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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 identified multiple SI stem cell populations, marked by Lgr5, Bmi1 or HopX expression, that 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. Intestines were harvested 3 h post-etoposide injection, flushed with PBS, and then flushed with ice-cold Methacarn (60% methanol, 30% chloroform, 10% acetic acid) to fix the tissue while preserving RNA integrity. Fixed intestines were rinsed with 70% ethanol, embedded in 2% agar followed by paraffin embedding. Sections were deparrafinized and dehydrated through xylene and an increasing gradient of ethanol. Laser capture microdissection was performed using the Acturus PixCell IIe, with the following settings: spot size power 80, duration 1 ms, repeat 0.2 s, target 0.150 V, current 1.82 mA. RNA was isolated from the CapSure HS LCM Caps (Applied Biosystem) using the PicoPure RNA Isolation kit (Applied Biosystems). All RNA samples were treated with DNaseI (Qiagen).

ORGANISM(S): Mus musculus

PROVIDER: GSE72122 | GEO | 2015/11/23

SECONDARY ACCESSION(S): PRJNA293104

REPOSITORIES: GEO

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Project description: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 identified multiple SI stem cell populations, marked by Lgr5, Bmi1 or HopX expression, that 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. Intestines were harvested 3 h post-etoposide injection, flushed with PBS, and then flushed with ice-cold Methacarn (60% methanol, 30% chloroform, 10% acetic acid) to fix the tissue while preserving RNA integrity. Fixed intestines were rinsed with 70% ethanol, embedded in 2% agar followed by paraffin embedding. Sections were deparrafinized and dehydrated through xylene and an increasing gradient of ethanol. Laser capture microdissection was performed using the Acturus PixCell IIe, with the following settings: spot size power 80, duration 1 ms, repeat 0.2 s, target 0.150 V, current 1.82 mA. RNA was isolated from the CapSure HS LCM Caps (Applied Biosystem) using the PicoPure RNA Isolation kit (Applied Biosystems). All RNA samples were treated with DNaseI (Qiagen). Four biological replicates were used for each of the following treatments: Fasted Etoposide, Fed Etoposide. Each group had 2 male and 2 females samples. 4-6 week old Lgr5EGFP-IRES-CreERT2/+ mice were dosed with 80mg/kg etoposide (i.v.).

Project description:Obesity and insulin resistance are two major risk factors underlying the metabolic syndrome. To gain more insight in the role of the small intestine in the etiology of these metabolic disorders, a microarray study was performed on small intestines (SI) of C57BL/6J mice that were fed a high fat diet mimicking the fatty acid composition of a Western-style human diet. The mice became obese and developed dietary fat-induced glucose intolerance. For gene expression profiling, the small intestines were subdivided in three equal parts along the longitudinal axis. The most pronounced effects of dietary fat were detected in part 2 of the small intestine. The biological processes that were most extensively modulated on a high fat diet were related to lipid metabolism, especially Î²- and Ï-fatty acid oxidation seemed to play an important role, cell cycle and inflammation/immune response. An additional secretome analysis revealed differentially expressed secreted proteins, such as Il18, Ffgf15, Mif, Igfbp3 and Angptl4, which might provoke systemic effects in peripheral organs by influencing their metabolic homeostasis. Furthermore, many of the dietary fat-modulated genes and biological processes in small intestine were previously already associated with obesity and/or insulin resistance. Together, the data of this exploratory study provided various leads for an essential role of the small intestine in development of obesity and/or insulin resistance. Experiment Overall Design: After a run-in period of 3 weeks on a low-fat diet, 9 weeks old mice were fed a high- (HF) or a low-fat (LF) purified diet for 2, 4, and 8 weeks (n=6 per diet, per time point). Body weight was recorded weekly and after 7 weeks of diet intervention an oral glucose tolerance test was performed. At the end of the experiment, mice were anaesthetized with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%). The small intestines were excised and the adhering fat and pancreatic tissue were carefully removed. The small intestines were divided in three equal parts along the proximal to distal axis (SI 1, SI 2 and SI 3).

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

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