Axelrod 2022 Abstract Bioblast
Event: Bioblast 2022
The intestinal mucosa (IM) comprises the inner lining of the intestinal tract, largely consisting of enterocytes with smaller sub-populations of enteroendocrine, immune, goblet, and stem cells. Respiration in IM cells supports nutrient absorption, barrier function, production of mucus, antimicrobial molecules, and growth factors, as well as cellular regeneration. Despite this, little is known of the integrated respiratory functions of mitochondria along the gastrointestinal (GI) tract. The purpose of this study was to develop a procedure to analytically determine respiratory flux of IM derived from varying segments of the GI tract.
Whole, intact intestine was harvested from C57BL/6J mice euthanized by CO2. The GI tract was flushed with ice cold saline, segmented by the jejunum, duodenum, and ileum, and fileted to expose IM. IM were collected directly into MiR05 by scraping the brush border membrane vesicles from the tissue, and mitochondria were subsequently prepared using a glass tissue homogenizer. Prior to assay, total protein was determined by BCA assay and uniformly normalized. Mitochondrial function was determined using a SUIT protocol to determine NADH- and succinate-linked oxidative phosphorylation and electron transfer capacity as well as Complex IV activity. The enzymatic activity of citrate synthase was determined on cryopreserved specimens using spectrophotometry.
Respiration across segments was highly coupled and limited predominantly by OXPHOS, not electron transfer. Substrate coupling was similar between duodenum and jejunum, with depressed NADH- and increased succinate-linked flux in the ileum. Respiratory activity was highest in the duodenum and decreased in a stepwise fashion distally along the GI tract. Conversely, citrate synthase activity was highest in the ileum and decreased in a stepwise fashion proximally along the GI tract.
Mitochondria contained within the IM of the GI tract are energetically robust with differential activity and volume based upon proximity of the segment.
• Keywords: Gastrointestinal tract, Intestine, Mucosa, Mitochondria, OXPHOS
• O2k-Network Lab: US LA Baton Rouge Noland RC
- Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA. -Christopher.Axelrod@pbrc.edu
Labels: MiParea: Respiration, Instruments;methods
Organism: Mouse Tissue;cell: Endothelial;epithelial;mesothelial cell Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, CIV HRR: Oxygraph-2k