Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival

Abstract

Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

Keywords: DNA damage; chemotherapy; fasting; stem cells.

Fig. 1.

Effects of high-dose etoposide on integrity of SI in fed and fasted mice. Male and female wild-type mice (4–6 wk of age) were allowed to feed ad libitum or were fasted for 24 h. Etoposide (100 mg/kg) was administered by tail vein injection (day 1). Mice were returned to single-housed cages and access to food was restored immediately after treatment. (A) Overall schema and timeline are illustrated. (B) Survival was monitored daily for 2 wk up to day 15. (C) Food consumption was monitored daily beginning with the 24-h period before etoposide treatment for fed mice and the 24-h period postetoposide treatment for fasted mice. (D) Individual mouse body weights were measured daily and were normalized to starting weight. All error bars are ±SEM.

Fig. S1.

Survival and etoposide clearance studies. (A–C) Male and female Lgr5^{eGFP-IRES-CreER/+};R26R mice were fed or fasted for 24 h and then etoposide was administered intravenously at a dose of 110 mg/kg. Mice were returned to single-housed cages and food was replenished. (A) Survival was monitored daily for 2 wk up to day 15. (B) Food consumption was monitored daily beginning with the 24-h period before etoposide treatment for fed mice and the 24-h period postetoposide treatment for fasted mice. (C) Individual mouse body weights were measured daily and were normalized to starting weight. (D and E) Male and female Lgr5^{eGFP-IRES-CreER/+}mice were fed or fasted for 24 h and then etoposide was administered intravenously at a dose of 110 mg/kg. Mice were returned to single-housed cages and food was replenished. (D) Survival was monitored daily up to day 140. (E) Individual mouse body weights were measured daily for 7 d postetoposide and then once every 7 d up to 140 d postetoposide and were normalized to starting weight. All error bars are ±SEM (F) Four- to 6-wk-old male and female Bmi1^CreER/+;R26R mice were fed or fasted for 24 h and then etoposide was administered intravenously at a dose of 100 mg/kg. Mice were returned to single-housed cages and food was replenished. Whole blood was isolated immediately following etoposide injection (time 0) or at 0.75, 6, or 24 h postetoposide injection and processed for plasma and LC/MS/MS analysis (SI Materials and Methods). n = 3 mice per treatment per time point were analyzed for plasma concentrations of etoposide (μg/mL) plotted as mean ± SEM.

Fig. 2.

Fasting preserves SI architecture in the presence of high-dose etoposide. (A) Wild-type mice were treated as shown. Asterisks indicate day of killing for experiments in indicated panels. Mice were randomly assigned to four treatment groups (n = 6–7 mice per group). (B) Representative images of H&E-stained jejunum (day 5). (Scale bars, 200 μm.) Representative crypts shown in Insets. (Scale bars, 25 μm.) (C) Villi heights (n = 30 per mouse) were measured and average value per mouse plotted. (D) Number of crypts per length (∼20 mm) of SI was quantified for each day 5 sample and average number of crypts per millimeter of SI length plotted. (E) Number of cells per crypt was determined (n = 45 crypts per mouse) and average number of cells per crypt plotted. n.s., nonsignificant; *P < 0.05; ***P < 0.005 by Tukey posttest of a one-way ANOVA. Error bars are ±SEM. (F) Lgr5^{EGFP-IRES-CreER/+} mice were treated as in A. Representative images of H&E-stained jejunum are shown. Arrows indicate neutrophils. [Scale bars, 100 μm (Left) and 25 μm (Right).]

Fig. S2.

Effects of high dose etoposide on SI of mice that were fed or fasted before treatment. Wild-type mice were randomly assigned to four treatment groups. These included mice that were allowed to feed ad libitum (fed) and treated with either saline (n = 7) or etoposide (n = 7), and mice that were deprived of food for 24 h (fasted), and treated with either saline (n = 7) or etoposide (n = 6). Etoposide was administered intravenously at a dose of 100 mg/kg on day 1. Food was replenished immediately after treatment. Mice were killed and either the jejunum or the entire length of the SI was isolated 4 d posttreatment. (A) Length of the SI was measured. ns, nonsignificant; ***P < 0.005 by Tukey posttest of a one-way ANOVA. (B) Cells present within single SI crypts located in the jejunum were counted. Dots represent the number of cells counted in a single crypt. Cells present within single SI crypts located in the duodenum (C) and ileum (D) were counted. *P < 0.05; ****P < 0.0001 by two-tailed Student’s t test. (E and F) Male and female Lgr5^{eGFP-IRES-CreER/+} (4–6 wk of age) were allowed to feed ad libitum or were fasted for 24 h. Etoposide (110 mg/kg) was administered by tail vein injection. Mice were returned to single-housed cages and food was replenished immediately after treatment. Mice were killed and the entire length of the SI was isolated 10 d posttreatment. (E) Representative images of H&E-stained jejunum. (Scale bars, 200 μm.) Representative crypts shown (Insets). (Scale bar, 25 μm.) (F) Cells present within single SI crypts located in the jejunum were counted. n.s. is nonsignificant by two-tailed Student’s t test. All error bars are ±SEM.

Fig. S3.

Effects of high-dose etoposide on colons of fed and fasted mice. Wild-type mice were randomly assigned to four treatment groups. These included mice that were allowed to feed ad libitum (fed) and treated with either saline (n = 6) or etoposide (n = 6), and mice that were deprived of food for 24 h (fasted), and treated with either saline (n = 7) or etoposide (n = 4). Etoposide was administered intravenously at a dose of 100 mg/kg on day 1. Food was replenished immediately after treatment. Mice were killed and the entire colon was isolated 4 d posttreatment. Representative images of H&E-stained transverse colon are shown (A). [Scale bars, 200 μm and 30 μm (Inset).] The entire colonic length (B) and transverse colonic crypt depth (C) were measured and average values per mouse are plotted. n.s. is nonsignificant by one-way ANOVA and by Tukey posttest of a one-way ANOVA. Error bars are ±SEM.

Fig. S4.

Fasting protects stem cell reporter mice from high-dose etoposide. Male and female mice at 4- to 6-wk-old were allowed to feed ad libitum (fed) or were deprived of food for 24 h (fasted). Etoposide was administered intravenously at the indicated doses, mice were returned to single-housed cages, and food was replenished in all cases immediately after treatment. Survival was monitored daily for 2 wk up to day 15 (A, C, E, and G). Individual mouse body weights were measured daily and were normalized to starting weight (B, D, F, and H). All error bars are ±SEM.

Fig. 3.

Fasting protects SI stem cells from high-dose etoposide in vivo and ex vivo. Reporter mice were randomly assigned to four treatment groups and were administered two doses of tamoxifen (t) 1 and 3 h after etoposide. Mice were killed 4 d later (day 5) and SI were harvested and whole-mount tissue stained for LacZ expression (n = 5–6 mice per group). (A) Representative images of nuclear fast red-counterstained cross-sections of LacZ-stained jejunums. (Scale bars, 100 μm.) (B) Villi were removed from a 2-cm section of LacZ-stained whole-mount tissue for counting traced crypts. Representative images are shown. (Scale bars, 100 μm.) (C) The number of fully traced crypts per field of view in whole-mount images was quantified using a custom image analysis program for each day 5 sample and then was normalized to the average number of crypts per millimeter within each strain per treatment. **P < 0.01 by one-tailed, Student’s t test of normalized arcsine transformed data. (D) Bmi1^CreER/+;R26R mice were randomly assigned to four treatment groups (n = 3 mice per group). SI crypts were isolated and plated in Matrigel with 50% L-WRN–conditioned media to generate cultures of stem cell-enriched epithelial spheroids. Spheroids were counted after 2 d of culturing in vitro. Spheroid number was normalized to number of crypts originally plated. *P < 0.05 by two-tailed, Student’s t test. (E) Representative images of spheroids after 2 d in culture are shown (Upper) at passage 0. Cultures were trypsinized on day 3 and subcultured in fresh Matrigel. Representative images of passage 1 spheroid cultures (2 d posttrypsinization) are shown (Lower). (Scale bars, 250 μm.) All error bars are ±SEM.

Fig. S5.

Both CBC and +4 stem cells give rise to villus epithelial cells in fasted, etoposide-injected mice. The protocol and timeline schema used for tamoxifen-induced, Cre-mediated lineage tracing experiments shown in Fig. 3 A–C is shown (A). Stem cell reporter mice were allowed to feed ad libitum or were fasted for 24 h. Etoposide was then administered intravenously at the following doses: Lgr5^{eGFP-IRES-CreER/+};R26R (80 mg/kg), Bmi1^CreER/+;R26R (100 mg/kg) and HopX^CreER/+;R26R (100 mg/kg). Control groups received saline. Food was replenished immediately after treatment. Mice were administered two doses of tamoxifen (t) 1 and 3 h after etoposide. Mice were killed 4 d later (day 5) and SI were harvested and whole-mount tissue stained for LacZ expression n = 6 mice per group. (B) Number of crypts per length (∼20 mm) of SI were quantified in a blinded fashion for day 5 samples (n = 3 mice per group) and the average number of crypts per millimeter of SI was plotted. n.s., nonsignificant; **P < 0.01; ***P < 0.001 by Tukey posttest of a one-way ANOVA. (C) The number of crypts per length of SI in the fed etoposide treated group for each mouse strain taken from the data in B. ***P < 0.001 by Tukey posttest of a one-way ANOVA. (D) The normalized fully traced crypts in the fasted etoposide treated group for each strain of mouse taken from the data in Fig. 3C. n.s. is nonsignificant by a 1-way ANOVA. Error bars are ±SEM. (E) Mice were treated as in A and were killed 8 d later (day 9). SI were harvested and whole-mount tissue stained for LacZ expression. (Scale bars, 500 μm.)

Fig. S6.

Flowchart for MatLab image processing program. A flowchart is provided to explain the logic and display the processing of the images embedded within the program, as well as subsequent image analysis for graphing. (Magnification: 90×.)

Fig. S7.

Validation of MatLab program. Bmi1^CreER/+;R26R mice were randomly assigned to four treatment groups. These included mice that were allowed to feed ad libitum (fed) or mice that were deprived of food for 24 h (fasted) before receiving an intravenous injection of either saline or 100 mg/kg etoposide (day 1). Food was replenished immediately after treatment. Mice were administered two doses of tamoxifen 1 and 3 h after etoposide. Mice were killed 4 d later, SI isolated and whole-mount tissue stained for LacZ-expression. SI were stacked in agar, embedded in paraffin and 5-μm cross-sections generated. (A) The number of fully traced crypts present in 4 cm of SI length was manually counted in each sample. Error bars are ±SEM. (B) A Spearman correlation was computed using values obtained by MatLab and values obtained by manually counting LacZ stained cross-sections. P < 0.0001; n = 22.

Fig. 4.

Fasting alters early response of Lgr5⁺ stem cells to etoposide. Lgr5^{EGFP-IRES-CreERT/+} mice were randomly assigned to four treatment groups. Two-hundred crypts were evaluated per mouse (n = 4 mice per group). Crypts in which the base made direct contact with the lamina propria were analyzed and only GFP⁺ cells residing at the crypt base were counted. (A) The number of GFP⁺ cells per 200 crypts per mouse at the indicated time points is shown. (B) The percentage of cells that costained for GFP and BrdU is shown. BrdU was allowed to incorporate for 1 h before harvest. ***P < 0.001 by two-tailed, Student’s t test. (C–E) Percentage of cells that costained for GFP and γH2AX (C) and representative images, shown in D and E. *P < 0.05 by two-tailed, Student’s t test of arcsine-transformed data. (F and G) Percentage of cells costaining for GFP and cleaved caspase 3 (CC3) is shown (F) and representative images from the 3-h time point (G). *P < 0.05 by two-tailed, Student’s t test of arcsine transformed data. All error bars are ±SEM. (H) Number of apoptotic bodies in the transient amplifying cell zone per 50 crypts at 3-h postetoposide treatment. n.s. is nonsignificant by two-tailed, Student’s t test. All error bars are ±SEM.