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. 2010 May 25;107(21):9730-5.
doi: 10.1073/pnas.1002575107. Epub 2010 May 11.

Insulin/IGF-1 signaling mutants reprogram ER stress response regulators to promote longevity

Sivan Henis-Korenblit  1 , Peichuan Zhang, Malene Hansen, Mark McCormick, Seung-Jae Lee, Michael Cary, Cynthia Kenyon
Affiliations

Insulin/IGF-1 signaling mutants reprogram ER stress response regulators to promote longevity

Sivan Henis-Korenblit et al. Proc Natl Acad Sci U S A. .

Abstract

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response is activated. This ER stress response restores ER homeostasis by coordinating processes that decrease translation, degrade misfolded proteins, and increase the levels of ER-resident chaperones. Ribonuclease inositol-requiring protein-1 (IRE-1), an endoribonuclease that mediates unconventional splicing, and its target, the XBP-1 transcription factor, are key mediators of the unfolded protein response. In this study, we show that in Caenorhabditis elegans insulin/IGF-1 pathway mutants, IRE-1 and XBP-1 promote lifespan extension and enhance resistance to ER stress. We show that these effects are not achieved simply by increasing the level of spliced xbp-1 mRNA and expression of XBP-1's normal target genes. Instead, in insulin/IGF-1 pathway mutants, XBP-1 collaborates with DAF-16, a FOXO-transcription factor that is activated in these mutants, to enhance ER stress resistance and to activate new genes that promote longevity.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
 ire-1 and xbp-1 contribute to the longevity of daf-2(−) animals. (A) Reduction of ire-1 activity by the null mutation ok799 shortened the lifespan of daf-2(e1370) mutants more than it shortened the lifespan of wild type. (B) The xbp-1(zc12) mutation shortened the lifespan of daf-2(e1370) mutants more than it shortened wild-type lifespan. (C) The xbp-1(zc12) mutation did not further shorten the lifespan of ire-1(−); daf-2(−) double mutants, suggesting that these genes function in a common pathway. [Compare survival curves of ire-1(−); daf-2(−) and ire-1(−); daf-2(−) xbp-1(zc12) mutants.] Note that xbp-1(zc12) mutation shortened the lifespan of daf-2(e1370) mutants, but not as much as did an ire-1(ok799) deletion. [Compare survival curves of ire-1(−); daf-2(−) and daf-2(−) xbp-1(zc12) mutants.] As xbp-1(zc12) is likely to eliminate xbp-1 function, this finding implies that ire-1 contributes to the longevity of daf-2 mutants in part through an xbp-1–independent pathway. For additional lifespan data, see Table S1. Also, see Fig. S1 for data showing that other ER stress–response genes, namely abu-11, pek-1, and atf-6, are not involved in this pathway.
Fig. 2.
Fig. 2.
 The ire-1/xbp-1 pathway is set at a lower level in daf-2(−) mutants. (A) Bar graph representing the mean ratio of spliced/unspliced xbp-1 transcripts, multiplied by a factor of 100, in three independent biological experiments. Control lane (yellow bar) presents the xbp-1 transcripts ratio in wild-type animals treated with tunicamycin. The rest of the bars present steady-state xbp-1 transcripts ratio in day-1 wild-type, ire-1, daf-2, and daf-16; daf-2 animals. These animals have not been treated with tunicamycin (blue bars). Error bars represent SEM. Asterisks mark Student t test values of P < 0.01. See Fig. S2 for data showing a representative RT-PCR experiment. (B) Representative fluorescence micrographs (100-fold magnification) of day-3 adults harboring an integrated Phsp-4::gfp transgene in a wild-type or daf-2(e1370) background. daf-2 mutants expressed lower levels of the Phsp-4::gfp transgene than did wild type. wild type, n = 9, relative mean fluorescence intensity (m) = 1 ± 0.14; daf-2(e1370), n = 10, m = 0.34 ± 0.07, P = 0.0012. (C) daf-2(e1370) mutants expressed less Phsp-4::gfp transgene than did wild type even upon heat shock. Phsp-4::gfp expression at 20 °C or upon 3 h of heat shock at 30 °C. Arrows point at anterior and posterior ends of the animals.
Fig. 3.
Fig. 3.
 daf-2(−) mutants are resistant to ER stress. Eggs from wild-type or daf-2(e1370) animals containing xbp-1(zc12), ire-1(ok799), pek-1(ok275), or atf-6(ok551) mutations were grown in the presence of 0, 2, or 5 μg/mL tunicamycin (corresponding to the blue, red, and off-white bars in the graph). Percentage of eggs that developed into mature adults was scored. Student t test values were calculated between daf-2(+) and daf-2(−) backgrounds. Each experiment was repeated independently with similar effects. Each strain was scored on three independent plates. Error bars represent SEM for repeat plates within the experiment. Asterisks mark Student t test values of P < 0.01.
Fig. 4.
Fig. 4.
 dox-1 promotes longevity, but not ER stress resistance, in daf-2 mutants. (A) Reduction of dox-1 activity by RNAi shortened the lifespan of daf-2(mu150) mutants more than it shortened the lifespan of daf-2(+) animals. Animals were in a fer-15(b26); fem-1(hc17) background to prevent reproduction. Similar results were seen in daf-2(e1370) mutants (Table S1). (B) Reduction of dox-1 did not increase the set-point of the ire-1/xbp-1 branch of the UPR in daf-2 mutants. Bar graph showing the mean intensity of day-2 adults expressing a Phsp-4::gfp transgene. daf-2(e1370), n = 13, relative mean fluorescence intensity (m) = 21.9 ± 2.2; dox-1(RNAi); daf-2(e1370), n = 17, m = 23.3 ± 2.3, P = 0.677. See Fig. S4 for representative micrographs and wild-type data. (C) dox-1 expression was up-regulated in daf-2 mutants. Bar graph showing the fraction of animals expressing high levels of Pdox-1::gfp. Day-1 adults of each strain were classified as expressing high or low GFP levels (Fig. S4). The fraction of animals expressing high levels of Pdox-1::gfp was 1.35 fold higher in transgenic daf-2(e1370) mutants than in wild-type animals (P = 0.037), but was not significantly increased in daf-2(e1370) xbp-1(tm2457) double mutants. The fraction of animals expressing high levels of Pdox-1::gfp was 1.52 fold higher in transgenic daf-2(e1368) mutants than in wild-type animals (P = 0.007), but was not increased in daf-2(e1368) xbp-1(tm2457) double mutants. The fraction of animals expressing high levels of Pdox-1::gfp was higher by 1.8 fold in transgenic daf-2(e1370) mutants than in wild-type animals (P = 0.026), but was not significantly increased in daf-16(mu86); daf-2(e1370) double mutants. Error bars represent SEM of at least two independent trials. Asterisks mark Student t test values of P < 0.05.
Fig. 5.
Fig. 5.
 daf-16 contributes to the ER stress resistance in daf-2 mutants. (A) Representative fluorescence micrographs of day-1 adults harboring an integrated Phsp-4::gfp transgene in a daf-2(e1370) or daf-16(mu86); daf-2(e1370) background. daf-16 deletion (and inactivation of daf-16 by RNAi) increased the level of Phsp-4::gfp in daf-2(−) mutants. daf-2(e1370), n = 20, relative mean fluorescence intensity (m) = 1 ± 0.06; daf-16(mu86); daf-2(e1370), n = 28, m = 2.70 ± 0.2, P < 0.0001. Error bars represent SEM. Arrows point at anterior and posterior ends of the animals. (B) Tunicamycin resistance developmental assay. Eggs from wild-type, daf-2(e1370), and daf-16(mu86); daf-2(e1370) animals were grown in the presence of 0 or 5 μg/mL tunicamycin (corresponding to the blue and off-white bars in the graph). Percentage of eggs that developed into mature adults was scored. Student t test values were calculated relative to daf-16(−); daf-2(−). Asterisks mark Student t test values of P < 0.05. This experiment was repeated independently with similar effects. (C) Model for how XBP-1 extends lifespan, increases ER stress resistance, and also lowers XBP-1 levels when insulin/IGF-1 signaling is reduced. In daf-2 mutants, multiple transcription factors, such as DAF-16/FOXO, HSF-1, ELT-3, and SKN-1 are activated. These can act in a combinatorial fashion with XBP-1 to turn on new longevity genes and new ER stress response genes (both in purple), which, unlike the “normal” ER stress–protective genes (in red), cannot be induced by XBP-1 alone. This combinatorial control results in a wider array of longevity and ER stress–protective genes, both of which contribute to the longevity of daf-2 mutants. In addition, as part of a negative feedback loop, the wider array of ER stress protective genes expressed in daf-2 mutants by xbp-1 can better reduce the level of unfolded proteins in the ER and improve ER homeostasis. Improved ER homeostasis would, in turn, dictate a lower basal activity level of the ire-1/xbp-1 pathway. In this model, the enhanced ER stress resistance of daf-2 mutants is completely xbp-1–dependent, and yet, because the ER stress response is more efficient, the set point of XBP-1 and its normal downstream targets is lower than in wild type.
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