DA1877 diet represses lipid content and induces lipid metabolic reprogramming in C. elegans

(A) O.R.O staining results of wild-type worms on OP or DA diet. (B) TLC analysis of TAG level in wild-type animals feeding OP or DA. (C) Volcano plot of RNA-seq results. The expression is calculated based on TPM (transcripts per million) and each dot represents one gene. Black dots, red dots and blue dots represent genes with similar expression level, upregulated level, and downregulated level in DA-fed worms compared to OP-fed animals, respectively. (D) Principal component analysis of the expression of 471 lipid metabolic genes in OP- and DA-fed worms. Gene expression data was from RNA-seq analysis. (E) Heatmap for the differentially expressed metabolic genes involved in 13 lipid metabolism pathways between OP- and DA-fed worms. The graph was generated using ClustVis (https://biit.cs.ut.ee/clustvis/). (F) Fatty acid composition of OP- and DA-fed worms by GC-MS. The fatty acid content is shown as percentage of total fatty acids. (G) GC-MS analysis of fatty acids in OP and DA bacteria. Statistical analyses in (A), (B), (F) & (G) were performed using Two-tailed T tests and the data is presented as mean ± SEM. # indicates comparison of dietary effects between OP- and DA-fed wild-type worms. * indicates comparison of individual fatty acid percentage between different diets-fed worms or different bacteria. * / # P < 0.05, ** / ## P < 0.01, *** / ### P < 0.001, **** / #### P < 0.0001.

DA diet decreases fat-7 expression level and lipid content through B12-SAM-PC axis

(A) qRT-PCR results of fat-7 mRNA level in the worms feeding OP or DA. Experiment contained three biological repeats and n = 10 for each repeat. (B) Representative images and quantitative results of FAT-7::GFP in OP or DA-fed worms. n ≥ 10 for each sample. (C) O.R.O staining results of wild-type, fat-7(wa36) mutants and tpEx697 worms, where fat-7 is overexpressed by using intestine-specific promotor vha-6 under OP or DA feeding. Experiment contained 2-3 biological repeats and n ≥ 50 in each repeat. (D) Flowchart of forward genetic screen. (E) Upper panel images showing FAT-7::GFP expression of wild-type animals and the isolated mutants from forward genetic screen on DA diet. The lower scheme illustrating the amino acid changes in those identified alleles. (F) LC/MS/MS analysis of metabolites extracted from worms (gray bar) and bacteria (white bar). n.d. means undetectable. (G) Images and quantitative results of FAT-7::GFP signals in FAT-7 reporter strain DMS303(nIs590) feeding OP or DA with or without B12 supplementation. (H) Quantitative results of FAT-7::GFP signals in the control worms DMS303(nIs590) and mutants isolated from screen feeding OP, DA, or OP with B12 supplementation. (I) O.R.O staining results of wild-type worms feeding OP, DA or OP supplemented with 64nM B12. (J) Cartoon showing the mechanism of B12 in regulation of fat content under DA diet. The graphic work in (D), (E), (J) are created with BioRender.com. Statistical analysis was performed using Two-tailed T tests and the bar plot is presented as mean ± SEM. The centerline in the box of boxplot denotes the median value in the data. # indicates comparison of dietary effects between OP- and DA-fed worms with the same genotype. * indicates comparison between different strains feeding the same diet. * / # P < 0.05, ** / ## P < 0.01, *** / ### P < 0.001, **** / #### P < 0.0001.

DA promotes PC level to repress lipid content in a SBP-1-dependent manner

(A) TLC analysis showing PC level in wild-type animals feeding OP or DA. (B) O.R.O staining results of wild-type worms supplemented with different concentrations of choline feeding OP or DA. Experiment contains 3 biological repeats and the number at the bottom of each graphic bar indicates the n value of each sample. (C) Representative confocal images of PCYT-1::3xFLAG (Green) in the intestinal nucleus of worms fed different diets with DAPI staining (Blue). (D) Images of FAT-7::GFP signals in the mutants isolated from forward genetic screen under control (RNAi) or sbp-1 (RNAi) treatment. (E) O.R.O staining results of wild-type and sbp-1(ep79) worms feeding OP or DA diet. Experiment contains 3 biological repeats and n ≥ 50 for each repeat. (F) Representative confocal image of GFP::SBP-1 localization in the intestine of worms feeding either OP or DA. (G) Cartoon for the mechanism of B12 in regulation of fat content under DA diet. This graphic is created with BioRender.com. Statistical analysis was performed using Two-tailed T tests and the data is presented as mean ± SEM. # indicates comparison of dietary effects between OP- and DA-fed worms with the same genotype. * indicates comparison between different strains feeding the same diet. * / # P < 0.05, ** / ## P < 0.01, *** / ### P < 0.001, **** / #### P < 0.0001.

DA1877-fed worms display significantly less and smaller LDs, and have decreased SEIP-1 targeting to peri-LD cages

(A) Representative images presenting the number and size of intestinal LDs visualized by LD marker protein DHS-3::GFP in DA or OP-fed worms. Scale bar = 5μm. Insets are magnified views from white arrowheads and red arrowheads indicates the clusters of LD rings. (B, C) Quantitative results of numbers and the size of DHS-3-labeled LDs using MATLAB software. * indicates comparison between OP- and DA-fed wild-type worms. (D) Quantitative results showing percentage of LDs associated with SEIP-1::GFP in OP- or DA-fed worms. n ≥ 7 for each sample. (E) Representative images for visualization of SEIP-1::GFP (Green) in the intestine of wild-type worms. mRuby2::DGAT-2 was used as LDs marker (Red). (F) Quantitative results showing percentage of LDs associated with SEIP-1::GFP in OP- or DA-fed wild-type worms under control RNAi or pcyt-1 RNAi treatment. (G) Representative images of the intestine for visualization of SEIP-1::GFP (Green) in the control RNAi or pcyt-1RNAi treated wild-type worms feeding OP or DA. mRuby2::mDGAT-2 was used as LDs marker (Red). Statistical analysis was performed using Two-tailed T tests and the data is presented as mean ± SEM. Unless indicated, # indicates comparison of dietary effects between OP- and DA-fed worms with the same genotype. * indicates comparison between different strains feeding the same diet. * / # P < 0.05, ** / ## P < 0.01, *** / ### P < 0.001, **** / #### P < 0.0001.

asm-3 genetically functions together with PC synthesis pathway to regulate fat content in DA-fed worms and is transcriptionally upregulated through B12-SAM-PC axis

(A) O.R.O staining results of wild-type, asm-3(ok1744) mutants and asm-3(ok1744); tpEx941 mutants, which contains overexpressed asm-3 driven by asm-3 promoter. (B) O.R.O staining results of wild-type, pcyt-1(et9), asm-3(ok1744), and asm-3(ok1744); pcyt-1(et9) mutants. (C) Images and quantitative results of Pasm-3::GFP signals in worms under OP, DA, or OP with B12 supplementation diet. (D) Images and quantitative results of the signals of asm-3 transcriptional reporter in wild-type, sams-1(ok3033), pcyt-1(et9), and asm-3(ok1744) mutant background. Statistical analysis was performed using Two-tailed T tests and the data is presented as mean ± SEM. # indicates comparison of dietary effects between OP- and DA-fed worms with the same genotype. * indicates comparison between different strains feeding the same diet. * / # P < 0.05, ** / ## P < 0.01, *** / ### P < 0.001, **** / #### P < 0.0001.

ASM-3 transports to coelomocytes from intestine to regulate fat content in DA-fed worms

(A) Representative images revealing the localization of ASM-3::mCherry (Red) in YW2989 [tpEx941] worms where ASM-3 was driven by its own promoter. (B) Images of ASM-3 localization in the coelomocyte of YW3735 strain, which contains single-copy insertion of asm-3::mCherry (Red) and lysosomal marker LMP-1::GFP (Green). White dotted lines mark coelomocytes. Scale bar = 10µm (C) Images presenting the localization of intestine-specific expressed ASM-3::mCherry (Red) in YW3034 [asm-3(ok1744); tpEx944]. White dotted lines mark coelomocytes. (D) O.R.O staining results of wild-type worms, asm-3(ok1744) mutants, asm-3(ok1744) with intestinal asm-3 expression using intestine-specific promoter vha-6, asm-3 expression in coelomocytes using coelomocyte-specific promoter unc-122, or asm-3 expression driven by the endogenous asm-3 promoter. Statistical analysis was performed using Two-tailed T tests and the data is presented as mean ± SEM. # indicates comparison of dietary effects between OP- and DA-fed worms with the same genotype. * indicates comparison between different strains feeding the same diet. * / # P < 0.05, ** / ## P < 0.01, *** / ### P < 0.001, **** / #### P < 0.0001. (E) Model showing mechanism of how DA1877 diet regulates fat content in the worms. This graphic is created with BioRender.com.