Cell size and fat content of dietary-restricted Caenorhabditis elegans are regulated by ATX-2, an mTOR repressor

Abstract

Dietary restriction (DR) is a metabolic intervention that extends the lifespan of multiple species, including yeast, flies, nematodes, rodents, and, arguably, rhesus monkeys and humans. Hallmarks of lifelong DR are reductions in body size, fecundity, and fat accumulation, as well as slower development. We have identified atx-2, the Caenorhabditis elegans homolog of the human ATXN2L and ATXN2 genes, as the regulator of these multiple DR phenotypes. Down-regulation of atx-2 increases the body size, cell size, and fat content of dietary-restricted animals and speeds animal development, whereas overexpression of atx-2 is sufficient to reduce the body size and brood size of wild-type animals. atx-2 regulates the mechanistic target of rapamycin (mTOR) pathway, downstream of AMP-activated protein kinase (AMPK) and upstream of ribosomal protein S6 kinase and mTOR complex 1 (TORC1), by its direct association with Rab GDP dissociation inhibitor β, which likely regulates RHEB shuttling between GDP-bound and GTP-bound forms. Taken together, this work identifies a previously unknown mechanism regulating multiple aspects of DR, as well as unknown regulators of the mTOR pathway. They also extend our understanding of diet-dependent growth retardation, and offers a potential mechanism to treat obesity.

Keywords: Caenorhabditis elegans; TORC1; mTOR pathway; metabolism.

Fig. 1.

Size reduction in DR worms requires ATX-2. (A) Quantification of the length of WT (N2) and atx-2(tm3562) homozygous animals grown on a series of bacterial dilutions. Results are normalized per strain to maximal bacterial concentration. n = 146. (B) Quantification of the length of eat-2(ad1116) young adults subjected to atx-2(RNAi) after 24, 48, and 72 h. Results are normalized to age-matched controls. n = 66 (C) Stereomicroscope images showing stage-matched eat-2(ad1116) worms fed for 3 d with either EV or atx-2(RNAi). (D) Phalloidin staining of the body muscle cells of adult eat-2(ad1116) that were fed for 3 d with either EV or atx-2(RNAi). Fourteen animals and 103 nuclei were used for the analysis. P = 7 × 10⁻¹¹. (Scale bar: 10 µm.) (E) Quantification of size change of the muscle cells. P = 7 × 10⁻¹¹. (F) The effect of the transgenic expression levels of ATX-2^tg in WT background on the size of 1-d-old adult worms. Error bars represent mean ± SEM. *P < 0.01; **P < 10⁻⁴; ***P < 10⁻⁶.

Fig. S1.

atx-2(RNAi) efficiency and the effects on animal width, other DR model animals, and pumping rate. (A) Semiquantitative PCR of atx-2 cDNA derived from wild type (N2) and DR (eat-2) animals grown on atx-2(RNAi) or on EV (L4440). The pmp-3 gene served as a control. (B) ATX-2^tg protein blot analysis using αFLAG antibodies. (C) Confocal microscopy of ATX-2^tg of animals grown on atx-2(RNAi) or on EV (L4440). BF, bright field. (Scale bar = 10 µm.) (D) eat-2(ad1116) animals down-regulated for atx-2 show increase in width. n = 34. P = 0.0002. (E) Pharyngeal pumping rates do not change in WT (N2) and eat-2(ad1116) adult animals grown for 72 h on EV or atx-2(RNAi). (F) atx-2(RNAi) increases the length of eat-5(ad1402) and eat-6(ad467) animals. n = 41. P = 3 × 10⁻⁵ and 0.0029, respectfully. Animals were moved postdevelopmentally to atx-2(RNAi) plates, and length was measured after 48 h. Error bars represent mean ± SEM.

Fig. S2.

ATX-2^tg is present in both the cytoplasm and the nucleus and its expression in tm3562 homozygous animals rescues their sterile phenotype. (A and B) WT (A) and eat-2 (B) young adults expressing ATX-2^tg were stained with DAPI. ATX-2::GFP is shown in green; DNA, in red. Expression was observed in most or all tissues and developmental stages. Particularly strong expression was observed in embryos and gut cells. ATX-2 is present in both the nucleus and cytoplasm and absent from the nucleolus; gut nuclei are shown. (Scale bar: 10 µm.) (C) Transgenic expression of ATX-2^tg in null background (sterile) partially rescued the egg-laying phenotype with 50–130 eggs/animal. WT animals laid an average of 260 eggs/animal. Error bars represent mean ± SEM. (D) Expression of transgenic ATX-2 (ATX-2^tg) reduces the amount of fat accumulation in these animals. P = 0.001, two-tailed t test.

Fig. 2.

ATX-2 regulates fat accumulation, brood size, and pace of development, independent of lifespan, in eat-2(ad1116) animals. (A) Oil Red O staining of wild type (N2) and eat-2(ad1116) worms fed postdevelopmentally for 72 h with either EV or atx-2(RNAi). (Insets) Higher-magnification images showing the lipid droplets. (B) Quantification of the effect of atx-2(RNAi) on relative intensity of Oil Red O in eat-2 and in wild type animals. n = 90. Error bars represent mean ± SEM. **P < 10⁻⁴. (C) Quantification of fat levels by Sudan Black B staining in WT (N2) and eat-2(ad1116) 9-d-old animals fed with either EV or atx-2(RNAi). n = 160. Error bars represent mean ± SEM. ***P < 10⁻⁶. (D) Fecundity of animals overexpressing ATX-2^tg. Worms were divided into three groups (no, middle, and high expression) according to the GFP intensity of the ATX-2^tg transgene. n = 30. Error bars represent mean ± SEM. *P < 0.01; **P < 10⁻⁴. (E) Developmental stage reached by eat-2(ad1116) animals following down-regulation of atx-2 for 3 d posthatching. n = 200. P = 3 × 10⁻⁵, χ² test. L1-2, larval stages 1–2; L3-4, larval stages 3–4; YA, young adults. (F) Survival curve for WT (N2) and eat-2(da1116) worms fed with EV (blue and purple), atx-2 (red and black), or lmn-1 (green and orange) at 23 °C. The RNAi treatments were initiated at larval stage 4. atx-2(RNAi) treatment did not change the lifespan of N2 or eat-2 animals, whereas lmn-1(RNAi) significantly shortened the life span of both N2 and eat-2 animals. P < 0.0005. The lifespan assay was repeated five times for N2 and eat-2 and two times for lmn-1.

Fig. S3.

atx-2 regulates sbp-1 expression in dietary-restricted animals. (A) Florescence microscopy images of wild type and eat-2 animals expressing GFP driven by the sbp-1 promoter and fed with either EV or atx-2(RNAi). The RNAi treatments were provided for 72 h starting at larval stage 4. (B) Quantification of florescence. n = 110. **P < 10⁻⁴.

Fig. 3.

atx-2 regulates animal size via the mTOR pathway. (A) The size effect of atx-2(RNAi) on different strains mutated in key metabolism regulating genes. n = 194. (B) Down-regulation of daf-15 (RAPTOR) suppresses the increase in animal length size following atx-2(RNAi) treatment. n = 117. (C) SIPA-1 shows structural similarity to TSC2. sipa-1(RNAi) causes an increase in animal length. n = 56. (D) gdi-1(RNAi) treatment increases the length of eat-2 mutants. n = 57. In A–D, error bars represent mean ± SEM. *P < 0.01; **P < 10⁻⁴; ***P < 10⁻⁶.

Fig. S4.

Mapping atx-2 to the mTOR pathway. (A) Postdevelopmental down-regulation of key metabolic genes shows that S6K (rsks-1) is essential for the effect of atx-2 on animal length. n = 231. (B) Deletion of rsks-1 prevents a size increase in atx-2 null animals in an eat-2 background. n = 61. (C) Postdevelopmental down-regulation of mTOR pathway genes demonstrates that rheb-1, but not rict-1 (RICTOR), affects the length of eat-2 animals. n = 217. (D) atx-2(RNAi) further increases the length of lon-1 mutants. Shown is the length of lon-1(e185) animals grown on EV or atx-2(RNAi) plates. n = 61. P = 0.0003, two tailed t test. (E) Down-regulation of proteins known to bind ATX-2 in the gonad has no effect on animal size. The length of DR (eat-2; red) and WT (N2; blue) animals was measured following down-regulation of gld-1, mex-3, or K04F10.1. n = 225. (F) Down-regulation of pab-1 decreases animal size. The length of eat-2 and WT (N2) animals was measured following down-regulation of pab-1, atx-2, or both. n = 239. (G) gdi-1(RNAi) increases the length of eat-2 mutants. n = 124. The size measurements in B and E were normalized to the same strain fed with EV. *P < 0.01; **P < 10⁻⁴; ***P < 10⁻⁶. Error bars represent mean ± SEM.

Fig. 4.

ATX-2 regulatory model. (A) FRET indicates a direct interaction between GDI-1 and ATX-2. Animals expressing both ATX-2^tg and GDI-1::MYC in an atx-2(tm3562) background were stained with mouse anti-MYC-tag (9E10) and Cy3 anti-mouse. Using acceptor photobleaching, energy transfer was observed between donor (ATX-2::GFP) and acceptor (Cy3 bound to GDI-1::MYC). Control animals expressing only ATX-2^tg were stained with MYC antibody with minimal washes. (Scale bar: 5 µm.) (B) Schematic model of the control of cell size by an ATX-2 complex in the mTOR pathway.

Fig. S5.

GDI-1 and RHEB-1 localize to both the nucleus and the cytoplasm. (A) Protein blot analysis of two lines expressing GDI-1 fused to a MYC tag. (B and C) animals expressing GDI-1::MYC (B) or RHEB-1::GFP (C) were stained with DAPI. Images show gonad (B) and gut (B and C) of young adult animals. (Scale bars: 10 µm.) Control WT animals stained with anti-MYC antibodies did not show specific staining.

Fig. S6.

ATX-2 does not control animal size and body fat content through ribosome synthesis. (A) Northern blot analysis of total RNA extracted from worms. (Left) Digital capture of an ethidium bromide-stained denaturing agarose gel, showing the steady-state accumulation of mature 18S and 26S rRNAs. 26S/18S rRNA ratio calculated from Agilent electropherograms. (Middle and Right) Gel transferred to nylon and hybridized to probes to detect major pre-rRNA intermediates. (B) A pre-rRNA processing pathway in worms (35).