The NHR-8 nuclear receptor regulates cholesterol and bile acid homeostasis in C. elegans

Abstract

Hormone-gated nuclear receptors (NRs) are conserved transcriptional regulators of metabolism, reproduction, and homeostasis. Here we show that C. elegans NHR-8 NR, a homolog of vertebrate liver X and vitamin D receptors, regulates nematode cholesterol balance, fatty acid desaturation, apolipoprotein production, and bile acid metabolism. Loss of nhr-8 results in a deficiency in bile acid-like steroids, called the dafachronic acids, which regulate the related DAF-12/NR, thus controlling entry into the long-lived dauer stage through cholesterol availability. Cholesterol supplementation rescues various nhr-8 phenotypes, including developmental arrest, unsaturated fatty acid deficiency, reduced fertility, and shortened life span. Notably, nhr-8 also interacts with daf-16/FOXO to regulate steady-state cholesterol levels and is synthetically lethal in combination with insulin signaling mutants that promote unregulated growth. Our studies provide important insights into nuclear receptor control of cholesterol balance and metabolism and their impact on development, reproduction, and aging in the context of larger endocrine networks.

Figure 1. NHR-8 Structure and Expression

(A) Phylogenetic relationship of NHR-8 to other mammalian, Drosophila, and C. elegans nuclear receptors based on amino acid sequence of the entire proteins as determined by maximum likelihood from aligned sequences. (B) Location of the DNA-binding domain (DBD), ligand-binding domain (LBD), and deletion coordinates of alleles in both transcripts of nhr-8 (F33D4.1a and F33D4.1b) (C and D) Amino acid sequence alignment of the DBDs and LBDs of C. elegans NHR-8 and DAF-12 and the NHR-8 homolog from the pathogenic nematode Brugia malayi. (E) NHR-8 fused to GFP is expressed in the intestinal cells, and localized primarily in the nucleus. Animals shown are at the L3 larval stage. Abbreviations: Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Homo sapiens (Hs), Mus musculus (Mm), Brugia malayi (Bm). Scale bar = 20 µm.

Figure 2. nhr-8 Animals Display Phenotypes of Dafachronic Acid Deficiency

(A and B) Animals were grown from eggs at 25°C or 27°C on plates containing either 0 µg/ml or 5 µg/ml of pure cholesterol. The fractions of animals that formed dauer were scored. (C) Fraction of animals with gonad migration defects. (D) Representative pictures of (i) wild-type (N2) animals with normal gonad migration, (ii) nhr-8(hd117) animals with a gonad migration defect (Mig), and (iii) an nhr-8(hd117) dauer (inset shows dauer alae). (E) A complementing extrachromosomal nhr-8∷gfp expression construct (Figure 1B, E) is sufficient to rescue Daf-c phenotypes of nhr-8(hd117) animals. (F) nhr-8 animals display early L1/L2 larval arrest upon cholesterol deprivation at 27°C, independent of daf-12. Animals were grown from eggs at 27°C on plates containing either 0 µg/ml or 5 µg/ml of pure cholesterol. (G) L1/L2 larval arrested nhr-8 animals are rescued by addition of dietary cholesterol, but not DA. Animals grown at 27°C on plates supplemented with 250 nM of the indicated compound (~1 µg of each compound added to the surface of plate). (H) Dauer rescue of nhr-8 animals by excess cholesterol, sterols, and DA. Animals grown at 27°C on plates containing 5 µg/ml cholesterol plus 250 nM of each compound (~1 µg of each compound added to the surface of the plate). *p<0.05, **p<0.01. All values from n≥3 independent experiments show as mean ± SEM. See also Figure S1.

Figure 3. Genetic Epistasis Places nhr-8 Upstream of daf-12/NR and daf-16/FOXO

(A) Dauer formation in nhr-8 mutants is suppressed by mutations of daf-12 and two alleles of daf-16/FOXO, but not daf-5/SNO. (B) Gonad migration defects (Mig) of nhr-8 animals are suppressed by a daf-16 null mutation. (C) daf-16 expression is not changed in nhr-8(hd117) mutants. (D) In the absence of dietary cholesterol, sod-3 and dod-3 are upregulated in nhr-8 mutants. (E) Chronogram representation of the spaciotemporal expression of sod-3P∷gfp from egg to adult of animals grown at 25°C. sod-3P∷gfp is upregulated in nhr-8 mutants in the absence of dietary cholesterol across all developmental stages. Shown are averaged profiles of identical sized animals and oriented left to right as head to tail. GFP intensities and body length (measured as time of flight) were determined using a COPAS Biosorter. Developmental stages are indicated on the y-axis and verified by microscopy. The color scale indicates the expression level of a given position on the chronogram relative to all chronograms. (F) Quantification of GFP intensity from chronograms in (E). (G) DAF-12 microRNA target, miR-241, is down-regulated in nhr-8 animals. Expression is rescued by supplementation with the DAF-12 ligand Δ⁷-DA (100 nM). miR-241 expression also require daf-12 and daf-36. *p<0.05, **p<0.01, ***p<0.001, ns=not significant. Mean±SEM. See also Figure S2.

Figure 4. Loss of nhr-8 Influences Sterol Metabolism

(A) Schematic of Δ7-DA biosynthesis from dietary cholesterol. (B) Endogenous free cholesterol levels are reduced in nhr-8 animals grown in the absence of dietary cholesterol. Mutation of daf-16 generally increases cholesterol levels. (C) Production of 7-dehydrocholesterol requires nhr-8. Mutation of daf-16 increases 7-dehydrocholesterol production to greater than wild-type levels independently of nhr-8. (D) Δ⁷-DA levels are lower in nhr-8 animals in the absence of dietary cholesterol. Mutation of daf-16 does not rescue Δ⁷-DA production. (E and F) Micrographs and quantification of the fluorescence intensity of individual eggs from animals fed 25-NBD cholesterol (1 µg/ml). In the presence of 5 µg/ml cholesterol, N2 and nhr-8 mutants deposit the same amount of 25-NBD cholesterol in their eggs. In the absence of dietary cholesterol, nhr-8 mutants deposit half the amount of 25-NBD cholesterol in their eggs as N2. *p<0.05, **p<0.01. Mean±SEM. Values from n≥5 (B–D) or n=3 (F) independent experiments. Scale bar = 20 µm.

Figure 5. nhr-8 Affects Expression of daf-36

(A) daf-36 mRNA expression is down-regulated in nhr-8 at L3. (B) Micrographs showing reduced expression of DAF-36∷GFP protein in nhr-8 animals at L3. Bar = 100 µm. (C) DAF-36∷GFP expression is reduced throughout development in nhr-8 animals grown in the absence of dietary cholesterol. Data were quantified from GFP intensities of several thousand animals using a COPAS biosorter (similar to Figure 3E) (D) Overexpression of daf-36 partially rescues dauer formation of nhr-8 animals. *p<0.05, ***p<0.001, ns=not significant. Mean±SEM. See also Figure S3.

Figure 6. nhr-8 Affects Fatty Acid Metabolism

(A) fat-5 and fat-7 transcript levels are decreased in nhr-8 animals, independent of dietary cholesterol (B) fat-2 and elo-1 transcript levels are decreased in nhr-8 when grown in the absence of dietary cholesterol (C) Fatty acid profiles as determined by GC analysis show an increase in saturated and a decrease in mono and polyunsaturated fatty acids in nhr-8 animals at the L3 stage of development in 0 µg/ml cholesterol. Comparable changes were also seen in day 1 adult animals. (D) Quantification of the different classes of fatty acids summed from (C). (E) Triglyceride levels in nhr-8 animals are similar to wild-type. (F) A model of fatty acid synthesis. Genes implicated in various steps shown in italics. Bold type indicates those that are downregulated in nhr-8 in the absence of dietary cholesterol. Fatty acids indicated in green are increased, whereas those in red are decreased in nhr-8 animals. Data shown are from L3 animals grown at 25°C. *p<0.05, **p<0.01, ***p<0.001. Mean±SEM. See also Figure S5.

Figure 7. nhr-8 Brood Size and Lifespan and are Cholesterol Dependent

(A) Transcript levels of the Apolipoprotein-like vitellogenins, vit-1 and vit-2, are decreased in nhr-8 animals. (B) VIT-2∷GFP expression is reduced throughout development in nhr-8 animals grown in the absence of dietary cholesterol. Data were quantified from GFP intensities of several thousand animals using a COPAS Biosort (similar to Figure 3E). (C) nhr-8 animals have reduced brood size. Supplementation with high amounts of cholesterol significantly improves fecundity of nhr-8 animals relative to wild-type grown under the same conditions. (D) nhr-8 mutant animals show reduced lifespans compared to N2. Excess dietary cholesterol rescues the maximum, but not the mean nhr-8 lifespan. Representative lifespan assay of N2 and nhr-8 animals grown from day 1 adult under low (0 µg/ml), normal (5 µg/ml), or high (25 µg/ml) amounts of dietary cholesterol. Development from egg to adult of all animals was in the presence of 5 µg/ml dietary cholesterol. (E) Quantification of mean and maximum lifespan of wild-type and nhr-8 animals mutants supplemented with 0, 5, or 25 µg/ml dietary cholesterol. (F) Schematic representation of the metabolic processes regulated by NHR-8. n≥3 experiments. *p<0.05, **p<0.01, ***p<0.001. Mean±SEM. See also Figure S6.