Dietary restriction improves intestinal cellular fitness to enhance gut barrier function and lifespan in D. melanogaster

Abstract

Loss of gut integrity is linked to various human diseases including inflammatory bowel disease. However, the mechanisms that lead to loss of barrier function remain poorly understood. Using D. melanogaster, we demonstrate that dietary restriction (DR) slows the age-related decline in intestinal integrity by enhancing enterocyte cellular fitness through up-regulation of dMyc in the intestinal epithelium. Reduction of dMyc in enterocytes induced cell death, which leads to increased gut permeability and reduced lifespan upon DR. Genetic mosaic and epistasis analyses suggest that cell competition, whereby neighboring cells eliminate unfit cells by apoptosis, mediates cell death in enterocytes with reduced levels of dMyc. We observed that enterocyte apoptosis was necessary for the increased gut permeability and shortened lifespan upon loss of dMyc. Furthermore, moderate activation of dMyc in the post-mitotic enteroblasts and enterocytes was sufficient to extend health-span on rich nutrient diets. We propose that dMyc acts as a barometer of enterocyte cell fitness impacting intestinal barrier function in response to changes in diet and age.

Fig 1. Intestinal dMyc modulates lifespan in a diet-dependent manner.

(A-C) Kaplan-Meier survival analysis of tissue-specific knockdown of dMyc upon AL and DR. (A) Enteroblasts and enterocytes (5966-GS>dMyc RNAi), (B) Intestinal stem cells and enteroblasts (5961-GS>dMyc RNAi), (C) Fat bodies (S₁106-GS>dMyc RNAi). (D) Median lifespan calculated from A-C are shown. Statistical analysis of the survival curves and the number of flies are provided in S2, S3 and S4 Tables. See also S1 Fig.

Fig 2. dMyc regulates gut barrier function.

(A) Smurf gut permeability assay in 5966-GS>dMyc RNAi 28 and 35-day old flies. d28:–RU486 (AL: n = 335, DR: n = 422), +RU486 (AL: n = 308, DR: n = 380). d35:–RU486 (AL: n = 84, DR: n = 237), +RU486 (AL: n = 77, DR: n = 151). (B) Age-dependent changes in mRNA expression of Diptericin (Dpt) in dissected fat bodies of 5966-GS>dMyc RNAi flies. Young, middle and old represent day 7, 21 and 35 of adulthood, respectively. mRNA expression for flies at day 0 was set to 1. ‘nc’ represents samples that were not collected. (C) upd3 mRNA expression in dissected guts from 21 day old 5966-GS>dMyc RNAi flies. (D) Mitotic ISCs quantification in 21 day old 5966-GS>dMyc RNAi flies. Error bars indicate SEM of 38 guts. (** p < 0.01 by t-test). (B and C) Error bars indicate SD from three independent biological replicates. (** p < 0.01, * p < 0.05 by t-test). See also S2 Fig.

Fig 3. dMyc knockdown in the EBs/ECs causes apoptosis.

(A) TUNEL assay using dissected guts from 28 day old 5966-GS>dMyc RNAi flies. Representative image (n = 25). Scale bar indicates 40 μm. (A’) Quantification of TUNEL positive cells from 25 images. (*** p < 0.001 by t-test). (B) hid mRNA expression in dissected guts of 21 day old 5966-GS>dMyc RNAi flies. (C) Smurf gut permeability assay in 14 day old 5966-GS>UAS-reaper flies.–RU486 (AL: n = 367, DR: n = 377), +RU486 (AL: n = 184, DR: n = 383). Error bars indicate SEM of 17 different vials. (** p < 0.01 by t-test). (D) Smurf gut permeability assay in 5966-GS, dMyc RNAi; + flies and 5966-GS, dMyc RNAi; UAS-p35 flies at 14 days of age. 5966-GS, dMyc RNAi; + flies–RU486 (AL: n = 218, DR: n = 226), +RU486 (AL: n = 233, DR: n = 218), 5966-GS, dMyc RNAi; UAS-p35 +RU486 (AL: n = 260, DR: n = 264). Error bars indicate SD of 11 different vials. (* p < 0.05 by t-test). (E) Mitotic ISCs quantification in 14 day old 5966-GS, dMyc RNAi; + and 5966-GS, dMyc RNAi; UAS-p35 flies. Error bars indicate SEM of 22 guts. (** p < 0.01, * p < 0.05 by t-test). (F) Kaplan-Meier survival analysis of 5966-GS, dMyc RNAi; + flies and 5966-GS, dMyc RNAi; UAS-p35 flies upon AL and DR. Statistical analysis of the survival curves, number of flies are provided in S2 and S3 Tables. (B) Error bars indicate SD of three independent biological replicates. (** p < 0.01, * p < 0.05 by t-test). See also S3 and S4 Figs.

Fig 4. Antibiotic treatment partially rescues the decrease in lifespan but not enterocyte cell death due to loss of dMyc.

(A) Diptericin mRNA expression in dissected fat bodies of 21 day old 5966-GS>dMyc RNAi flies with/without antibiotic treatment. (B, C and D) puc mRNA (B), hid mRNA (C) and upd3 mRNA (D) expressions in dissected guts of 21 day old 5966-GS>dMyc RNAi flies with/without antibiotic treatment. (E) Mitotic ISCs quantification in 21 day old 5966-GS>dMyc RNAi flies with/without antibiotic treatment. Error bars indicate SEM of 13 guts. (F) Smurf gut permeability assay in 20 day old 5966-GS>dMyc RNAi flies with/without antibiotic treatment. Control (–RU486) without antibiotic (AL: n = 185), 5966-GS>dMyc RNAi (+RU486) without antibiotic (AL: n = 222, DR: n = 199), Control (–RU486) with antibiotic (AL: n = 233), 5966-GS>dMyc RNAi (+RU486) with antibiotic (AL: n = 173, DR: n = 238) Error bars indicate SEM of 12 different vials. (G) Kaplan-Meier survival analysis of 5966-GS>dMyc RNAi flies with/without antibiotic treatment upon AL and DR. (A-D) Error bars indicate SD from 3 independent biological replicates. ‘AB’ represents antibiotic. Statistical analysis of the survival curves and the number of flies are provided in S2 and S3 Tables.

Fig 5. dMyc mediates improved intestinal cellular fitness upon dietary restriction.

(A) dMyc mRNA expression in dissected guts from w1118 upon AL and DR was measured with age. mRNA expression from flies at day 0 was set to 1. (B) Immunostaining of dMyc-GFP in dissected guts. dMyc:GFP flies were fed AL and DR diets for 7 days (Young) and 35 days (Old). Representative image (n = 10–13). Scale bar indicates 40 μm. (B’) Quantification of dMyc-GFP-positive cells from 10–13 images. (** p < 0.01 by t-test). (C) GFP-positive flip-out EBs/ECs were observed at 48 hours (Top panels) and 7 days after flip-out (AFO) (Bottom panels) in the posterior midgut upon DR. (Left) WT flip-out EBs/ECs. (Center) dMyc RNAi flip-out EBs/ECs. (Right) dMyc RNAi, UAS-DIAP1 flip-out EBs/ECs. Scale bar indicates 40 μm. (C’) Quantification of the size of GFP positive cells. (*** p < 0.001 by t-test). (D) SYTOX orange staining in the WT flip-out EBs/ECs (Top) and dMyc RNAi EBs/ECs (Bottom) in the posterior midgut upon DR at 48 hours AFO. (Left) merged image. (Middle) GFP-positive flip-out EBs/ECs. (Right) SYTOX staining. White arrow head indicates SYTOX positive cell. Nuclei were stained with Hoechst 33342. Scale bar indicates 20 μm. (E) Immunostaining of mitotic ISCs using dissected guts from dMyc RNAi flip-out flies upon DR at 48 hours AFO. (Top-left) merged image. (Top-right) GFP-positive dMyc RNAi EBs/ECs with pH3 staining. Bottom panels are magnified images of the yellow square in the top-left image. Scale bars of top and bottom panels indicate 40 and 20 μm, respectively. (E’) Mitotic ISCs quantification in the gut from WT flip-out and dMyc RNAi flip-out flies (n = 15). See also S5 Fig.

Fig 6. The modest activation of dMyc in EBs/ECs extends health-span on the rich nutrient diets.

(A and B) Kaplan-Meier survival analysis of enteroblasts and enterocytes specific dMyc overexpression (5966-GS>UAS-dMyc) upon AL and DR. (A) RU486 was administrated from day 21 of age. (B) RU486 was administrated every Monday and Tuesday during the adult stage. Statistical analysis of the survival curves and the number of flies are provided in S2 Table. See also S6 Fig.

Fig 7. The role of dMyc in the fly gut under different nutrient conditions.

Schematic diagram showing how intestinal dMyc regulates gut homeostasis and lifespan under different nutrient conditions. dMyc depletion induces EC cell death by cell competition due to loss of cellular fitness and its increases gut permeability. AL conditions enhance cellular damage and reduce levels of dMyc leading to loss of cellular fitness and causing EC cell death, increased ISC proliferation as a consequence of Upd3 secretion and increased gut permeability which shortens lifespan. These changes are reversed by DR which upregulates dMyc levels to improve intestinal homeostasis in the gut.