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. 2018;49(3):947-960.
doi: 10.1159/000493226. Epub 2018 Sep 5.

C. Elegans Fatty Acid Two-Hydroxylase Regulates Intestinal Homeostasis by Affecting Heptadecenoic Acid Production

Yuanbao Li  1 , Chunxia Wang  1 , Yikai Huang  2 , Rong Fu  1 , Hanxi Zheng  2 , Yi Zhu  1 , Xiaoruo Shi  2 , Prashanth K Padakanti  3 , Zhude Tu  3 , Xiong Su  2   4 , Huimin Zhang  1
Affiliations

C. Elegans Fatty Acid Two-Hydroxylase Regulates Intestinal Homeostasis by Affecting Heptadecenoic Acid Production

Yuanbao Li et al. Cell Physiol Biochem. 2018.

Abstract

Background/aims: The hydroxylation of fatty acids at the C-2 position is the first step of fatty acid α-oxidation and generates sphingolipids containing 2-hydroxy fatty acyl moieties. Fatty acid 2-hydroxylation is catalyzed by Fatty acid 2-hydroxylase (FA2H) enzyme. However, the precise roles of FA2H and fatty acid 2-hydroxylation in whole cell homeostasis still remain unclear.

Methods: Here we utilize Caenorhabditis elegans as the model and systemically investigate the physiological functions of FATH-1/C25A1.5, the highly conserved worm homolog for mammalian FA2H enzyme. Immunostaining, dye-staining and translational fusion reporters were used to visualize FATH-1 protein and a variety of subcellular structures. The "click chemistry" method was employed to label 2-OH fatty acid in vivo. Global and tissue-specific RNAi knockdown experiments were performed to inactivate FATH-1 function. Lipid analysis of the fath-1 deficient mutants was achieved by mass spectrometry.

Results: C. elegans FATH-1 is expressed at most developmental stages and in most tissues. Loss of fath-1 expression results in severe growth retardation and shortened lifespan. FATH-1 function is crucially required in the intestine but not the epidermis with stereospecificity. The "click chemistry" labeling technique showed that the FATH-1 metabolites are mainly enriched in membrane structures preferable to the apical side of the intestinal cells. At the subcellular level, we found that loss of fath-1 expression inhibits lipid droplets formation, as well as selectively disrupts peroxisomes and apical endosomes. Lipid analysis of the fath-1 deficient animals revealed a significant reduction in the content of heptadecenoic acid, while other major FAs remain unaffected. Feeding of exogenous heptadecenoic acid (C17: 1), but not oleic acid (C18: 1), rescues the global and subcellular defects of fath-1 knockdown worms.

Conclusion: Our study revealed that FATH-1 and its catalytic products are highly specific in the context of chirality, C-chain length, spatial distribution, as well as the types of cellular organelles they affect. Such an unexpected degree of specificity for the synthesis and functions of hydroxylated FAs helps to regulate protein transport and fat metabolism, therefore maintaining the cellular homeostasis of the intestinal cells. These findings may help our understanding of FA2H functions across species, and offer potential therapeutical targets for treating FA2H-related diseases.

Keywords: 2-hydroxylation; C. elegans; FA2H; Fatty acids; Heptadecenoic acid.

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Conflict of interest statement

Disclosure Statement

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Figures

Figure 1.
Figure 1.. The spatial-temporal distribution patterns of FATH-1 in C. elegans.
(A) A diagram shows the FATH-1 protein domains, including four transmembrane domains and one Cyt-b5 domain. GFP was inserted at the N-terminus of FATH-1. (B-F) The distribution patterns of fusion protein GFP::FATH-1 in the C. elegans at embryonic (B), L1 larvae (C), L4 larvae (D) and adult (E, F) stage. (D) is a composite confocal image showing GFP::FATH-1 localization in the epidermal cells. (E-E”) shows the GFP::FATH-1 distribution in the intestine (solid arrowhead) and nervous system (open arrowhead). (F-F”) shows the GFP::FATH-1 distribution in the intestine (solid arrowhead) but not the gonad (open arrow). Scale bar in D, 20 μm; Scale bars in B,C,E and F, 10 μm.
Figure 2.
Figure 2.. The systemic and tissue-specific effect of fath-1 inactivation.
(A) Bright-field images of worms fed with HT115 control or fath-1 RNAi bacteria from L1 to young adult stage. Scale bar, 400 μm. (B) Changes of body lengths of worms treated with HT115 or fath-1 RNAi from L1 stage. N ≥ 45 for each data set. (C) Survival curves of the worms treated with HT115 or fath-1 RNAi from L1. N≥ 75 for each data set. (D) Survival curves of dauer-like animals caused by starvation (control) or by fath-1 RNAi treatment. N≥ 81 for each data set. (E) Body length measurements of wildtype, epidermis-specific RNAi (epi KD) and intestine-specific RNAi (int KD) worms treated with fath-1 RNAi, dpy-7 RNAi (positive control for epidermal-specific RNAi) or HT115 (negative control). N ≥ 52 for each data set.
Figure 3.
Figure 3.. The stereospecificity and subcellular localization pattern of 2-OH FAs.
(A) Body length measurement of fath-1 RNAi worms rescued by BSA control, 350 μM (R)-2-OH FA (2R) or 350 μM (S)-2-OH FA (2S) supplementation. N ≥ 50 for each data set. (B) Body length measurements showing the rescuing effects of 5, 20, 35, 50, 100 or 200 μM alkynyl 2-OH-PA supplementation on the growth arrest defect caused by fath-1 RNAi. N ≥ 50 for each data set. (C) Distribution of fatty acids and derivatives in the C. elegans intestine labeled by “Click chemistry” approach. PA, palmitic acid; OH-PA, 2-hydroxy plamitic acid; alk, alkynyl; az, azide. Scale bar, 5 μm.
Figure 4.
Figure 4.. The subcellular defects caused by fath-1 inactivation.
(A) Representative fluorescent confocal images showing intestinal cellular organelles of young adult C. elegans treated with HT115 or fath-1 RNAi. The organelles include: lipid droplets (DHS-3::GFP, BODIPY 493/503); peroxisomes (mRFP::PTS1); late endosomes (GFP::RAB-7); apical recycling endosomes (mCherry::RAB-11); basolateral recycling endosomes (mRFP::RME-1); early endosomes (mCherry::RAB-5); endoplasmic reticulum (GFP::TRAM-1); mitochondria (GES-1::GFP) or Golgi (MANS-1::GFP). Lysosomes were marked by lysotracker green. BODIPY 500/510 visualizes the process of fatty acid uptake. The scale bar represents 10 μm. (B, C, D, E, F) Quantification of the numbers of endosome and peroxisome aggregates or lipid droplets in worms lacking fath-1 expression, corresponding to results shown in A. N ≥ 40 for each data set. Error bars, ± SEM; ***p<0.001.
Figure 5.
Figure 5.. The fatty acid profiles affected by fath-1 inactivation.
(A-C) Representative mass spectra of AEPP derivatives of total FAs from wildtype animals fed with HT115 control (A), fath-1 RNAi (B) or fath-1 RNAi plus 350 μM (R)-2-OH FA (2R) supplementation (C). (D) Representative product-ion MS spectrum of m/z 494 ion by ESI-MS/MS. (E) The structure of Δ10C17:1 AEPP derivative and fragment ion peak assignment.
Figure 6.
Figure 6.. Δ10C17:1 mediates FATH-1 function in growth and development.
(A) Relative FA abundance normalized to HT115 control group. (three biological replicates, N >1000/condition). Error bars, ± SEM; *p<0.05, **p<0.01. (B) Bright-field images of worms fed with HT115 control or fath-1 RNAi treated with BSA, Δ10C17:1 or Δ9C18:1. Scale bar, 400 μm. (C) Body length measurement of worms fed with HT115 control or fath-1 RNAi treated with BSA, Δ10C17:1 or Δ9C18:1. N≥ 51 for each data set.
Figure 7.
Figure 7.. Δ10C17:1 rescues subcellular defects of fath-1 deficient worms
(A) Representative fluorescent confocal images showing intestinal cellular organelles of C. elegans treated with HT115, HT115 plus 100 μM Δ10C17:1, HT115 plus 100 μM Δ9C18:1, fath-1 RNAi, fath-1 RNAi plus 100 μM Δ10C17:1 or fath-1 RNAi plus 100 μM Δ9C18:1. The organelles include: lipid droplets (DHS-3::GFP); peroxisomes (mRFP::PTS1); late endosome (GFP::RAB-7) and apical recycling endosome (mCherry::RAB-11). The scale bar represents 10 μm. (B, C, D, E) Quantification of the numbers of endosome and peroxisomes aggregates or lipid droplets in worms fed with HT115 or fath-1 RNAi treated with Δ10C17:1 or Δ9C18:1, corresponding to results shown in A. N=20 for each data set. Error bars, ± SEM; **p<0.01, ***p<0.001.
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