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. 2010 Sep 8;12(3):260-72.
doi: 10.1016/j.cmet.2010.08.004.

Insulin-like signaling determines survival during stress via posttranscriptional mechanisms in C. elegans

Gawain McColl  1 , Aric N Rogers, Silvestre Alavez, Alan E Hubbard, Simon Melov, Christopher D Link, Ashley I Bush, Pankaj Kapahi, Gordon J Lithgow
Affiliations

Insulin-like signaling determines survival during stress via posttranscriptional mechanisms in C. elegans

Gawain McColl et al. Cell Metab. .

Abstract

The insulin-like signaling (ILS) pathway regulates metabolism and is known to modulate adult life span in C. elegans. Altered stress responses and resistance to a wide range of stressors are also associated with changes in ILS and contribute to enhanced longevity. The transcription factors DAF-16 and HSF-1 are key effectors of the longevity phenotype. We demonstrate that increased intrinsic thermotolerance, due to lower ILS, is not dependent on stress-induced transcriptional responses but instead requires active protein translation. Translation profiling experiments reveal genes that are posttranscriptionally regulated in response to altered ILS during heat shock in a DAF-16-dependent manner. Furthermore, several novel proteins are specifically required for ILS effects on thermotolerance. We propose that lowered ILS results in metabolic and physiological changes. These DAF-16-induced changes precondition a translational response under acute stress to modulate survival.

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Figures

Figure 1
Figure 1. ILS increases Resistance and Survival to Heat Shock
(A) daf-2(e1370) mutants have significantly increased thermotolerance (median survival 920 mins, n=42, p<0.0001) compared to wild type (630 min, n=53). Mutation of daf-16 suppresses thermotolerance increase (p<0.0001), daf-16(m26);daf-2(e1370) median survival 500 min, n=50. (B) daf-2(e1370) mutants have significantly increased survival (p<0.0001) following a 7h 35°C heat shock, compared to wild type or daf-16(m26);daf-2(e1370).
Figure 2
Figure 2. ILS effects on Heat Shock Induced HSP mRNA levels
Average expression levels of (A) hsp-12.6, (B) hsp-16.2 and (C) hsp-70. Relative expression levels were derived from Calibrated Normalized Relative Quantities using the geometric mean of 2 ‘house keeping’ genes, gpd-1 and gpd-4 and are plotted as arbitrary units (A.U) ±S.E.M. Data from n=15 individual worms for wild type and daf-16(m26);daf-2(e1370) groups, and n=14 for the daf-2(e1370) groups. For all genes and in all genotypes the Heat shock (HS) treatment (2h at 35°C) significantly increased relative mRNA levels (p<0.001) compared to genotype matched controls (CTL). Asterisks above CTL bars mark significance compared to wild type CTL, while those above HS bars are against wild type HS values (* p<0.05, ** p<0.01 and *** p<0.001).
Figure 3
Figure 3. ILS Does Not Alter HSF-1 DNA-Binding Activity and HSF-1 is Not Required for Thermotolerance
(A) Validation of EMSA of HSF-1-HSE DNA binding following a HS. Lane 1 free ds-DNA probe; lane 2 control nuclear protein extract (NPE); lane 3 NPE from samples treated with a 4h 35°C HS. Lane 4, NPE from HS with molar excess of cold HSE, lane 5 NPE from HS with molar excess of cold HSE-mut1, lane 6 NPE from HS with molar excess of cold HSE-mut2 (see experimental procedures). Data shown is representative of replicate assays. (B) ILS does not alter HS induced DNA binding activity of HSF-1. Lane 1 free ds-DNA probe; lane 2 wild type NPE; lane 3 daf-2(e1370) NPE; lane 4 daf-16(m26) NPE. No heat shock controls (-), 1h of 35°C HS (+1) and 4h of 35°C HS (+4). Representative image of duplicate experiments. (C) Fluorometric analysis showing Relative Fluorescence Units (R.F.U) during recovery at 25°C of wild type and daf-2(e1370) mutants, which show unaltered GFP induction of a hsp-16.2:gfp transcriptional reporter and hence increased hsp-16.2 promoter activity following a HS compared to wild type. Treatments were controls (CTL), and HS of 33°C for 2h (HS). Error bars ± 2× S.E.M.. (D) Loss of HSF-1 activity abolishes mean GFP induction following a HS. Error bars ± 2× S.E.M. (E) Immunoblot detection of HSP-16.2 in wild type and hsf-1(sy441) mutants following an inducing HS. hsf-1(sy441) mutants express greatly reduced HSP-16.2. No HS control (-) and 2h 33°C HS with 6h recovery (+). Equivalent loading of total protein per lane demonstrated via detection of β-ACTIN. (F) Loss of HSF-1 does not affect intrinsic thermotolerance to 35°C heat stress, but does limit acquired thermotolerance. Kaplan-Meier survival curves show wild type and hsf-1(sy441) mutants have equivalent thermotolerance, median survival 420 min (wild type, n= 53) and 430 min (hsf-1(sy441), n=52) respectively (not significant). Pretreatment (30°C for 6h, followed by 12h recovery at 20°C, shown by dashed lines) significantly increases wild type thermotolerance, p<0.0001 (700 min, n=56). hsf-1(sy441) mutants also show a gain of thermotolerance following pretreatment, p<0.0001 (660 min, n=54), however, this gain is reduced compared to that observed in wild type, p<0.0001. (G) Loss of wild type HSF-1 activity does not affect daf-2(RNAi) gains in thermotolerance. Wild type and hsf-1(sy441) mutants have equivalent thermotolerance, median survival 540 min (wild type, n= 47) and 600 min (hsf-1(sy441), n=40) respectively (not significant). Pretreatment with daf-2(RNAi) significantly increases (p<0.0001) both wild type (1020 min, n=62) and hsf-1(sy441) thermotolerance (1020 min, n=45).
Figure 4
Figure 4. Inhibition of Transcription Does Not Alter Thermotolerance
(A) Effects of inhibition of transcription by α-amanitin pretreatment prior to inducing HS. Increasing concentrations of α-amanitin blocked HS induced GFP induction. Plotted is the Relative Fluorescence (R.F) versus time during recovery at 25°C. Error bars ±2× S.E.M. (B) Average expression levels of hsp-12.6, hsp-16.1 and hsp-16.2 following a heat shock (HS, 2h at 35°C), with and without a 1h pretreatment with α-amanitin (α-aman). Relative expression levels were derived from Calibrated Normalized Relative Quantities using the geometric mean of 2 ‘house keeping’ genes, gpd-1 and gpd-4 and are plotted as arbitrary units (A.U) ±S.E.M. Data from n=11 individuals. For all genes the heat shock (+, HS) treatment significantly increased relative mRNA levels (p<0.001) compared to controls (-). α-amanitin pretreatment significant reduced stress induced expression for all genes (p<0.001). (C) Immunoblot using anti-HSP-16.2 antibody of untreated (-) and heat shocked (+, HS) (33°C for 2h with a 12h recovery, HS) wild type populations with and without a pretreatment of 100ug/ml α-amanitin (as previously described). (D) Inhibition of transcription does not affect intrinsic thermotolerance in wild type, daf-2(e1370) or daf-16(m26) mutants. Kaplan-Meier survival curves show daf-2(e1370) mutants have significantly increased thermotolerance compared to wild type (p<0.0001) and daf-16(m26) mutants (p<0.0001), respectively. Pretreatment with 100 mg/ml α-amanitin (α-aman) did not alter thermotolerance of any genotype. Median survival were 480 min (wild type, n=50), 480 min (wild type + α-amanitin, n=50), 590 min (daf-2(e1370), n=51), 590 min (daf-2(e1370) + α-amanitin, n=52), 480 min (daf-16(m26), n=52), 590 min (daf-16(m26) + α-amanitin, n=50). (E) Inhibition of transcription significantly reduces resistance to the oxidative stressor juglone. Median survival of wild type was significantly reduced following pretreatment with 100 mg/ml α-amanitin (α-aman), p<0.0001. Median survival were 195 min (wild type, n=46) versus 135 min (wild type + α-amanitin, n=45). Median survival of daf-16(mu86) (185 min, n=47) was also significantly reduced compared to wild type, p<0.05.
Figure 5
Figure 5. Thermotolerance of ILS Mutants Determined by de novo Translation
(A) Effects of inhibition of translation by cycloheximide pretreatment prior to inducing HS. Increasing concentrations of cycloheximide reduced HS induced GFP induction. Plotted is the Relative Fluorescence (R.F) versus time during recovery at 25°C. Error bars ±2× S.E.M. (B) Inhibition of translation suppresses daf-2(e1370) gains of thermotolerance. Kaplan-Meier survival curve shows daf-2(e1370) mutants compared to wild type have significantly increased thermotolerance, median survival 360 min (wild type, n=53) versus 480 min (daf-2(e1370), n=57), p<0.0001. However, treatment with 10 mM cycloheximide (CHX), significantly reduces (daf-2(e1370) survival (360 min, n=55), p<0.0001. No effect was observed in wild type (360 min, n=54), not significant. (C) Inhibition of translation suppresses age-1(hx546) gains of thermotolerance. Kaplan-Meier survival curve shows age-1(hx546) mutants compared to wild type have significantly increased thermotolerance, median survival 360 min (wild type, n=53) versus 480 min (age-1(hx546), n=54), p<0.0001. Treatment with 10 mM cycloheximide (CHX), significantly reduces (age-1(hx546) survival (360 min, n=57), p<0.0001.
Figure 6
Figure 6. Translation State Array Analysis of Heat Shock Responses in C. elegans
(A) Schematic of Translation State Array experimental design. (B) Scatter plot of log2 wild type HS F3 versus daf-2(e1370) log2 HS F3 versus. Transcripts with significantly altered abundance were identified via t-tests and a FDR ≤ 0.05. Transcripts significantly increased in daf-2(e1370) shown in orange (n = number of data points) and those decreased shown in purple, r2 denotes the correlation coefficient and ** p<0.01. (C) Plot of Relative Abundance of the sdz-28 transcript in each fraction (F1-F3) under control (CTL) and HS treatments (HS). Each bar represent mean of 4 biological replicates. (D) Plot of Relative Abundance of the phat-4 transcript in each fraction (F1-F3) under control (CTL) and HS treatments (HS). Each bar represent mean of 4 biological replicates. (E) Scatter plot of log2 wild type CTL Ti (i.e. F3/F2) versus log2 wild type HS Ti.
Figure 7
Figure 7. C08H9.1 is post-transcriptionally regulated by ILS and required for increased ILS-mediated thermotolerance
(A) Venn diagram of transcripts identified as having significantly elevated Ti under HS compared to CTL. Shown is number of transcripts shared and unique to each genotype respectively. (B) Graph of the HS Ti / CTL Ti ratio for transcripts F46F5.4 and C08H9.1 in each genotype (as shown). (C) Relative abundance of C08H9.1 transcripts in each fraction, where F1, F2 and F3 represent unbound mRNA, light polysomes and heavy polysomes during control (CTL) and HS conditions (HS). Each bar represents the mean relative abundance measured in four biological replicates. (D) Schematic of the predicted domain organization of the C08H9.1 protein. (E) C08H9.1 is essential for increased daf-2(e1370) thermotolerance at 35°C. Kaplan-Meier survival curves showing daf-2(e1370) mutants have significantly increased thermotolerance compared to wild type, p<0.0001. Median survival: 540 min (wild type + empty vector (EV), n=50) versus 990 min (daf-2(e1370) + EV, n=42). (C08H9.1) RNAi suppression of elevated daf-2(e1370) thermotolerance (p<0.0001) is comparable to daf-16(RNAi). Median survival 540 min (daf-2(e1390) + daf-16(RNAi), n=50) and 540 min (daf-2(e1390) + C08H9.1(RNAi), n=44). (F) C08H9.1 does not modulate wildtype thermotolerance at 35°C. Kaplan-Meier survival curves showing daf-2(e1370) mutants have significantly increased thermotolerance compared to wild type, p<0.0001. Median survival: 360 min (wild type + empty vector (EV), n=50) versus 580 min (daf-2(e1370) + EV, n=45). Neither daf-16(RNAi) nor (C08H9.1) RNAi altered wild type survival. Median survival 360 min (wild type + daf-16(RNAi), n = 46) and 360 min (wild type + C08H9.1(RNAi), n=50). (G) C08H9.1 is required for daf-2(e1370) but not wild type longevity at 25°C. Kaplan-Meier survival curves showing daf-2(e1370) mutants are long lived compared to wild type, p<0.0001. Median lifespan: 17 (days) (wild type + empty vector (EV), n=72) versus 27 (daf-2(e1370) + EV, n=42). daf-16(RNAi) reduced daf-2(e1370) lifespan, p<0.0001, median life span: 14 (daf-2(e1390) + daf-16(RNAi), n=75). (C08H9.1)RNAi moderately reduced daf-2(e1370) lifespan, p<0.0026, median life span: 22 (daf-2(e1390) + C08H9.1(RNAi), n=65). In contrast, C08H9.1(RNAi) had no effect on wild type lifespan, median lifespan: 16 (wild type + C08H9.1(RNAi), n=72).
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References

    1. Benjamini Y, Hochberg Y. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. (B).Journal of the Royal Statistical Society. 1995;57:289–300.
    1. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. - PMC - PubMed
    1. Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing activities protect against age-onset proteotoxicity. Science. 2006;313:1604–1610. - PubMed
    1. Cypser JR, Johnson TE. Multiple stressors in Caenorhabditis elegans induce stress hormesis and extended longevity. J Gerontol A Biol Sci Med Sci. 2002;57:B109–B114. - PubMed
    1. Dinkova TD, Keiper BD, Korneeva NL, Aamodt EJ, Rhoads RE. Translation of a small subset of Caenorhabditis elegans mRNAs is dependent on a specific eukaryotic translation initiation factor 4E isoform. Mol Cell Biol. 2005;25:100–113. - PMC - PubMed

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