previously observed by Klass (1977), the life span of wildtype (N2) worms grown at 12 8C was much longer than when grown at 25 8C. N2 living at 18.5 8C (the mean of 12 8C and 25 8C) had an intermediate life span. In our hands, N2 worms constantly incubated at 12 8C, 18.5 8C and 25 8C had mean lifetimes of 33.7 days, 19.2 days and 12.6 days, respectively (Fig. 1A). When temperature was shifted between 12 8C and 25 8C at 10-min intervals, the same strain had a mean life span of 31.0 days, which was very close to the mean life span of worms constantly living at 12 8C (Fig. 1A). Most significantly, this was much longer than the 19.2 day mean life span obtained for worms grown constantly at 18.5 8C the temperature half way between 12 8C and 25 8C. N2 worms were also shifted at 1-min and 1-h intervals between 12 8C and 25 8C and had life spans of 19.4 days and 24.5 days (versus 31.0 days for 10-min shifts), respectively. Therefore, 10min intervals prolonged life spans to a greater extent than periodicities of 1 min or 1 h. Furthermore, wild-type animals that spent 10 min at 12 8C and 50 min at 25 8C or 50 min at 12 8C and
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intervals, the life span was similar to the 18.5 8C control, with a mean life span of 16.7 days (Fig. 1B). Therefore, the life span extension afforded by temperature shifts was absent in a daf-16 genetic background. Life span determinations were conducted on the daf-16 experiment on three occasions giving consistant results (Table 1). This result was not limited to the mgDf50 allele of daf-16, but was also observed in a second daf-16 allele (m27). These thermocycled animals had a significantly shorter 50% reduction in life span compared to those that were reared constantly at 12 8C. Specifically, they had mean life spans of 11.5 days, 16.1 days and 27.2 days when grown at 25 8C, in a thermocycler and at 12 8C, respectively (data not shown). In a similar fashion, a mutation in daf-18 eliminated the life span extension afforded by thermocycling animals, having life spans of 13.5 days, 18.8 days and 28.7 days when grown at 25 8C, in a thermocycler and at 12 8C, respectively (data not shown). 3.2. HSP expression analyses
Fig. 1. (A) Survival plot of N2 (wild-type) nematodes. Wild-type shifted every 10 min between 12 8C and 25 8C (N2 10-min cycles), and three control groups of N2 constantly incubated at 12 8C, 18.5 8C or 25 8C. (B) Survival plot of a daf-16 mutant. daf-16 shifted every 10 min between 12 8C and 25 8C (daf-16 10-min cycles), and three control groups of daf-16 constantly incubated at 12 8C, 18.5 8C or 25 8C. The arrows indicate the 50% survival time (median life span) at each temperature regimen.
10 min at 25 8C yielded mean life spans of 15.4 days and 29.8 days, respectively (Table 1). Thus, even though most of these treatments extended the survival time of N2 worms, the greatest effect was observed with the 10-min thermocycling treatment. 3.1.2. Long-lived mutants The same experimental protocol was carried out on long-lived clk-1, eat-2, and daf-2 mutants. The clk-1 and eat-2 mutants displayed basically the same overall pattern as observed with the wild-type. Specifically, animals were short lived at 25 8C and long lived at 12.5 8C. Most importantly, their life spans were most definitely not intermediate to 12 8C and 25 8C when they were shifted from 12 8C to 25 8C at 10-min intervals (Fig. 2A and B). In fact, these animals live slightly longer than those grown constantly at 12 8C, despite the fact that they spend one-half of their adult lives at 25 8C. Conversely, the daf-2 mutant showed a temperatureindependent life span, living almost the same time under all three temperature regimens (Fig. 2C and Table 1). This was observed previously by Gems and associates (1998). 3.1.3. daf-16 mutant Similar to wild-type worms, a daf-16 mutant had a longer life span at 12 8C, an intermediate one at 18.5 8C and a much shorter one at 25 8C. These were 29.9 days, 18.6 days and 10.8 days, respectively. As reported in the literature (Libina et al., 2003; Hartman and Ishii, 2007), the life span of the daf-16 mutant was slightly shorter than that of wild type. Most importantly, when the daf-16 mutant was shifted between 12 8C and 25 8C at 10-min
3.2.1. HSP-4 transgenic worms The experiments reported in the previous section indicate the life span extension caused by temperature shifting is mediated through the insulin-like signaling pathway controlled by daf-16. In an effort to address the mechanism by which this pathway prolongs life span, two transgenic mutants were employed that expressed green fluorescent protein (GFP) under the control of the heat shock protein (HSP) promoters HSP-4 and HSP-16. HSPs are activated downstream of daf-16 in response to environmental stresses. As such, they are good genetic markers to identify the activation of the daf-16 pathway. Although life spans were not rigorously determined for these two transgenic strains, animals grown at 12 8C and those shifted at 10-min intervals were observed to live significantly longer than animals maintained at 25 8C; that is, their life spans approximated that of N2. These worms showed similar physical characteristics to the N2 worms and produced similar amounts of progeny. Transgenic worms with GFP driven by a HSP-4 promoter were subjected to the same experimental protocol as employed for life span determinations. The expression of GFP was periodically quantified via fluorescent microscopy and digital photography (Fig. 3). The worms incubated at 12 8C had little activation of HSP-4 protein as evidenced by minimal expression of GFP. On the contrary, worms incubated at 25 8C expressed high levels of GFP, more so towards the latter part of their lifetime. The thermocycled worms (at 10-min intervals between 12 8C and 25 8C) also expressed high levels of GFP, approximating levels of the worms incubated at 25 8C and at significantly higher levels than the worms incubated at 12 8C (Figs. 3 and 4A). The time needed for significant HSP-4 induction was greater than 12 h, as animals that were thermocycled for 12 h had GFP levels only 1.2 higher than those held at 12 8C (data not shown). Therefore, GFP levels were measured at 3-day intervals at first, and followed by 6-day intervals. As indicated by the relatively small standard error bars, there was relatively little variation from animal to animal for each treatment. 3.2.2. HSP-16 transgenic worms A second set of transgenic worms with GFPs driven by a HSP-16 promoter was subjected to similar experimentation. Though GFP expression was slightly lower than those of the worms having GFP driven by the HSP-4 promoter, they showed the same pattern of expression, having minimal levels expressed at 12 8C and much higher levels expressed at 25 8C and in the thermocycled worms (Fig. 4B). As with HSP-4, the induction of HSP-16 was a gradual process, as animals that were thermocycled for 12 h had GFP levels only 1.4 higher than those held at 12 8C (data not shown).
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Table 1 A summary of all experimental results listing the strains used (N2, daf-16, clk-1, eat-2 and daf-2), temperature regimens considered and individually listing each experiments result (indicating repeated trials for consistency) Strain
Temperature regiment
Mean lifespan S.D. (days)
50% survival (days)
Maximum life span (days)
Total number of worms
N2 (wild-type)
12 8C
33.0 6.9 33.7 6.4 31.2 7.5 34.0 7.7 19.2 4.7 17.4 4.1 15.1 3.7 12.6 3.0 13.2 3.7 11.5 2.1 30.6 6.5 31.0 7.5 28.8 8.5 24.5 9.2 19.4 6.0 29.8 9.7 24.8 6.1 15.4 5.4 14.3 3.3
33.0 32.5 30.5 33.5 19.0 17.0 15.0 13.0 13.5 11.0 31.5 32.0 29.0 27.0 20.0 29.0 24.0 15.0 14.5
44 47 48 45 27 24 21 17 18 15 42 44 41 36 32 49 40 24 18
50 25 20 15 25 15 50 25 20 15 50 25 15 50 30 25 20 25 20
30.0 7.9 29.9 8.0 28.9 6.9 18.6 5.3 16.2 5.5 13.4 3.5 10.8 2.9 9.5 1.3 16.0 3.2 16.7 4.2 11.3 2.6
32.0 30.5 29.0 18.5 15.0 13.5 11.0 9.0 15.5 16.0 10.0
42 43 40 28 25 19 15 12 23 25 17
50 25 20 25 25 50 25 15 50 25 15
40.7 8.4 38.2 10.3 39.2 8.9 18.7 4.3 15.5 3.8 42.0 12.1 41.4 12.5
43.0 40.5 41.0 18.0 15.0 44.5 43.5
53 51 50 26 22 58 56
50 25 20 25 15 25 15
1-h cycles
36.6 12.6 34.7 8.9 17.3 4.7 16.6 3.8 37.1 15.2 36.5 13.3 25.8 15.5
41.5 36.5 16.5 16.0 42.5 39.5 29.0
58 48 28 23 61 55 51
50 15 25 15 50 15 50
12 8C 25 8C 10-min cycles (at 120c and 250c) 1-h cycles
38.9 12.3 38.7 15.2 39.7 14.9 32.6 18.3
43.0 42.0 43.0 40.5
64 60 67 58
50 50 50 50
18.5 8C 25 8C
10-min cycles (at 12 8C and 25 8C)
1-h cycles 1-min cycles 50–10-min cycles (at 12 8C and 25 8C) 10–50-min cycles (at 12 8C and 25 8C)
daf-16
12 8C
18.5 8C 25 8C
10-min cycles (at 12 8C and 25 8C)
clk-1
12 8C
25 8C 10-min cycles (at 12 8C and 25 8C)
eat-2
12 8C 25 8C 10-min cycles (at 12 8C and 25 8C)
daf-2
The results are reported with the mean life span standard deviation, median life span (50% survival), longest lived worm (maximum life span) and the total number of worms used for each experiment.
4. Discussion Many factors contribute to altering the life span of organisms. One of them is temperature, which plays an especially important role in determining the lifetime of cold-blooded organisms (poikilotherms) including the nematode C. elegans (Klass, 1977; Hosono et al., 1982). Klass (1977) measured the life spans of wildtype C. elegans at six different temperatures ranging from 10 8C to 26 8C. Interpolating his data, the mean life span was 9 days at 25 8C, 17 days at 18.5 8C (the temperature one-half way between 12 8C and 25 8C, the high and low temperatures employed in the current study) and 27.5 days at 12 8C. In a similar fashion, Hosono and associates (1982) found life spans were 8.2 days, 12.6 days and 19.7 days when grown at 25 8C, 20 8C and 15 8C, respectively. These values compare favorably with our data (Table 1), indicating that
we have not generated aberrant data using the culture conditions developed for our experiments. In addition, all three data sets indicate that animals grown at intermediate temperatures respond with intermediate life spans. This predicts that, in the absence of some sort of metabolic change, alternating animals systemically between the two extreme temperatures should have yielded a life span roughly equivalent to that when animals were grown at the intermediate temperature. Contrary to this prediction, wild-type C. elegans lived only 8% shorter (rather than 57% or 62% shorter predicted by Klass’ and Hosono’s data, respectively) when ‘‘thermocycled’’ between 12 8C and 25 8C at 10-min intervals (Fig. 1A and Table 1). Therefore, the animals cycled between 12 8C and 25 8C are clearly physiologically different than those grown constantly at the intermediate temperature.
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Fig. 2. (A) Survival plot of a clk-1 mutant. clk-1 shifted every 10 min between 12 8C and 25 8C (clk-1 10-min cycles), and two control groups of clk-1 constantly incubated at 12 8C or 25 8C. (B) Survival plot of a eat-2 mutant. eat-2 shifted every 10 min between 12 8C and 25 8C (eat-2 10-min cycles), and two control groups of eat-2 constantly incubated at 12 8C or 25 8C. (C) Survival plot of a daf-2 mutant. daf-2 shifted every 10 min between 12 8C and 25 8C (daf-2 10-min cycles), and two control groups of daf-2 constantly incubated at 12 8C or 25 8C. The arrows indicate the 50% survival time (median life span) at each temperature regimen.
To elucidate the genetic requirements of this physiological difference, we examined the responses of a number of C. elegans mutants known to affect life span through different mechanisms. First, we measured life spans in an eat-2 strain that alters pharyngeal pumping, thus limiting consumption in a fashion that mimic caloric restriction (McKay et al., 2004; Lakowski and Hekimi, 1998). We observed that, as is consistent with the literature, eat-2 mutant animals were long lived. In addition, the ‘‘thermocycled’’ animals lived much longer than did animals grown constantly at the intermediate temperature (Fig. 2B and Table 1). Therefore, the physiological alteration provoked by alternating between 12 8C and 25 8C was not mediated via caloric restriction. Second, we determined the life spans in a clk-1 mutant background. clk-1 encodes a highly conserved mitochondrial protein that when mutated results in pleiotropic effects that affect physiological traits, including life span extension (Wong et al., 1995). We were particularly intrigued with clk-1 because Branicky and associates (2001) demonstrated that the defecation cycle length was significantly affected when wild-type worms were reared at 20 8C and shifted to another temperature. Conversely, clk-1 animals did not adjust the length of their defecation cycles under the same conditions. This indicates that in wild-type C. elegans adaptation to temperature is an active process mediated by Clk-1. However, as was the case with eat-2, clk-1
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mutants lived significantly longer than predicted when alternated between 12 8C and 25 8C (Fig. 2A and Table 1). Third, we explored whether the insulin-like signaling pathway, which plays a prominent role in regulating C. elegans life span (Guarente and Kenyon, 2000), might mediate the physiological change triggered by alternating temperature between 12 8C and 25 8C. We initially examined the effects of the daf-2 allele e1370. The resultant decrease in daf-2 signaling caused a significant life span extension. We observed that daf-2 animals lived roughly the same length under three temperature regimens (constant at 12 8C, constant at 25 8C and ‘‘thermocycled’’) (Fig. 2C and Table 1). A similar temperature-independence was observed by Gems and associates (1998). Therefore, these results did not inform as to the role of the insulin-like signaling pathway in C. elegans’ response to ‘‘thermocycling.’’ We then determined the life spans in a daf-16 genetic background. daf-16 is negatively regulated by daf-2 and encodes a fork-head transcription factor that induces expression of a number of genes in response to insulin-like signaling. Unlike with eat-2, clk-1, and daf-2, a loss-of-function mutation in daf-16 clearly abrogated the life span extension provoked by regularly alternating temperature between 12 8C and 25 8C. Specifically, the daf-16 animals grown in the thermocycler lived over 70% shorter than at 12 8C. In fact, unlike wild type, eat-2, and clk-2, the ‘‘thermocycled’’ daf-16 animals lived slightly shorter than did the animals constantly maintained at 18.5 8C. Therefore, this effect is mediated through the insulin-like signaling pathway. Both SKN-1 (Wilson et al., 2006) and AAK-2 (Apfeld et al., 2004) have been shown to function in parallel and downstream of daf-2 in the insulin-like signaling pathway. The fact that the daf-16 mutation completely abrogated the life span extension afforded by growth in a thermocycler indicates that neither gene plays a role in this effect. Johnson and Lithgow (Johnson et al., 1996; Lithgow, 1996) proposed that the insulin-like signaling pathway controls life span by regulating expression of stress-response genes that are downstream targets of daf-16. In particular, a number of studies (Walker and Lithgow, 2003; Rea et al., 2005) have provided evidence that heat shock proteins, notably HSP-16, play an important role in this process. We therefore examined expression of HSP-16 as well as that of heat-shock protein HSP-4 using transgenic strains in which the GFP gene was placed under the control of either the HSP-16 or HSP-4 promoter (Figs. 3 and 4). These data indicate that the same 10-min fluctuations that extended life span also increased expression of both heat shock proteins. This is consistent with the notion that the physiological response to temperature fluctuation is ultimately mediated by heat-shock proteins whose expression is regulated in a daf-16-dependent fashion. We do not necessarily posit that these two HSPs are solely responsible for the life span extension afforded by thermocycling worms. There are a number of downstream targets of daf-16 (Lee et al., 2003; Murphy et al., 2003; Jensen et al., 2006), including HSP-16 and HSP-4. Interestingly, the duration of the temperature shifts strongly influenced the extent of the life span extension. Specifically, 10min intervals yielded the longest observed life span extensions of the wild-type nematodes, while temperature shifts of shorter time intervals (e.g., 1 min), longer time intervals (e.g., 1 h) or even combinations of time intervals did not have as a strong effect as did the 10-min thermocycling (Table 1). The fact that 10-min shifts elicited a stronger response than 1-min shift is particularly noteworthy because it indicates that the life span extensions were not the consequence of temperature change per se. That is, they were not the result of the transitions between 12 8C and 25 8C, which were approximately eight times more frequent with the 1min versus the 10-min shifts. Though the temperature-shifted worms had life spans similar to the worms living constantly at 12 8C, they used a different
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Fig. 3. Digital images of the transgenic worms (HSP-4) expressing GFP. The absolute intensities of GFP expressed driven by the HSP-4 promoter is shown as a function of survival time. The 12 8C and 25 8C controls are shown on either side of the temperature-shifted worms (10-min cycles). Images were initially taken every 3 days and then taken every 6 days. 25 8C controls had a shorter life span and were imaged up to the 11th day, while the 12 8C controls and temperature-shifted worms lived almost twofold longer and were imaged up to the 29th day of survival.
molecular mechanism to achieve the extended life spans. The temperature-shifted worms had relatively high levels of two heat shock proteins (HSPs) that acted downstream of daf-16 and whose expression is elevated in a daf-2(1370) genetic background (Halaschek-Wiener et al., 2005). These were also expressed at high levels in worms constantly incubated at 25 8C, but the expression was significantly lower in the worms incubated at 12 8C. This suggests that the temperature-shifted worms used a molecular mechanism similar to that of the worms incubated at 25 8C, but showed life spans similar to nematodes incubated at 12 8C. The molecular mechanism used by the thermocycled worms are further shown to be different to those incubated at 12 8C by a clk-1 mutant, which had a life span even slightly greater for the thermocycled worms than the same mutant incubated constantly at 12 8C. We propose that the short time spent at the higher temperature (10 min at 25 8C) in the temperature-shifted worms induced a stress-relieving response and led to partial activation of the insulin-like signaling pathway and its downstream genes including HSPs. Thereafter, for the short time at the lower temperature, the proteins encoded by these genes acted to reduce
the stress caused by the heat. This cycle continued in the worms that were thermocycled and thereby were able to live for an extended time. Moreover, we suggest that 1-min intervals were not long enough to efficiently induce this gene expression. Conversely, while 1-h temperature shifts provided sufficient time for HSP induction, the time spent at 25 8C was sufficiently long to have resulted in enough oxidative stress to have counterbalanced the life span extension afforded by HSP induction. Our results demonstrate a specific type of hormesis, a phenomenon in which exposure to stress for a brief time induces the expression of genes that can counteract that stress. In most cases hormesis has been demonstrated using a single exposure (e.g., high temperature) to a stressor, although Olsen and associates (2006) examined the effects of repeated 4-h treatments at 30 8C or 33 8C. In our case, although 25 8C is well within the normal growth range of C. elegans (e.g., 25 8C is typically employed as the restrictive temperature for temperature-sensitive mutants), it was more stressful than was the growth at 12 8C. Finally, our work shows an application for thermocyclers that was not likely envisioned by Kary Mullis when he conceived and
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Fig. 4. (A) Plot of relative GFP expression in transgenic worms carrying GFP driven by the HSP-4 promoter. (B) Plot of relative GFP expression in transgenic worms carrying GFP driven by the HSP-16 promoter. Each datum point indicates the average intensities of three worms subjected to the same temperature treatment and the intervals depict a standard deviation.
developed the technique of polymerase chain reaction (PCR). But it is one we think he might find amusing.
Acknowledgements We thank David Straughan, Texas Christian University, for generating the preliminary data that gave direction to this project. This work was funded in part by two intramural sources at Texas Christian University, the URGP Fund and the URCAI Fund.
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Contents lists available at ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Repeated temperature fluctuation extends the life span of Caenorhabditis elegans in a daf-16-dependent fashion Thushara Galbadage, Phil S. Hartman * Texas Christian University, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 December 2007 Received in revised form 21 March 2008 Accepted 12 April 2008 Available online 2 May 2008
Thermocyclers were utilized to regularly shift nematodes between 12 8C and 25 8C throughout their life spans. When wild-type worms (N2) were ‘‘thermocycled’’ between 12 8C and 25 8C at 10-min intervals they lived almost as long as those that were incubated constantly at 12 8C. Shifting at 1-min or 1-h intervals lessened this effect. Similar results were observed for the long-lived mutants daf-2, eat-2 and clk-1, each of which prolongs life span through different mechanisms. In contrast, the life span of a daf-16 mutant was not prolonged by thermocycling worms, indicating that the effect is mediated by an insulinlike signaling pathway. To elucidate the molecular basis for the life span extension, two transgenic strains were employed in which heat shock proteins (HSPs) drove expression of the green fluorescent protein (GFP) gene. As expected, both HSPs were expressed at significantly higher levels in animals grown at 25 8C. Moreover, HSP expression in the thermocycled worms approximated that of animals grown at 25 8C more so than animals grown at 12 8C. This suggests that incubation at the higher temperatures for short time intervals induced stress-responsive gene expression that led to significant life span extension. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Temperature Life span Daf-16 HSP Caenorhabditis elegans Aging PCR
1. Introduction Caenorhabditis elegans, Drosophila and fish have been used extensively as model systems to study the temperature dependence of life span. The life spans of most cold-blooded organisms (poikilotherms) are strongly temperature-dependant. For example, C. elegans live much longer at lower temperatures as opposed to higher ones, with intermediate life spans at intermediate temperatures (e.g., Klass, 1977; Hosono et al., 1982). Drosophila shows similar variations in life spans (Vermeulen and Bijlsma, 2004). Several hypotheses have been advanced to explain these life span relationships. The most prominent of these is the ‘‘rate-ofliving’’ theory, first advanced by Pearl in 1928 and subsequently modified by Shaw and Bercaw (1962). This theory posits that life spans decrease as a function of increased temperature because of overall increases in the rate of metabolism. However, it has been documented that lower body temperature does not always depress metabolism (reviewed by Liu and Walford, 1972). In addition, it has been suggested that metabolism may be fundamentally
* Corresponding author at: Texas Christian University, Biology Department TCU, Box 298930, Fort Worth, TX 76129, United States. Tel.: +1 817 257 6176; fax: +1 817 257 6177. E-mail address: [email protected] (P.S. Hartman). 0047-6374/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.04.012
different at various temperatures, such that a ‘‘metabolic reorganization’’ might occur as organisms are passed from one temperature to another (Fry and Hochachka, 1970; Somero, 1972; Yen et al., 2004). Considerable insights have been derived from shifting animals from one temperature to another and assaying life span. For example, while fish live longer at low temperatures than at high temperatures, they live even longer when transferred from a higher temperature to a lower one midway through their lifetime (Liu and Walford, 1972, 1975). This follows the classic experiments of Maynard Smith (1962), who showed that Drosophila alternated between 30 8C (for 1 day) and 20 8C (for 3 days) lived almost as long as those kept continuously at 20 8C. He postulated that the adult fruit fly’s life span can be divided into two phases (‘aging or induction’ and ‘dying or development’). A similar phenomenon was observed with C. elegans. Specifically, animals reared at high temperatures lived much longer when shifted to lower temperature after they had developed into adults, but reversing this sequence did not prolong life span (Hosono et al., 1982). A slightly different experimental regimen has been employed to elucidate the molecular basis of hormesis. Hormesis can be defined as life span extensions that result from transient exposure to stressful conditions (Johnson et al., 2002; Cypser and Johnson, 2003). Exposure presumably induces a battery of genes that better protect against the normal ‘‘wear-and-tear’’ of aging. A variety of
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stressors including heat can promote hormesis in C. elegans (Lithgow et al., 1995; Butov et al., 2001; Cypser and Johnson, 2001, 2002, 2003; Yashin et al., 2001, 2002; Olsen et al., 2006). Olsen et al. (2006) have recently demonstrated that repeated thermal stress throughout the worm’s life span enabled animals to live longer than a single treatment. They also showed that levels of two heat shock proteins correlated well with the life span data; that is, both HSP-4 and HSP-16 were dramatically induced by the mild thermal treatment offered animals. We were interested in exploring the effects of more regular heat fluctuation on C. elegans life span. To do so, we employed thermocyclers in a novel fashion. Specifically, rather than amplifying DNA, we developed conditions enabling worms to be grown and maintained in microfuge tubes in a thermocycler. This allowed us to systemically shift temperatures between 12 8C and 25 8C, the upper and lower bounds for C. elegans growth. We discovered that shifting animals at 10-min increments resulted in a life span extension that was dependent upon a functional daf-16 gene. We also observed the induction of HSP-4 and HSP-16 in the ‘‘thermocycled’’ worms. Collectively, these data suggest that incubation at the higher temperature induced a daf-16-dependent increase in expression of stress-responsive genes, which in turn led to significant life span extension. 2. Materials and methods 2.1. Life span analyses 2.1.1. Strains The following strains were employed in this study: N2 (wild type), CB1370 [daf2(e1370)], CB1375 [daf-18(e1375)]CB4876 [clk-1(e2519)], DA465 [eat-2(ad465)], DR27 [daf-16(m27)], GR1307 [daf-16(mgDf50)], CL2070 [dvIs70 (hsp-16.2::GFP; rol6(su1006)] and SJ4005 [zcIs4(hsp-4::GFP)]. All were obtained from the Caenorhabditis Genetics Center and were maintained on NGM agar plates (Brenner, 1974) seeded with Escherichia coli OP50. 2.1.2. Synchronization of worms Synchronous populations of embryos were obtained by lysing gravid hermaphrodites in alkaline bleach as described by Emmons et al. (1979). Once washed free of the alkaline bleach by centrifugation, the embryos were inoculated on to NGM agar plates (Brenner, 1974) seeded with E. coli OP50. The plates were then incubated at 16 8C for 3 days before being transferred to liquid culture and subjected to a specific temperature regimen. This ensured that all worms, including the temperaturesensitive mutant daf-2, developed into adults rather than arresting as dauer larvae. 2.1.3. Life span determinations So that animals could be incubated in thermocyclers, microfuge tubes were employed for all experimental protocols. After incubating the synchronous populations at 16 8C for 3 days, larvae were counted and transferred into S medium (Lewis and Fleming, 1995) in these microfuge tubes. Different environmental conditions were tested in order to establish optimal growth conditions. These included comparisons of: (i) microfuge tubes of 500 ml and 600 ml volumes; (ii) inoculations of five worms versus ten worms per microfuge tube; (iii) different volumes of S medium, varying from 50 ml to 120 ml; (iv) different amounts of E. coli OP50 as a food source, from 1 ml to 10 ml at concentrations of between 2 109 and 1010 bacteria per milliliter (with bacteria first grown in LB broth and then resuspended after centrifugation in S buffer); and (v) either two or four holes drilled into the microfuge tubes to maintain a steady gas exchange. After a number of trials, 600 ml microfuge tubes with two holes having five worms per microfuge tube, in 100 ml of S-medium and 5 ml of E. coli (2 109 bacteria/ml) proved to be optimal in that life spans were both consistent from experiment to experiment and in keeping with reports in the literature. Therefore this set of conditions was employed for the remainder of the study. Worms were transferred into autoclaved, sterile microfuge tubes either every other day or once every 3 days, dependent upon the strain and stage of life of the worms used. Two-day transfers occurred during the fecund period to minimize contamination with progeny and fungi. Worms were monitored, counted and fed on a daily basis. In addition, 10–30 ml of S medium was added to compensate for loss owing to evaporation. These transactions typically took about 10 min and occurred at ambient temperature, which presumably had little effect on life span. Five microliters of E. coli was added daily. In reporting the life spans of the nematodes the events of ‘‘bagging’’ were not censored; on the contrary all the deaths of a cohort of worms were considered to be natural. Bagging occurs when larvae hatch internally and eventually kill a gravid
adult, creating a ‘‘bag’’ of larvae constrained by the remnant cuticle of the adult. Bagging was not censored because the overall rates observed were either less than or in the range of 10–15% for a cohort, with the exception of daf-16 worms grown at 18.5 8C, which had bagging rates of 20–25%. Thus, it was judged that premature deaths owing to bagging did not significantly alter the mean life spans in these experiments. The first day each worm was observed dead was considered to be its maximum life span. Death was assumed when no movement was observed, either of a spontaneous nature or in response to mechanical stimulation. 2.1.4. Thermocylers Two thermocyclers (PCR Sprint – SPRT001 and GeneMate – FPR0G05G) were employed for shifting the temperature at constant time intervals between 12 8C and 25 8C. The thermocyclers were programmed in such a way that they fluctuated indefinitely between these two temperatures. The ramp-times from 12 8C to 25 8C and vice versa were about 35–50 s, and were dependent on the thermocycler used. Each thermocycler could accommodate up to 20 microfuge tubes, thus allowing incubation of up to 100 worms at a given time. All temperatures were measured within 0.3 8C using a wire probe attached to a calibrated thermometer. The probe was placed in a microfuge tube containing S medium, which was positioned in the thermocyclers or incubators. Experimental worms were incubated in thermocyclers for their entire lifetimes, save the first 3 days, when they were incubated at 16 8C. Controls from the same population of synchronized worms were maintained at constant temperatures of 12 8C, 18.5 8C or 25 8C to match to the number of experimental worms. 2.2. HSP expression analyses Transgenic animals were synchronized and grown as described above, with thermocyclers utilized as described previously. One cohort of each strain was temperature shifted every 10 min between 12 8C and 25 8C. Each strain was also maintained at constant temperatures of 12 8C and 25 8C. As for the life span experiments, these worms were also transferred to fresh S medium once every 3 days. During the transfer process, three worms from each treatment were removed and observed under a fluorescent microscope (Zeiss Axioskop equipped with Nomarski optics and epifluorescence). Still images were taken using a digital camera (Sony DSC S-75). To reduce the movement of the worms a ceramic plate precooled to 20 8C was placed below the glass slide for a few seconds before obtaining the images. The process of imaging the transgenic worms was completed in less than 5 min to reduce any external stresses. Worms that were observed under the fluorescent microscope were then discarded. Fluorescent images of three worms from each of the three temperature treatments (i.e., 10-min shifts in a thermocycler, constant incubation at 12 8C, constant incubation at 25 8C) were obtained and quantified using the NIH Image (also known as ImageJ) software. In reporting the observed intensities, the worms were quantified, and the background ‘‘noise’’ was subtracted from the absolute intensities, yielding the relative GFP intensities. This procedure was carried out throughout the lifetimes of these worms and images were taken, first at 3-day intervals and then at 6-day intervals. This experimental procedure was repeated twice and the combined results are presented.
3. Results 3.1. Life span analyses 3.1.1. Wild-type (N2) worms As previously observed by Klass (1977), the life span of wildtype (N2) worms grown at 12 8C was much longer than when grown at 25 8C. N2 living at 18.5 8C (the mean of 12 8C and 25 8C) had an intermediate life span. In our hands, N2 worms constantly incubated at 12 8C, 18.5 8C and 25 8C had mean lifetimes of 33.7 days, 19.2 days and 12.6 days, respectively (Fig. 1A). When temperature was shifted between 12 8C and 25 8C at 10-min intervals, the same strain had a mean life span of 31.0 days, which was very close to the mean life span of worms constantly living at 12 8C (Fig. 1A). Most significantly, this was much longer than the 19.2 day mean life span obtained for worms grown constantly at 18.5 8C the temperature half way between 12 8C and 25 8C. N2 worms were also shifted at 1-min and 1-h intervals between 12 8C and 25 8C and had life spans of 19.4 days and 24.5 days (versus 31.0 days for 10-min shifts), respectively. Therefore, 10min intervals prolonged life spans to a greater extent than periodicities of 1 min or 1 h. Furthermore, wild-type animals that spent 10 min at 12 8C and 50 min at 25 8C or 50 min at 12 8C and
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intervals, the life span was similar to the 18.5 8C control, with a mean life span of 16.7 days (Fig. 1B). Therefore, the life span extension afforded by temperature shifts was absent in a daf-16 genetic background. Life span determinations were conducted on the daf-16 experiment on three occasions giving consistant results (Table 1). This result was not limited to the mgDf50 allele of daf-16, but was also observed in a second daf-16 allele (m27). These thermocycled animals had a significantly shorter 50% reduction in life span compared to those that were reared constantly at 12 8C. Specifically, they had mean life spans of 11.5 days, 16.1 days and 27.2 days when grown at 25 8C, in a thermocycler and at 12 8C, respectively (data not shown). In a similar fashion, a mutation in daf-18 eliminated the life span extension afforded by thermocycling animals, having life spans of 13.5 days, 18.8 days and 28.7 days when grown at 25 8C, in a thermocycler and at 12 8C, respectively (data not shown). 3.2. HSP expression analyses
Fig. 1. (A) Survival plot of N2 (wild-type) nematodes. Wild-type shifted every 10 min between 12 8C and 25 8C (N2 10-min cycles), and three control groups of N2 constantly incubated at 12 8C, 18.5 8C or 25 8C. (B) Survival plot of a daf-16 mutant. daf-16 shifted every 10 min between 12 8C and 25 8C (daf-16 10-min cycles), and three control groups of daf-16 constantly incubated at 12 8C, 18.5 8C or 25 8C. The arrows indicate the 50% survival time (median life span) at each temperature regimen.
10 min at 25 8C yielded mean life spans of 15.4 days and 29.8 days, respectively (Table 1). Thus, even though most of these treatments extended the survival time of N2 worms, the greatest effect was observed with the 10-min thermocycling treatment. 3.1.2. Long-lived mutants The same experimental protocol was carried out on long-lived clk-1, eat-2, and daf-2 mutants. The clk-1 and eat-2 mutants displayed basically the same overall pattern as observed with the wild-type. Specifically, animals were short lived at 25 8C and long lived at 12.5 8C. Most importantly, their life spans were most definitely not intermediate to 12 8C and 25 8C when they were shifted from 12 8C to 25 8C at 10-min intervals (Fig. 2A and B). In fact, these animals live slightly longer than those grown constantly at 12 8C, despite the fact that they spend one-half of their adult lives at 25 8C. Conversely, the daf-2 mutant showed a temperatureindependent life span, living almost the same time under all three temperature regimens (Fig. 2C and Table 1). This was observed previously by Gems and associates (1998). 3.1.3. daf-16 mutant Similar to wild-type worms, a daf-16 mutant had a longer life span at 12 8C, an intermediate one at 18.5 8C and a much shorter one at 25 8C. These were 29.9 days, 18.6 days and 10.8 days, respectively. As reported in the literature (Libina et al., 2003; Hartman and Ishii, 2007), the life span of the daf-16 mutant was slightly shorter than that of wild type. Most importantly, when the daf-16 mutant was shifted between 12 8C and 25 8C at 10-min
3.2.1. HSP-4 transgenic worms The experiments reported in the previous section indicate the life span extension caused by temperature shifting is mediated through the insulin-like signaling pathway controlled by daf-16. In an effort to address the mechanism by which this pathway prolongs life span, two transgenic mutants were employed that expressed green fluorescent protein (GFP) under the control of the heat shock protein (HSP) promoters HSP-4 and HSP-16. HSPs are activated downstream of daf-16 in response to environmental stresses. As such, they are good genetic markers to identify the activation of the daf-16 pathway. Although life spans were not rigorously determined for these two transgenic strains, animals grown at 12 8C and those shifted at 10-min intervals were observed to live significantly longer than animals maintained at 25 8C; that is, their life spans approximated that of N2. These worms showed similar physical characteristics to the N2 worms and produced similar amounts of progeny. Transgenic worms with GFP driven by a HSP-4 promoter were subjected to the same experimental protocol as employed for life span determinations. The expression of GFP was periodically quantified via fluorescent microscopy and digital photography (Fig. 3). The worms incubated at 12 8C had little activation of HSP-4 protein as evidenced by minimal expression of GFP. On the contrary, worms incubated at 25 8C expressed high levels of GFP, more so towards the latter part of their lifetime. The thermocycled worms (at 10-min intervals between 12 8C and 25 8C) also expressed high levels of GFP, approximating levels of the worms incubated at 25 8C and at significantly higher levels than the worms incubated at 12 8C (Figs. 3 and 4A). The time needed for significant HSP-4 induction was greater than 12 h, as animals that were thermocycled for 12 h had GFP levels only 1.2 higher than those held at 12 8C (data not shown). Therefore, GFP levels were measured at 3-day intervals at first, and followed by 6-day intervals. As indicated by the relatively small standard error bars, there was relatively little variation from animal to animal for each treatment. 3.2.2. HSP-16 transgenic worms A second set of transgenic worms with GFPs driven by a HSP-16 promoter was subjected to similar experimentation. Though GFP expression was slightly lower than those of the worms having GFP driven by the HSP-4 promoter, they showed the same pattern of expression, having minimal levels expressed at 12 8C and much higher levels expressed at 25 8C and in the thermocycled worms (Fig. 4B). As with HSP-4, the induction of HSP-16 was a gradual process, as animals that were thermocycled for 12 h had GFP levels only 1.4 higher than those held at 12 8C (data not shown).
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Table 1 A summary of all experimental results listing the strains used (N2, daf-16, clk-1, eat-2 and daf-2), temperature regimens considered and individually listing each experiments result (indicating repeated trials for consistency) Strain
Temperature regiment
Mean lifespan S.D. (days)
50% survival (days)
Maximum life span (days)
Total number of worms
N2 (wild-type)
12 8C
33.0 6.9 33.7 6.4 31.2 7.5 34.0 7.7 19.2 4.7 17.4 4.1 15.1 3.7 12.6 3.0 13.2 3.7 11.5 2.1 30.6 6.5 31.0 7.5 28.8 8.5 24.5 9.2 19.4 6.0 29.8 9.7 24.8 6.1 15.4 5.4 14.3 3.3
33.0 32.5 30.5 33.5 19.0 17.0 15.0 13.0 13.5 11.0 31.5 32.0 29.0 27.0 20.0 29.0 24.0 15.0 14.5
44 47 48 45 27 24 21 17 18 15 42 44 41 36 32 49 40 24 18
50 25 20 15 25 15 50 25 20 15 50 25 15 50 30 25 20 25 20
30.0 7.9 29.9 8.0 28.9 6.9 18.6 5.3 16.2 5.5 13.4 3.5 10.8 2.9 9.5 1.3 16.0 3.2 16.7 4.2 11.3 2.6
32.0 30.5 29.0 18.5 15.0 13.5 11.0 9.0 15.5 16.0 10.0
42 43 40 28 25 19 15 12 23 25 17
50 25 20 25 25 50 25 15 50 25 15
40.7 8.4 38.2 10.3 39.2 8.9 18.7 4.3 15.5 3.8 42.0 12.1 41.4 12.5
43.0 40.5 41.0 18.0 15.0 44.5 43.5
53 51 50 26 22 58 56
50 25 20 25 15 25 15
1-h cycles
36.6 12.6 34.7 8.9 17.3 4.7 16.6 3.8 37.1 15.2 36.5 13.3 25.8 15.5
41.5 36.5 16.5 16.0 42.5 39.5 29.0
58 48 28 23 61 55 51
50 15 25 15 50 15 50
12 8C 25 8C 10-min cycles (at 120c and 250c) 1-h cycles
38.9 12.3 38.7 15.2 39.7 14.9 32.6 18.3
43.0 42.0 43.0 40.5
64 60 67 58
50 50 50 50
18.5 8C 25 8C
10-min cycles (at 12 8C and 25 8C)
1-h cycles 1-min cycles 50–10-min cycles (at 12 8C and 25 8C) 10–50-min cycles (at 12 8C and 25 8C)
daf-16
12 8C
18.5 8C 25 8C
10-min cycles (at 12 8C and 25 8C)
clk-1
12 8C
25 8C 10-min cycles (at 12 8C and 25 8C)
eat-2
12 8C 25 8C 10-min cycles (at 12 8C and 25 8C)
daf-2
The results are reported with the mean life span standard deviation, median life span (50% survival), longest lived worm (maximum life span) and the total number of worms used for each experiment.
4. Discussion Many factors contribute to altering the life span of organisms. One of them is temperature, which plays an especially important role in determining the lifetime of cold-blooded organisms (poikilotherms) including the nematode C. elegans (Klass, 1977; Hosono et al., 1982). Klass (1977) measured the life spans of wildtype C. elegans at six different temperatures ranging from 10 8C to 26 8C. Interpolating his data, the mean life span was 9 days at 25 8C, 17 days at 18.5 8C (the temperature one-half way between 12 8C and 25 8C, the high and low temperatures employed in the current study) and 27.5 days at 12 8C. In a similar fashion, Hosono and associates (1982) found life spans were 8.2 days, 12.6 days and 19.7 days when grown at 25 8C, 20 8C and 15 8C, respectively. These values compare favorably with our data (Table 1), indicating that
we have not generated aberrant data using the culture conditions developed for our experiments. In addition, all three data sets indicate that animals grown at intermediate temperatures respond with intermediate life spans. This predicts that, in the absence of some sort of metabolic change, alternating animals systemically between the two extreme temperatures should have yielded a life span roughly equivalent to that when animals were grown at the intermediate temperature. Contrary to this prediction, wild-type C. elegans lived only 8% shorter (rather than 57% or 62% shorter predicted by Klass’ and Hosono’s data, respectively) when ‘‘thermocycled’’ between 12 8C and 25 8C at 10-min intervals (Fig. 1A and Table 1). Therefore, the animals cycled between 12 8C and 25 8C are clearly physiologically different than those grown constantly at the intermediate temperature.
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Fig. 2. (A) Survival plot of a clk-1 mutant. clk-1 shifted every 10 min between 12 8C and 25 8C (clk-1 10-min cycles), and two control groups of clk-1 constantly incubated at 12 8C or 25 8C. (B) Survival plot of a eat-2 mutant. eat-2 shifted every 10 min between 12 8C and 25 8C (eat-2 10-min cycles), and two control groups of eat-2 constantly incubated at 12 8C or 25 8C. (C) Survival plot of a daf-2 mutant. daf-2 shifted every 10 min between 12 8C and 25 8C (daf-2 10-min cycles), and two control groups of daf-2 constantly incubated at 12 8C or 25 8C. The arrows indicate the 50% survival time (median life span) at each temperature regimen.
To elucidate the genetic requirements of this physiological difference, we examined the responses of a number of C. elegans mutants known to affect life span through different mechanisms. First, we measured life spans in an eat-2 strain that alters pharyngeal pumping, thus limiting consumption in a fashion that mimic caloric restriction (McKay et al., 2004; Lakowski and Hekimi, 1998). We observed that, as is consistent with the literature, eat-2 mutant animals were long lived. In addition, the ‘‘thermocycled’’ animals lived much longer than did animals grown constantly at the intermediate temperature (Fig. 2B and Table 1). Therefore, the physiological alteration provoked by alternating between 12 8C and 25 8C was not mediated via caloric restriction. Second, we determined the life spans in a clk-1 mutant background. clk-1 encodes a highly conserved mitochondrial protein that when mutated results in pleiotropic effects that affect physiological traits, including life span extension (Wong et al., 1995). We were particularly intrigued with clk-1 because Branicky and associates (2001) demonstrated that the defecation cycle length was significantly affected when wild-type worms were reared at 20 8C and shifted to another temperature. Conversely, clk-1 animals did not adjust the length of their defecation cycles under the same conditions. This indicates that in wild-type C. elegans adaptation to temperature is an active process mediated by Clk-1. However, as was the case with eat-2, clk-1
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mutants lived significantly longer than predicted when alternated between 12 8C and 25 8C (Fig. 2A and Table 1). Third, we explored whether the insulin-like signaling pathway, which plays a prominent role in regulating C. elegans life span (Guarente and Kenyon, 2000), might mediate the physiological change triggered by alternating temperature between 12 8C and 25 8C. We initially examined the effects of the daf-2 allele e1370. The resultant decrease in daf-2 signaling caused a significant life span extension. We observed that daf-2 animals lived roughly the same length under three temperature regimens (constant at 12 8C, constant at 25 8C and ‘‘thermocycled’’) (Fig. 2C and Table 1). A similar temperature-independence was observed by Gems and associates (1998). Therefore, these results did not inform as to the role of the insulin-like signaling pathway in C. elegans’ response to ‘‘thermocycling.’’ We then determined the life spans in a daf-16 genetic background. daf-16 is negatively regulated by daf-2 and encodes a fork-head transcription factor that induces expression of a number of genes in response to insulin-like signaling. Unlike with eat-2, clk-1, and daf-2, a loss-of-function mutation in daf-16 clearly abrogated the life span extension provoked by regularly alternating temperature between 12 8C and 25 8C. Specifically, the daf-16 animals grown in the thermocycler lived over 70% shorter than at 12 8C. In fact, unlike wild type, eat-2, and clk-2, the ‘‘thermocycled’’ daf-16 animals lived slightly shorter than did the animals constantly maintained at 18.5 8C. Therefore, this effect is mediated through the insulin-like signaling pathway. Both SKN-1 (Wilson et al., 2006) and AAK-2 (Apfeld et al., 2004) have been shown to function in parallel and downstream of daf-2 in the insulin-like signaling pathway. The fact that the daf-16 mutation completely abrogated the life span extension afforded by growth in a thermocycler indicates that neither gene plays a role in this effect. Johnson and Lithgow (Johnson et al., 1996; Lithgow, 1996) proposed that the insulin-like signaling pathway controls life span by regulating expression of stress-response genes that are downstream targets of daf-16. In particular, a number of studies (Walker and Lithgow, 2003; Rea et al., 2005) have provided evidence that heat shock proteins, notably HSP-16, play an important role in this process. We therefore examined expression of HSP-16 as well as that of heat-shock protein HSP-4 using transgenic strains in which the GFP gene was placed under the control of either the HSP-16 or HSP-4 promoter (Figs. 3 and 4). These data indicate that the same 10-min fluctuations that extended life span also increased expression of both heat shock proteins. This is consistent with the notion that the physiological response to temperature fluctuation is ultimately mediated by heat-shock proteins whose expression is regulated in a daf-16-dependent fashion. We do not necessarily posit that these two HSPs are solely responsible for the life span extension afforded by thermocycling worms. There are a number of downstream targets of daf-16 (Lee et al., 2003; Murphy et al., 2003; Jensen et al., 2006), including HSP-16 and HSP-4. Interestingly, the duration of the temperature shifts strongly influenced the extent of the life span extension. Specifically, 10min intervals yielded the longest observed life span extensions of the wild-type nematodes, while temperature shifts of shorter time intervals (e.g., 1 min), longer time intervals (e.g., 1 h) or even combinations of time intervals did not have as a strong effect as did the 10-min thermocycling (Table 1). The fact that 10-min shifts elicited a stronger response than 1-min shift is particularly noteworthy because it indicates that the life span extensions were not the consequence of temperature change per se. That is, they were not the result of the transitions between 12 8C and 25 8C, which were approximately eight times more frequent with the 1min versus the 10-min shifts. Though the temperature-shifted worms had life spans similar to the worms living constantly at 12 8C, they used a different
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Fig. 3. Digital images of the transgenic worms (HSP-4) expressing GFP. The absolute intensities of GFP expressed driven by the HSP-4 promoter is shown as a function of survival time. The 12 8C and 25 8C controls are shown on either side of the temperature-shifted worms (10-min cycles). Images were initially taken every 3 days and then taken every 6 days. 25 8C controls had a shorter life span and were imaged up to the 11th day, while the 12 8C controls and temperature-shifted worms lived almost twofold longer and were imaged up to the 29th day of survival.
molecular mechanism to achieve the extended life spans. The temperature-shifted worms had relatively high levels of two heat shock proteins (HSPs) that acted downstream of daf-16 and whose expression is elevated in a daf-2(1370) genetic background (Halaschek-Wiener et al., 2005). These were also expressed at high levels in worms constantly incubated at 25 8C, but the expression was significantly lower in the worms incubated at 12 8C. This suggests that the temperature-shifted worms used a molecular mechanism similar to that of the worms incubated at 25 8C, but showed life spans similar to nematodes incubated at 12 8C. The molecular mechanism used by the thermocycled worms are further shown to be different to those incubated at 12 8C by a clk-1 mutant, which had a life span even slightly greater for the thermocycled worms than the same mutant incubated constantly at 12 8C. We propose that the short time spent at the higher temperature (10 min at 25 8C) in the temperature-shifted worms induced a stress-relieving response and led to partial activation of the insulin-like signaling pathway and its downstream genes including HSPs. Thereafter, for the short time at the lower temperature, the proteins encoded by these genes acted to reduce
the stress caused by the heat. This cycle continued in the worms that were thermocycled and thereby were able to live for an extended time. Moreover, we suggest that 1-min intervals were not long enough to efficiently induce this gene expression. Conversely, while 1-h temperature shifts provided sufficient time for HSP induction, the time spent at 25 8C was sufficiently long to have resulted in enough oxidative stress to have counterbalanced the life span extension afforded by HSP induction. Our results demonstrate a specific type of hormesis, a phenomenon in which exposure to stress for a brief time induces the expression of genes that can counteract that stress. In most cases hormesis has been demonstrated using a single exposure (e.g., high temperature) to a stressor, although Olsen and associates (2006) examined the effects of repeated 4-h treatments at 30 8C or 33 8C. In our case, although 25 8C is well within the normal growth range of C. elegans (e.g., 25 8C is typically employed as the restrictive temperature for temperature-sensitive mutants), it was more stressful than was the growth at 12 8C. Finally, our work shows an application for thermocyclers that was not likely envisioned by Kary Mullis when he conceived and
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Fig. 4. (A) Plot of relative GFP expression in transgenic worms carrying GFP driven by the HSP-4 promoter. (B) Plot of relative GFP expression in transgenic worms carrying GFP driven by the HSP-16 promoter. Each datum point indicates the average intensities of three worms subjected to the same temperature treatment and the intervals depict a standard deviation.
developed the technique of polymerase chain reaction (PCR). But it is one we think he might find amusing.
Acknowledgements We thank David Straughan, Texas Christian University, for generating the preliminary data that gave direction to this project. This work was funded in part by two intramural sources at Texas Christian University, the URGP Fund and the URCAI Fund.
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