Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB102

Poster Communications

Investigating the effect of hyperglycaemia on the airway surface liquid proteome.

M. G. Biggart1, D. L. Baines1

1. Institute for Infection and Immunity, St George's, University of London, LONDON, United Kingdom.

Diabetes is a disease in which the homeostatic pathways for the safe storage of glucose fail. The resultant rise in blood glucose (hyperglycaemia) is known to affect the expression and function of proteins. The complement of proteins produced by a cell type is its proteome. In diabetes hyperglycaemia is known to alter the synthesis and induce aberrant post-translation modifications to the proteomes of varied cell and tissue types. Hyperglycaemia increases glucose flux across the lung epithelium raising the glucose levels of the liquid interface separating the cells from the environment, airway surface liquid (ASL). ASL glucose is usually maintained at ~0.4mM. However, in diabetes ASL glucose can rise as high as ~3mM when comorbid with respiratory diseases such as Cystic Fibrosis or Chronic obstructive pulmonary disease1. Raised glucose in the ASL has been associated with an increased susceptibility to lung infections1. The effect of hyperglycaemia on the ASL proteome remains comparatively unexplored. We hypothesised that acute and chronic hyperglycaemia would modify the ASL proteome and have used an in vitro model to test this. Calu3 and H441 cell were grown at airway-liquid interface and exposed to hyperglycaemic media once the cells reach a transepithelial resistance (Rt) of 600 and 350Ω cm2 respectively. Cells were then washed with Ca2+ and Mg2+ enriched Dulbecco's phosphate buffered saline (DPBS) and placed in hyperglycaemic (25mM glucose) or normoglycaemic conditions (Calu3: 5.5mM, H441:11mM glucose) osmotically balanced with mannitol. Rt was measured and samples of the media and ASL were taken at 0, 24, 48 and up to 120 hours. Glucose concentration was measured with Amplex red glucose assay (Life Technologies). Samples for protein analysis were quantified using a BCA assay (Pierce) and either flash frozen for future analysis or subjected to SDS PAGE on pre-cast Bis-Tris (4-12%) gels and 16% Tricine gel (Novex). The separated proteins were then examined using silver staining (Pierce). The stained gels were imaged and the pixel density of each lane analysed and molecular weights extrapolated from LI-COR molecular weight marker (928-40000) via the GelAnalyzer 2010a program. Glucose analysis indicated that hyperglycaemic conditions were maintained in the cell cultures. Rt declined between 120hrs and 148hrs. This data helped form a time frame for future experiments. Data from the BCA assays showed ASL in acute (24-48 hours) and chronic hyperglycaemia (120 hours) have higher protein concentrations than normoglycaemia. Several protein bands were observed to change their prominence or appear in hyperglycaemiacompared with normoglycaemia. This early data will now inform next generation shot-gun proteomic sequencing using a nano-electrospray ionisation-LC-MS/MS. ASL from primary human airway cells will also be introduced for analysis.

Where applicable, experiments conform with Society ethical requirements