Impact of experimental colitis on mitochondrial bioenergetics in intestinal epithelial cells

Abstract

Intestinal homeostasis is highly dependent on optimal epithelial barrier function and permeability. Intestinal epithelial cells (IEC) regulate these properties acting as cellular gatekeepers by selectively absorbing nutrients and controlling the passage of luminal bacteria. These functions are energy demanding processes that are presumably met through mitochondrial-based processes. Routine methods for examining IEC mitochondrial function remain sparse, hence, our objective is to present standardized methods for quantifying mitochondrial energetics in an immortalized IEC line. Employing the murine IEC4.1 cell line, we present adapted methods and protocols to examine mitochondrial function using two well-known platforms: the Seahorse Extracellular Flux Analyzer and Oxygraph-2 k. To demonstrate the applicability of these protocols and instruments, IEC were treated with and without the murine colitogenic agent, dextran sulfate sodium (DSS, 2% w/v). Profound impairments with DSS treatment were found with both platforms, however, the Oxygraph-2 k allowed greater resolution of affected pathways including short-chain fatty acid metabolism. Mitochondrial functional analysis is a novel tool to explore the relationship between IEC energetics and functional consequences within the contexts of health and disease. The outlined methods offer an introductory starting point for such assessment and provide the investigator with insights into platform-specific capabilities.

Alkaline sphingomyelinase deficiency impairs intestinal mucosal barrier integrity and reduces antioxidant capacity in dextran sulfate sodium-induced colitis

“…Additionally, during the ultrastructural examination, we observed common mitochondrial swelling in intestinal epithelial cells following DSS-induced inflammation, particularly in the context of alk-SMase deletion, indicating more pronounced mitochondrial degeneration. DSS-induced murine colitis has been previously reported to affect mitochondrial function, resulting in reduced intestinal barrier function and increased permeability[ 38 - 40 ].…”

Effects of alimentary-derived bacterial metabolites on energy metabolism in colonic epithelial cells and inflammatory bowel diseases

“…Of note, all the 13mitochondria-encoded genes were found to be related to ATP production. Interestingly, using colonic biopsies from ulcerative colitis patients, Sünderhauf and collaborators [65] demonstrated that mucosal energy deficiency is linked to impaired goblet cell differentiation, thus pointing out an impaired production and secretion of mucin-producing goblet cells in ulcerative colitis.…”

Protocol for measuring respiratory function of mitochondria in frozen colon tissue from rats

“… Timing: 60 min Prepare mitochondrial respiration MiR05 1 solution following materials and equipment section. Calibrate the respirometry following Oxygraph-2k respirometer 1 , 2 user manual for specific procedures. Note: Calibration of the Oxygraph is an essential step to minimize confounding effects of instrumental oxygen consumption.…”

“…Calibrate the respirometry following Oxygraph-2k respirometer 1 , 2 user manual for specific procedures.…”

“…The tissue amount needs to be determined for each tissue types such as cardiac fibers, 1 hippocampus 8 , 12 , 13 and epithelial cells. 2 The method is not able to measure proton leak-related respiration. In this protocol, tissues frozen for 48 h or 4 months were used.…”