HIF-1 modulates dietary restriction-mediated lifespan extension via IRE-1 in Caenorhabditis elegans

Abstract

Dietary restriction (DR) extends lifespan in various species and also slows the onset of age-related diseases. Previous studies from flies and yeast have demonstrated that the target of rapamycin (TOR) pathway is essential for longevity phenotypes resulting from DR. TOR is a conserved protein kinase that regulates growth and metabolism in response to nutrients and growth factors. While some of the downstream targets of TOR have been implicated in regulating lifespan, it is still unclear whether additional targets of this pathway also modulate lifespan. It has been shown that the hypoxia inducible factor-1 (HIF-1) is one of the targets of the TOR pathway in mammalian cells. HIF-1 is a transcription factor complex that plays key roles in oxygen homeostasis, tumor formation, glucose metabolism, cell survival, and inflammatory response. Here, we describe a novel role for HIF-1 in modulating lifespan extension by DR in Caenorhabditis elegans. We find that HIF-1 deficiency results in extended lifespan, which overlaps with that by inhibition of the RSKS-1/S6 kinase, a key component of the TOR pathway. Using a modified DR method based on variation of bacterial food concentrations on solid agar plates, we find that HIF-1 modulates longevity in a nutrient-dependent manner. The hif-1 loss-of-function mutant extends lifespan under rich nutrient conditions but fails to show lifespan extension under DR. Conversely, a mutation in egl-9, which increases HIF-1 activity, diminishes the lifespan extension under DR. This deficiency is rescued by tissue-specific expression of egl-9 in specific neurons and muscles. Increased lifespan by hif-1 or DR is dependent on the endoplasmic reticulum (ER) stress regulator inositol-requiring protein-1 (IRE-1) and is associated with lower levels of ER stress. Therefore, our results demonstrate a tissue-specific role for HIF-1 in the lifespan extension by DR involving the IRE-1 ER stress pathway.

Figure 1. HIF-1 functions downstream of S6K to modulate C. elegans lifespan.

(A) The hif-1(ia04) deletion mutant extends lifespan, whereas the egl-9(sa307) deletion mutant does not affect lifespan significantly under standard lab culture conditions. (B)–(D) Inhibition of hif-1 by RNAi extends lifespan of (B) wild-type N2, (C) daf-16(mgDf47), and (D) daf-2(e1370) animals. (E) Inhibition of hif-1 by RNAi does not further extend lifespan of an unc-24(e138) daf-15(m634)/nT1 heterozygous mutant. (F) egl-9 suppresses lifespan extension by the rsks-1(ok1255) mutation, and hif-1 does not further increase lifespan of rsks-1. Each of the lifespan experiments was performed twice with consistent results. Quantitative data and statistical analyses for the experiments shown here and the repeated experiments are included in Table 1 and Table S1, respectively.

Figure 2. DR extends lifespan, increases stress resistance and decreases fecundity in C. elegans.

(A) Survival curves of N2 animals fed with E. coli at different concentrations (1.0×10⁸ to 1.0×10¹² cfu/ml) during adulthood. Mean lifespan was 12.2 days for 1.0×10¹² cfu/ml, 14.0 days for 1.0×10¹¹ cfu/ml, 18.9 days for 1.0×10¹⁰ cfu/ml, 21.2 days for 1.0×10⁹ cfu/ml, and 18.0 days for 1.0×10⁸ cfu/ml. (B) Heat stress resistance of adults after maintenance under different nutrient conditions. Mean survival was 9.5 hours for 1.0×10¹² cfu/ml, 10.9 hours for 1.0×10¹¹ cfu/ml, 14.3 hours for 1.0×10¹⁰ cfu/ml, 15.9 hours for 1.0×10⁹ cfu/ml, and 13.8 hours for 1.0×10⁸ cfu/ml. (C) Egg production after maintenance under different nutrient conditions. Average brood sizes were 203±37 for 1.0×10¹² cfu/ml, 254±30 for 1.0×10¹¹ cfu/ml, 234±60 for 1.0×10¹⁰ cfu/ml, 178±32 for 1.0×10⁹ cfu/ml, and 61±15 for 1.0×10⁸ cfu/ml. Fifteen animals were scored for each food concentration.

Figure 3. HIF-1 modulates lifespan extension by DR.

(A–E) Survival curves and (F) mean lifespan of N2, hif-1 and egl-9 animals fed with E. coli at different concentrations (1.0×10⁸ to 1.0×10¹² cfu/ml) during adulthood. The hif-1 mutant shows lifespan extension under AL and higher bacterial concentrations but not under DR. egl-9 animals show diminished lifespan extension under DR, but they show lifespan similar to that of N2 animals under AL conditions. Each of the lifespan experiments was performed three times with consistent results. Quantitative data and statistical analyses for the experiments shown here and the repeated experiments are included in Table 2 and Table S2, respectively.

Figure 4. egl-9 suppresses lifespan extension by the eat-2 mutation.

The lifespan experiments were performed twice with consistent results. Quantitative data and statistical analyses for the experiments shown here and the repeated experiments are included in Table 2 and Table S2, respectively.

Figure 5. HIF-1 functions in specific neurons and muscles to regulate lifespan extension by DR.

Lifespan of wild-type N2, JT307 egl-9(sa307) and egl-9 animals with various tissue-specific promoters driving egl-9 cDNA (CX strains) was measured under DR to determine the tissues where expression of egl-9 rescues the lack of full lifespan extension by DR. Tissues where egl-9 expression is restored were (A) all cells, (B) pan-neuronal and uv1 cells, (C) pan-neuronal cells, (D) uv1 cells, (E) ADF, NSM neurons, (F) URX, AQR, PQR neurons, (G) body wall and vulval muscles, (H) vulval muscle, and (I) pharyngeal muscle. Each of the lifespan experiments was performed twice with consistent results. Quantitative data and statistical analyses for the experiments shown here and the repeated experiments are included in Table 3 and Table S3, respectively.

Figure 6. HIF-1 functions through the IRE-1 ER stress pathway to modulate lifespan.

(A) IRE-1 modulates lifespan extension by DR. ire-1(v33) mutant animals show significantly reduced changes in lifespan upon nutrient manipulation. (B) ire-1(v33) fully suppresses lifespan extension by hif-1 under both AL and DR conditions. Each of the lifespan experiments was performed multiple times with consistent results. Quantitative data and statistical analyses for the experiments shown here are included in Table 2 and Table 4. Analyses for the repeated experiments are included in Table S2 and Table S5. (C) hsp-4 mRNA levels are regulated by DR and HIF-1. DR reduces hsp-4 transcription in N2, hif-1 and egl-9 (p<0.05, t-test). Overexpression of HIF-1 due to the egl-9(sa307) mutation results in increased hsp-4 transcription under both AL and DR conditions (p<0.01, t-test). qRT-PCR experiments were performed twice with consistent results using two independent RNA preparations.

Figure 7. A genetic model depicting the modulation of lifespan by nutrients, HIF-1 and ER stress in C. elegans.

High nutrients activate HIF-1 through the TOR-S6K pathway, which leads to increased ER stress and shortened lifespan. Other regulators such as PHA-4, SKN-1, AAK-2, DAF-16 and HSF-1 may function in parallel to HIF-1 to modulate DR-induced longevity phenotypes.