Conditions and workflow for INT1- and INT2-9-specific translatomics.

(A) Representative images of wild-type animals bearing integrated INT1::GFP and INT2- 9::mCherry transgenes. Upper panel, GFP expression in INT1 cells and mCherry expression in INT2-9 cells; lower panel, merge with DIC. Scale bar, 100 μm. (B) Representative images of wild-type animals in fed, 30 min fasted, and 30 min refed states fixed and stained with Oil Red O. (C) Fat content was quantified for each condition and expressed as a percentage of wild-type animals in fed state ± SEM (n=20). nsp>0.05 by one-way ANOVA (Tukey). (D) Representative images of wild-type animals in fed, 180 min fasted, 30 min refed, 90 min refed, and 180 min refed states fixed and stained with Oil Red O. (E) Fat content was quantified for each condition and expressed as a percentage of wild-type animals in fed state ± SEM (n=18-20). ***p<0.001, nsp>0.05 by one-way ANOVA (Sidak). (F) 100000 C. elegans expressing INT1::RPL-22::3xHA or INT2-9::RPL-22::3xHA were grown to day 1 adults. Worms were fasted and refed for the time indicated and harvested for TRAP-Seq. Panel created with BioRender.com/rm8ylcm.

Identification of the fundamental differences between INT1 and INT2-9 translatomes.

(A) Principal component analysis showing translatomic variability across the first two principle components of TRAP-Seq data from INT1 and INT2-9 promoters under both acute and chronic conditions (fed, fasted, and refed). (B) Cluster dendrogram produced by average linkage hierarchical clustering in WGCNA. The color row under the dendrogram indicates the module assignment. (C) Pearsons correlations of TRAP-seq conditions with WGCNA modules. Each row represents a WGCNA module and its means correlation to the indicated trait across replicates.The color of each cell indicates the mean pearsons correlation coefficient between the module and the condition; red represents a positive correlation and blue represents a negative correlation. (D) Average expression of red modules genes in the TRAP-seq conditions. The y-axis shows mean variance stabilized expression across all genes and replicates per condition with standard deviation indicated by the error bars. (E) WormCat visualization of categories enriched in genes of module red. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value. (F) Volcano plot of differentially expressed genes for INT1 fed versus INT2-9 fed. High confidence differentially expressed genes are labeled. The horizontal dashed line indicates an adjusted p-value cutoff of 0.05. (G) WormCat visualization of categories enriched in genes upregulated in INT1 cells versus INT2-9 cells in the fed state. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value. (H) A hierarchical clustering tree of significantly enriched GO terms (Biological Process, BP) in the upregulated genes for INT1 fed versus INT2-9 fed by ShinyGO. GO terms with many shared genes are clustered together. Larger dots indicate more significant adjusted p-values. (I) Fluorescent images of transgenic animals bearing PB0024.4::mNeonGreen, Pclec-86::mNeonGreen, Pclec-160::mNeonGreen, PF01D5.5::mNeonGreen or PF01D5.1::mNeonGreen transgenes. The white arrowhead indicates mNeonGreen expression in INT1 cells. Scale bar, 100 μm. (J) Fluorescent image of a transgenic animal bearing Pnpr-28::mNeonGreen transgene. Scale bar, 100 μm.

Identification of the differences between INT1 and INT2-9 translatomes under acute and chronic conditions.

(A, D, G, J) Volcano plot of differentially expressed genes for INT1 versus INT2-9 in acute fasted, acute refed, chronic fasted and chronic refed states. High confidence differentially expressed genes. The horizontal dashed line indicates an adjusted p-value cutoff of 0.05. (B, E, H, K) WormCat visualization of categories enriched in genes upregulated in INT1 cells versus INT2-9 cells in acute fasted, acute refed, chronic fasted and chronic refed states. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value. (C, F, I, L) A hierarchical clustering tree of significantly enriched GO terms (Biological Process, BP) in the upregulated genes for INT1 versus INT2-9 in acute fasted, acute refed, chronic fasted and chronic refed states by ShinyGO. GO terms with many shared genes are clustered together. Larger dots indicate more significant adjusted p-values.

Comparisons of InterPro categories for INT1 versus INT2-9 under acute and chronic conditions.

(A-H) Venn diagrams showing the number of upregulated or downregulated genes in specific categories for INT1 versus INT2-9 under the three acute conditions (fed, fasted, and refed). (I-Q) Venn diagrams showing the number of genes in specific categories for INT1 versus INT2-9 under the three chronic conditions (fed, fasted, and refed). (R) Venn diagram showing the overlap between overlapped upregulated ShKT proteins for INT1 versus INT2-9 under acute and chronic conditions.

Comparison of fasting and refeeding responses between INT1 and INT2-9 cells under acute and chronic conditions.

(A, B) Scatterplots comparing the acute and chronic fasting response genes in INT1 vs INT2-9 differential expression. The top 1000 DEGs by significance were selected for each line and fasting response to be compared, with a regression line fitted to compared INT1 vs INT2-9 response. (C) Table of differentially expressed genes (FDR < 0.05) under acute and chronic conditions for INT1 and INT2-9 datasets. (D) Venn diagrams showing the number of differentially expressed genes (FDR < 0.05) in fasting and refeeding conditions for INT1 and INT2-9 datasets. (E, F) WormCat visualization of categories enriched in upregulated genes in INT1 cells and INT2-9 cells under chronic fasting or refeeding conditions. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.

INT1-specific genes modulate behavior and metabolism.

(A) The vector, clec-86, and clec-160 RNAi treated sid-1;INT1::sid-1 animals after 8 hr exposure to PA14 lawns are shown. (B) The distribution of indicated RNAi-treated sid-1;INT1::sid-1 animals at hour 8 after being transferred to PA14 plates. At hour 0, a total of 30 animals were placed near the PA14 lawn. Data are expressed as a percentage of the animals staying inside PA14 lawn at hour 8 ± SEM (n=3, 3 plates per RNAi treatment, 30 animals on each plate). **p<0.01 by one-way ANOVA (Dunnett). (C) The lifespan of each indicated RNAi-treated animal after being transferred to PA14 lawn was determined by counting the number of live and dead animals every 6hr until all animals had died. Data are plotted as the percentage of animals that survived on any given hour relative to the number of live animals at hour 0. The number of animals for each RNAi treatment is indicated in the figure panels. ***p<0.001 by Log-rank Test. (D-F) Average expression of brown, magenta, and turquoise modules genes in the TRAP-seq conditions. The y-axis shows mean variance stabilized expression across all genes and replicates per condition and module with standard deviation indicated by the error bars. (G, I, K) sid-1;INT1::sid-1 animals bearing integrated INT1::INS-7mCherry and CLM::GFP transgenes were treated with vector, aakg-4 or fmo-2 RNAi. INS-7 secretion levels were measured after 30 min fasting and after 30 min refeeding. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each RNAi treatment. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of fed animals treated with indicated RNAi ± SEM (n=21-27). *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA (Dunnett T3). (H, J) Normalized expression plots of aakg-4 and fmo-2 under all conditions in INT1 and INT2-9 cells by replicate.

Pyruvate is critical for maintaining INS-7 secretion dynamics in INT1 cells.

(A) INS-7 secretion dynamics during fasting and re-feeding with OP50, latex beads, Day 1 OP50 supernatant, Day 1 OP50 supernatant with latex beads, and UV-killed OP50 were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=20-26). **p<0.01, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (B) INS-7 secretion dynamics during fasting and re-feeding with OP50, 2% glucose and 100 mM pyruvate were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=14-26). **p<0.01, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (C-F) INS-7 secretion dynamics in INT1-specific vector RNAi, mpc-1 and mpc-2 RNAi, pyc-1 RNAi, pdha-1 and pdhb-1 RNAi-treated animals during fasting and re-feeding with 100 mM pyruvate were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of vector RNAi fed animals ± SEM (n=13-24). *p<0.05, **p<0.01, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (G) INS-7 secretion dynamics during fasting and fasting with the presence of 100 mM pyruvate were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of fed animals ± SEM (n=16-19). ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3).

The genes differentially expressed in INT1 cells.

(A) Module-trait relationships for selected conditions in WGCNA. Rows indicate the WGCNA module and the columns comparative traits. The color indicates the strength of the pearsons correlation and bracketed number the significance. The color red represents a positive correlation; the color green represents a negative correlation. (B) GO enrichment of selected modules in the WGCNA analysis. Size indicates the strength of the significance and color the significance. (C-E) Plots showing the fold change of the intestinal anterior, middle and posterior genes from Ghaddar et al. in our INT1 vs. INT2-9 Fed datasets. (F-J) Expression profiles of B0024.4, clec-86, clec-160, F01D5.5 and F01D5.1 under all conditions in INT1 and INT2-9 cells.

InterPro annotations of differentially expressed genes for INT1 Fed versus INT2-9 Fed.

(A, B) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 fed versus INT2-9 fed.

InterPro annotations of differentially expressed genes for INT1 versus INT2-9 under acute conditions.

(A, B) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 30 min fasted versus INT2-9 30 min fasted. (C, D) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 30 min refed versus INT2-9 30 min refed.

InterPro annotations of differentially expressed genes for INT1 versus INT2-9 under chronic conditions.

(A, B) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 180 min fasted versus INT2-9 180 min fasted. (C, D) Bar charts of the upregulated or downregulated genes categorized by the protein domains predicted by InterPro for INT1 90 min refed versus INT2-9 90 min refed.

WormCat annotations of differentially expressed genes under acute fasting and refeeding conditions for INT1 and INT2-9 cells.

(A, B) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under acute fasting condition. (C, D) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under acute refeeding condition. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.

WormCat annotations of differentially expressed genes under chronic fasting condition for INT1 and INT2-9 cells.

(A, B) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under chronic fasting condition. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.

WormCat annotations of differentially expressed genes under chronic refeeding condition for INT1 and INT2-9 cells.

(A, B) WormCat visualization of categories enriched in differentially expressed genes in INT1 cells and INT2-9 cells under chronic refeeding condition. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.

WormCat annotations of undulating genes in INT1 and INT2-9 cells.

(A, B) WormCat visualization of categories enriched in genes of module brown and turquoise. Categories 1 are all bold uppercase; Categories 2 are capitalized; Categories 3 are gray italics. The size of the bubbles indicates the gene counts in the category and the color of the bubble represents the adjusted p-value.

The role of pyruvate in refeeding responses of INS-7 secretion from INT1 cells.

(A) INS-7 secretion dynamics during fasting and re-feeding with different concentrations of pyruvate (0.01 mM -100 mM) were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=15-23). ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3). (B) INS-7 secretion dynamics during fasting and re-feeding with 10 mM pyruvate, 10 mM 3-FP, and 10 mM pyruvate and 3-FP were determined at the indicated time points. The intensity of INS-7mCherry fluorescence within a single coelomocyte was quantified and normalized to the area of CLM::GFP expression for each time point. Data are expressed as a percentage of the normalized INS-7mCherry fluorescence intensity of wild-type fed animals ± SEM (n=17-20). *p<0.05, ***p<0.001, nsp>0.05 by one-way ANOVA (Dunnett T3).

C. elegans strains used in this study.

Plasmids used in this study.

Cloning primers used in this study.