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

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.

Protamine sulfate induced bladder injury protects from distention induced bladder pain.

PURPOSE: Bladder pain is a debilitating symptom of many urological conditions. There is no generally effective treatment. Abnormal urothelial turnover is common to multiple disease states but the specific components of urothelial injury and the resulting molecular signals that lead to bladder pain are unknown. We examined mouse models of bladder injury induced by uropathogenic Escherichia coli, protamine sulfate (Sigma®) and bacterial lipopolysaccharide to identify cellular and molecular correlates underlying pain sensitization in response to the stimuli. MATERIALS AND METHODS: C57BL/6 female mice (Jackson Laboratory, Bar Harbor, Maine) were given intravesicular protamine sulfate, lipopolysaccharide or uropathogenic E. coli. The impact of each on nociception was determined by measuring the evoked visceromotor response to bladder distention 24 hours after inoculation. Levels of pyuria and tissue inflammation were examined by urinary cytology and tissue histology. Quantitative polymerase chain reaction and gene expression analysis were used to identify injury profiles associated with nociception. RESULTS: Protamine sulfate treatment was significantly analgesic upon bladder distention. Protamine treated bladders did not show pyuria or extensive tissue damage. Protamine injury was associated with a global decrease in the expression of inflammation associated genes. In contrast, uropathogenic E. coli injury significantly increased the nociceptive response to bladder distention. Lipopolysaccharide treatment did not affect nociception. Finally, injury induced expression of inflammation associated genes correlated with nociceptive responses. CONCLUSIONS: Protamine treatment of the bladder is analgesic and tissue protective, and it suppresses the inflammatory cytokine expression normally associated with nociception. Also, the injury modalities that result in differential tissue response patterns provide an innovative method for identifying mediators of visceral pain.

Metabotropic glutamate receptor 5 (mGluR5) regulates bladder nociception.

BACKGROUND: Interstitial cystitis/painful bladder syndrome (IC/PBS), is a severely debilitating chronic condition that is frequently unresponsive to conventional pain medications. The etiology is unknown, however evidence suggests that nervous system sensitization contributes to enhanced pain in IC/PBS. In particular, central nervous system plasticity of glutamatergic signaling involving NMDA and metabotropic glutamate receptors (mGluRs) has been implicated in a variety of chronic pain conditions. Here, we test the hypothesis that mGluR5 mediates both non-inflammatory and inflammatory bladder pain or nociception in a mouse model by monitoring the visceromotor response (VMR) during graded bladder distention. RESULTS: Using a combination of genetic and pharmacologic approaches, we provide evidence indicating that mGluR5 is necessary for the full expression of VMR in response to bladder distention in the absence of inflammation. Furthermore, we observed that mice infected with a uropathogenic strain of Escherichia coli (UPEC) develop inflammatory hyperalgesia to bladder distention, and that the selective mGluR5 antagonist fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl) urea], reduces the VMR to bladder distention in UPEC-infected mice. CONCLUSIONS: Taken together, these data suggest that mGluR5 modulates both inflammatory and non-inflammatory bladder nociception, and highlight the therapeutic potential for mGluR5 antagonists in the alleviation of bladder pain.

Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.

ß-hydroxybutyrate (ß-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine ß-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5+ stem cell-enriched epithelial spheroids treated with ß-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of ß-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.

Fasting Reduces Intestinal Radiotoxicity, Enabling Dose-Escalated Radiation Therapy for Pancreatic Cancer.

PURPOSE: Chemotherapy combined with radiation therapy is the most commonly used approach for treating locally advanced pancreatic cancer. The use of curative doses of radiation in this disease setting is constrained because of the close proximity of the head of the pancreas to the duodenum. The purpose of this study was to determine whether fasting protects the duodenum from high-dose radiation, thereby enabling dose escalation for efficient killing of pancreatic tumor cells. METHODS AND MATERIALS: C57BL/6J mice were either fed or fasted for 24 hours and then exposed to total abdominal radiation at 11.5 Gy. Food intake, body weight, overall health, and survival were monitored. Small intestines were harvested at various time points after radiation, and villi length, crypt depth, and number of crypts per millimeter of intestine were determined. Immunohistochemistry was performed to assess apoptosis and double-strand DNA breaks, and microcolony assays were performed to determine intestinal stem cell regeneration capacity. A syngeneic KPC model of pancreatic cancer was used to determine the effects of fasting on the radiation responses of both pancreatic cancer and host intestinal tissues. RESULTS: We demonstrated that a 24-hour fast in mice improved intestinal stem cell regeneration, as revealed by microcolony assay, and improved host survival of lethal doses of total abdominal irradiation compared with fed controls. Fasting also improved survival of mice with orthotopic pancreatic tumors subjected to lethal abdominal radiation compared with controls with free access to food. Furthermore, fasting did not affect tumor cell killing by radiation therapy and enhanced γ-H2AX staining after radiation therapy, suggesting an additional mild radiosensitizing effect. CONCLUSIONS: These results establish proof of concept for fasting as a dose-escalation strategy, enabling ablative radiation in the treatment of unresectable pancreatic cancer.

Stem cell enriched-epithelial spheroid cultures for rapidly assaying small intestinal radioprotectors and radiosensitizers in vitro.

Radiation therapy is one of the main treatment options for many cancer patients. Although high doses of radiation may maximize tumor cell killing, dose escalation is limited by toxicity to neighboring normal tissues. This limitation applies particularly to the small intestine, the second most radiosensitive organ in the body. Identifying small intestinal (SI) radioprotectors could enable dose escalation in the treatment of abdominopelvic malignancies. However, the only assay currently available to identify effects of radiomodulating drugs on the regenerating capacity of SI stem cells is the Withers-Elkind microcolony assay, which requires large numbers of mice, making it a costly and low throughput method. Here, we describe a novel spheroid formation assay (SFA) that utilizes SI stem cell-enriched three-dimensional epithelial spheroid cultures to identify gastrointestinal radiomodulators ex vivo. The SFA is scalable for high throughput screening and can be used to identify both radioprotectors and radiosensitizers.