Monomethyl branched-chain fatty acids play an essential role in Caenorhabditis elegans development

Abstract

Monomethyl branched-chain fatty acids (mmBCFAs) are commonly found in many organisms from bacteria to mammals. In humans, they have been detected in skin, brain, blood, and cancer cells. Despite a broad distribution, mmBCFAs remain exotic in eukaryotes, where their origin and physiological roles are not understood. Here we report our study of the function and regulation of mmBCFAs in Caenorhabditis elegans, combining genetics, gas chromatography, and DNA microarray analysis. We show that C. elegans synthesizes mmBCFAs de novo and utilizes the long-chain fatty acid elongation enzymes ELO-5 and ELO-6 to produce two mmBCFAs, C15ISO and C17ISO. These mmBCFAs are essential for C. elegans growth and development, as suppression of their biosynthesis results in a growth arrest at the first larval stage. The arrest is reversible and can be overcome by feeding the arrested animals with mmBCFA supplements. We show not only that the levels of C15ISO and C17ISO affect the expression of several genes, but also that the activities of some of these genes affect biosynthesis of mmBCFAs, suggesting a potential feedback regulation. One of the genes, lpd-1, encodes a homolog of a mammalian sterol regulatory element-binding protein (SREBP 1c). We present results suggesting that elo-5 and elo-6 may be transcriptional targets of LPD-1. This study exposes unexpected and crucial physiological functions of C15ISO and C17ISO in C. elegans and suggests a potentially important role for mmBCFAs in other eukaryotes.

Figure 1. Structure of mmBCFAs of 15 and 17 Carbons

C15ISO, 13-methyl myristic acid; C17ISO, 15-methyl hexadecanoic acid; C17anteISO, 14-methyl hexadecanoic acid. Other mmBCFAs mentioned in the text are the following: C13ISO, 11-methyl lauric acid; C15anteISO, 12-methyl tetradecanoic acid. C15ISO and C17ISO are readily detectable in C. elegans.

Figure 2. The Expression of elo-5Prom::GFP and elo-6Prom::GFP Constructs in Wild-Type Worms

(A, C, E, and G) DIC images; (B, D, F and H) fluorescence images. (A–D) Strong expression of the elo-5Prom::GFP construct in the gut and in the head is shown. (E–H) The expression of the elo-6Prom::GFP construct in the gut, vulvae (white arrows), and nerve ring is shown. Scale bars, 100 μm.

Figure 3. RNAi Treatment of elo-5 and elo-6 Significantly Alters the FA Composition

(A and B) GC profiles showing the FA composition in the wild-type strain (Bristol N2) containing the RNAi feeding control vector and in the elo-5(RNAi) feeding strain. Arrowheads point to the peaks corresponding to C15ISO and C17ISO. (C) Comparison of FA composition in three strains: wild type, elo-5(RNAi), and elo-6(RNAi). C17ISO is decreased in both RNAi strains, while C15ISO is only decreased in elo-5(RNAi). (D) Suggested elongation reactions catalyzed by ELO-5 and ELO-6 in C15ISO and C17ISO biosynthesis. FAs are elongated by an addition of two carbon groups at a time. Combined data presented in this figure and in the text suggest that ELO-6 acts at the elongation step from C15 to C17, whereas ELO-5 may be involved in the production of both C15ISO and C17ISO.

Figure 4. The C. elegans BCKAD Homolog Is Involved in mmBCFA Biosynthesis

(A) Early steps of mmBCFA biosynthesis in bacteria, based on Smith and Kaneda (1980), Oku and Kaneda (1988), and Toal et al. (1995). IVD, isovaleryl-CoA dehydrogenase. Predicted corresponding C. elegans genes encoding predicted orthologs were identified (shown in italicized names of reading frames). (B) GC profiles reveal differences in the FA composition in the wild-type animals and animals treated with RNAi of E1 alpha subunit of BCKAD encoded by Y39E4A.3. Black arrowheads point to C15ISO and C17ISO. (C) A summary of several independent preparations shows a significant decrease in both mmBCFAs in the Y39E4A.3 dsRNA-treated animals (p = 0.001 and 0.008 for C15ISO and C17ISO, respectively).

Figure 5. RNAi Treatment of elo-5 Causes L1 Arrest and Other Physiological Defects

(A–C) Nomarski images of worms grown from eggs placed on RNAi plates. Scale bars, 100 μm. (A) Young adults had normal morphology and growth rates. (B) On the second day of adulthood, these animals displayed an egg-laying defect; eggs hatched inside the worms. Arrows point to the late embryos and hatched larvae inside a worm. (C) F1 generation arrested uniformly at the first larval stage (L1), and larvae arrested for 4–5 d died. (D–F) Images of worms derived from late larvae (L2–L4) placed on the RNAi plates. (D) The F1 progeny of worms developed from the treated larvae had smaller size and a scrawny morphology compared to the wild type shown in (A). Scale bar, 100 μm. (E) These animals produced very few oocytes, some of which gave rise to embryos and L1 worms. White arrows indicate embryos. Some oocytes remained unfertilized (black arrow). Scale bar, 10 μm. (F) The proximal part of the gonads undergoes deterioration resulting in sterility. The white arrow indicates spermatica, the black arrow shows an abnormally amorphous oocyte, and the two-way arrow points to the clumsy gonad arm that is finely ordered in wild-type animals. Scale bar, 10 μm.

Figure 6. The FA Composition in Worms Maintained on elo-5 RNAi Plates Supplemented with FA or with S. maltophilia Enriched with C15ISO and C15anteISO FA

Black arrowheads indicate positions of mmBCFAs. (A) Animals grown with C15ISO supplements were partially rescued to the wild-type phenotype; however, no accumulation of C15ISO or its elongation to C17ISO was detectable. (B and C) Animals grown with the (B) C17ISO or (C) C17anteISO supplements were fully rescued. Peaks corresponding to C17ISO and C17anteISO are prominent. (D) The FA composition in S. maltophilia. Arrowheads point to the major FAs, C15ISO and C15anteISO. (E) The elo-5(RNAi) animals are able to elongate dietary C15ISO and C15anteISO into C17ISO and C17anteISO. Arrowheads indicate mmBCFAs. The horizontal arrow illustrates the elongation from C15 to C17 mmBCFA.

Figure 7. A Fluctuation of the C17ISO Amounts in Development

(A) Relative amounts of C15ISO and C17ISO in the worm samples collected at different developmental stages. The amount of the mmBCFA molecule is presented as the percentage of total FA in each sample. Grey bars, C15ISO; black bars, C17ISO. (B) Proposed relationship between the levels of mmBCFA during development and the RNAi effects. Depending on the time of RNAi onset, the amount of C17ISO in F1 eggs varies. If elo-5 is suppressed in parental animals after they have begun to synthesize mmBCFA, then their eggs will have a reduced C17ISO level that is still above the critical low level, which permits these animals to grow but causes them to display gonadal defects. These worms produce a small number of progeny that is then arrested in L1. If parental animals are treated with elo-5(RNAi) right after hatching, they are unable to initiate mmBCFA biosynthesis and the levels of C15ISO and C17ISO in their eggs are reduced to below the critical low level, resulting in L1 arrest of their progeny.

Figure 8. Correlation between the Level of C17ISO and the Levels of Linoleic and Vaccenic Acids during Development

Graphical illustrations of the correlation between the levels of C17ISO and (A) vaccenic acid and (B) linoleic acid. Data were obtained by GC analysis of synchronized populations of worms. Combined with the GC measurements generated from 50 additional samples (see Materials and Methods), these data were used to calculate correlation coefficients: CORREL _{C17ISO/C18:2 n6} = 0.82772, T-TEST = 6.54814 × 10⁻⁷, and CORREL _{C17ISO/C18:1 n7} = −0.85162, T-TEST = 4.74094 × 10⁻⁵. Black bars, C17ISO; white bars, vaccenic acid; grey bars, linoleic acid.

Figure 9. RNAi of the C. elegans SREBP Homolog Alters the FA Composition

(A and B) The GC profiles of (A) wild-type and (B) lpd-1(RNAi)-treated worms. (C) A summary of several independent GC runs. Bars represent the percentages of total FAs. The levels of C15ISO, C17ISO, and C16:0 are significantly altered by the RNAi treatment. Black arrowheads point to differences in the C15ISO and C17ISO amounts. Grey arrowheads indicate the changes in palmitic acid, C16:0.

Figure 10. The Expressions of elo-5 and lpd-1 Reporter Constructs Are Spatially Similar

(A and B) Nomarski and GFP-filtered images of an adult animal containing the lpd-1Prom::GFP construct, showing strong expression in two symmetrical head neurons, each of which has processes to the nose and around a nerve ring. Scale bars, 10 μm. (C) DiI staining of amphid neurons in lpd-1Prom::GFP (dsRed filter). Arrows indicate neuronal nuclei shown in (D). Scale bar, 10 μm. (D) GFP expression in the animal shown in (C). Scale bar, 10 μm. (E and F) Nomarski and GFP-filtered images of an animal containing elo-5Prom::GFP, revealing fluorescence in the similar amphid neuron. Scale bar, 7.5 μm. (G and H) The intestinal and intestinal-muscle GFP expression in (G) lpd-1Prom::GFP and (H) elo-5Prom::GFP constructs. Scale bar, 7.5 μm.

Figure 11. The Expression of GFP Fusion Constructs Suggests the Involvement of lpd-1, acs-1, and pnk-1 in mmBCFA Biosynthesis

(A–D) elo-5Prom::GFP expression is downregulated in the lpd-1(RNAi) background. Scale bars, 100 μm. (A and C) GFP-filtered images of (A) elo-5Prom::GFP and (C) elo-6Prom::GFP in wild-type animals, showing the characteristic bright intestinal fluorescence. (B and D) GFP-filtered images of (B) elo-5Prom::GFP and (D) elo-5Prom::GFP in lpd-1(RNAi) animals, revealing diminished fluorescence in the gut. (E–H) lpd-1 expression is upregulated in neurons of the elo-5(RNAi) animals deficient for C15ISO and C17ISO. Scale bars, 15 μm. (E and F) Nomarski and GFP images of wild-type L1 larvae containing lpd-1Prom::GFP. (G and H) Nomarski and GFP images of elo-5(RNAi)-treated animals (L1 arrested) containing lpd-1Prom::GFP, showing a visibly brighter fluorescence than that seen in (E) and (F). Circles are centered on the pharyngeal back bulb. (I–K) acs-1Prom::GFP expression is upregulated in the elo-5(RNAi) animals deficient for C15ISO and C17ISO. Panels show GFP images of acs-1Prom::GFP animals grown on the (I) control, (J) elo-5(RNAi), and (K) lpd-1(RNAi) plates. The fluorescence from acs-1Prom::GFP in (J) is significantly stronger than that in (I). Scale bars, 100 μm. (L–N) pnk-1Prom::GFP expression is upregulated by elo-5(RNAi) but downregulated by lpd-1(RNAi). Panels show GFP images of pnk-1Prom::GFP animals grown on the (L) control, (M) elo-5(RNAi), and (N) lpd-1(RNAi) plates. The fluorescence of the fusion construct is stronger in (M) but weaker in (N) than that in the control (L). Scale bars, 100 μm.

Figure 12. RNAi of Four Candidate Genes with Altered Expression in elo-5(RNAi) Worms Affects the FA Composition

(A) GC profile of the wild type. (B–E) GC profiles of the RNAi-treated worms. (B–D) RNAi of the three genes resulted in a decrease of the C17ISO or both C15ISO and C17ISO levels, indicated by black arrowheads. In addition, a significant elevation in straight-chain saturated FA levels, indicated by grey arrowheads, is observed in K10C3.6(RNAi). (E) C27H6.2(RNAi) does not cause significant changes in mmBCFA but results in an elevation of straight-chain monounsaturated FA and C18:1 n7, indicated by white arrowheads. Statistical analysis of several GC runs on each of the samples was also carried out (unpublished data).