Glucose consumption of vascular cell types in culture; toward optimization of experimental conditions

Abstract

Any experimental outcomes are potentially influenced by extracellular glucose availability and its cellular metabolism. However, there is a lack of attention paid to the supply and utilization of glucose in many cultured experiments. Surveillance of vascular‐related journals for the past 5 years demonstrated that less than 20% of published articles declared the medium glucose concentration. The present studies were designed to seek ideal medium glucose concentration(s) in various cell types with particular attention paid to changes in glucose consumption. By using distinct glucose concentrations in Dulbecco's Modified Eagle's medium (DMEM), we identified ideal glucose media for stimulation experiments. We have compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without angiotensin II stimulation. In all cell types after a 48‐hour incubation in relatively low glucose media (1 g/L), medium glucose concentration was reduced to almost 0 (EC 0.01±0.01 g/L, VSMC 0.13±0.05 g/L, AF 0.02±0.01 g/L). Whereas medium glucose concentration remained significantly higher (EC 2.77±0.05 g/L, VSMC 3.87±0.05 g/L, AF 3.32±0.01 g/L) when cells were incubated for 48 hours in high glucose (4.5 g/L) media. In middle glucose (2.75 g/L) media, medium glucose concentration remained in physiological ranges (EC 0.62±0.18 g/L, VSMC 1.98±0.07 g/L, AF 1.17±0.17 g/L). AngII treatment enhanced glucose consumption in AF and VSMC but not in EC. Enhanced extracellular acidification rate by AngⅡ was observed in AF as well as VSMC. In AF, AngII induction of target proteins at 48 hours varied depending on the glucose concentration used. In low glucose media induction of Grp78 or hexokinase II was highest, whereas induction of vascular cell adhesion molecule‐1 was lowest. However, no glucose preference was seen in AngII‐induced c‐Fos induction at 1 hour in AF. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AngII‐induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo‐ or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Medium glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.