The corneal epithelial tissue is a layer of rapidly growing cells
The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. nitroxyl stimulate glucose uptake in L929 cells 3 to 4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself. Keywords: Glucose uptake, HCLE cells, L929 fibroblast cells, GLUT1, acute regulation 1. Introduction Corneal epithelial cells are rapidly growing cells that have a life cycle of 7-10 days. They originate from stem cells in the limbal basal region at the edge of the cornea and migrate across basement membrane of the anterior cornea forming a basal corneal epithelial layer. Cell division occurs in the basal layer and the daughter cells migrate anteriorly, differentiating to wing cells and squamous superficial cells that are eventually shed from the ocular surface, thereby maintaining an epithelium that is 5-7 cell layers thick . Corneal epithelial cells are reported to have few mitochondria and are known to be heavily dependent on glycolysis. The predominant or only 447407-36-5 IC50 glucose transporter responsible for glucose uptake by corneal epithelial cells is GLUT1 [2-5]. GLUT1 expression and glucose uptake are enhanced during the corneal epithelial wound healing process, but little else is known about the regulation of GLUT1 activity [4, 6]. While GLUT1 is responsible for a basal level of glucose uptake in a wide variety of cells, data from cells that exclusively or predominately express GLUT1 reveal that this transporter can be acutely activated, that can be, triggered within 15 mins, 3rd party of fresh GLUT1 biosynthesis. Circumstances such as blood sugar starvation [7, 8], hyperposmolarity[9, 10] or publicity to azide[11, IL22R 12], methylene blue , C-peptide , or berberine, and thiol energetic real estate agents such as cinnamaldehye, phenylarsine oxide , and nitroxyl all activate blood sugar subscriber base via GLUT1. An immortalized human being cornealClimbal epithelial (HCLE) cell range offers been created [19-21], that forms stratified levels like the in vivo 447407-36-5 IC50 corneal epithelium and states the mucins known to become indicated by shallow corneal epithelial cells. These cells possess been utilized by us to investigate the protecting results of potassium ions against UVB harm [22, 23]. The HCLE cell range can be fairly new and glucose uptake has not been measured, nor has its response to acute stress been determined. Regulation of glucose uptake in corneal cells is relevant to diabetic patients where the disease is associated with an increased fragility of the corneal epithelium and a slowing of wound healing [24-26]. Therefore, the purpose of this study was to measure glucose uptake in HCLE cells, to confirm the expression of GLUT1,and to determine if glucose uptake is acutely regulated in a similar fashion to the regulation of GLUT1 in L929 fibroblast cells [11, 13, 27].The GLUT1 protein is recognized by the same antibody, which indicates that the transporter is very similar in the two species. Therefore, this suggests any differences in the regulation of GLUT1 are more likely a function of different cell types than of different types. 2. 447407-36-5 IC50 Methods and Materials 2.1 Chemical substances Angelis sodium (AS) was a ample present of Dr. Mark G. Toscano (Johns Hopkins College or university) and was kept at ?4 C under nitrogen. Phenylarsineoxide (PAO), cinnamaldehyde (California), berberine, cytochalasin T, quercetin, 2-deoxy-D-glucose-[1,2-3H] (2DG) and D-mannitol-1-14C had been bought from the Sigma-Aldrich Chemical substance Business (St. Louis, MO, USA). 2.2 Cell lifestyle The immortalized individual cornealClimbal epithelial (HCLE) cell range was attained from Dr. Ilene Gipson (Section of Opthalmology, Harvard Medical College) and taken care of asmonolayer civilizations in Keratinocyte-Serum Totally free moderate (K-SFM)(Invitrogen, Carlsbad, California), as described previously. The D929 mouse fibroblast cells had been attained from the American Type.