title: Betaine stabilizes cell volume and protects against apoptosis in human corneal epithelial cells under hyperosmotic stress.
abstract: Elevated tear osmolarity is one of the key pathological factors in dry eye leading to ocular discomfort associated with damage to the ocular surface and inflammation. The aim of this study was to determine the capacity of the organic osmolyte, betaine, to act as an osmoprotectant against hypertonic stress-induced human corneal epithelial cell shrinkage and apoptosis using in vitro cell culture models. Human corneal limbal epithelial (HCLE) cells exposed to culture medium for 16 h at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) in the presence or absence of betaine (5 or 10 mM) were evaluated for cell volume changes; cell viability; and apoptosis. Betaine (10 mM) ameliorated hyperosmotically induced reduction of cell volume (from 27% reduction to 11%) and resulted in increased mitochondrial activity (by 17%) and an increase in viable cell numbers (by 12%) compared to controls (exposure to hyperosmotic medium without betaine). Hyperosmotically shocked HCLE cells in the presence of betaine (10 mM) halved the number of damaged cells (apoptotic/necrotic) compared to cells in the absence of betaine. The presence of betaine (at 5 or 10 mM) significantly reduced the activity of caspase-8, -9 and -3/7 and release of TNF-α was also reduced by 34% or 55% after exposure of HCLE to 500 mOsm in the presence of 5 or 10 mM betaine, respectively. Using polyclonal antibody against Betaine/GABA transporter 1 (BGT-1), we detected the presence of BGT-1 in HCLE. We demonstrated that the transport of betaine was facilitated by increased osmolarity. In conclusion, betaine stabilized corneal epithelial cell volume under hyperosmotic stress and limited hyperosmotic stress-induced HCLE apoptosis.
authors.affiliation: Brien Holden Vision Institute, Sydney, Australia. q.garrett@brienholdenvision.org
authors.forename: Qian
authors.initials: Q
authors.lastname: Garrett
authors.forename: Neeta
authors.initials: N
authors.lastname: Khandekar
authors.forename: Sharon
authors.initials: S
authors.lastname: Shih
authors.forename: Judith L
authors.initials: JL
authors.lastname: Flanagan
authors.forename: Peter
authors.initials: P
authors.lastname: Simmons
authors.forename: Joseph
authors.initials: J
authors.lastname: Vehige
authors.forename: Mark D P
authors.initials: MD
authors.lastname: Willcox
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001622:Betaine / Q000378:metabolism / Q000494:pharmacology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009997:Osmotic Pressure; D012462:Saline Solution, Hypertonic / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D012462:Saline Solution, Hypertonic; D014409:Tumor Necrosis Factor-alpha; C072207:betaine plasma membrane transport proteins; D001622:Betaine; D020169:Caspases