TY - JOUR
T1 - Glucose transport and phosphorylation in single cardiac myocytes
T2 - Rate- limiting steps in glucose metabolism
AU - Manchester, J.
AU - Kong, X.
AU - Nerbonne, J.
AU - Lowry, O. H.
AU - Lawrence, J. C.
PY - 1994
Y1 - 1994
N2 - Microanalytic methods were used to investigate the regulation of glucose metabolism by insulin in single myocytes isolated from adult rat ventricles. Cultured myocytes were incubated with or without insulin and, with either glucose or 2-deoxyglucose (2-DG), rinsed, and freeze-dried. Individual cells were weighed and levels of 2-DG-6-phosphate (2-DG-6-P) or glucose and glucose 6-phosphate (G-6-P) were determined after enzymatic amplification. In cells incubated with 2-DG, insulin increased the level of 2-DG-6-P by as much as 30-fold, indicative of dramatic activation of glucose transport. In cells incubated with glucose, insulin increased the levels of G-6-P by approximately threefold. Increasing extracellular glucose without insulin also increased G-6-P; however, intracellular glucose concentrations were not increased, indicating that glucose transport is rate limiting in nonstimulated myocytes. In contrast, intracellular glucose concentrations were increased by over an order of magnitude by insulin, reaching 60% of the extracellular glucose concentration. Measurements of glucose and G-6-P in the same insulin-treated cells revealed that accumulation of G-6-P reached a plateau when extracellular glucose was increased >2 mM. At this point the estimated intracellular glucose concentration was 300 μM, or approximately 10 times the Michaelis constant of hexokinase for glucose. These results indicate that in the presence of insulin and physiological concentrations of glucose, hexokinase is saturated with glucose. Consequently, the rate- limiting step for insulin-stimulated glucose utilization is glucose phosphorylation rather than glucose transport.
AB - Microanalytic methods were used to investigate the regulation of glucose metabolism by insulin in single myocytes isolated from adult rat ventricles. Cultured myocytes were incubated with or without insulin and, with either glucose or 2-deoxyglucose (2-DG), rinsed, and freeze-dried. Individual cells were weighed and levels of 2-DG-6-phosphate (2-DG-6-P) or glucose and glucose 6-phosphate (G-6-P) were determined after enzymatic amplification. In cells incubated with 2-DG, insulin increased the level of 2-DG-6-P by as much as 30-fold, indicative of dramatic activation of glucose transport. In cells incubated with glucose, insulin increased the levels of G-6-P by approximately threefold. Increasing extracellular glucose without insulin also increased G-6-P; however, intracellular glucose concentrations were not increased, indicating that glucose transport is rate limiting in nonstimulated myocytes. In contrast, intracellular glucose concentrations were increased by over an order of magnitude by insulin, reaching 60% of the extracellular glucose concentration. Measurements of glucose and G-6-P in the same insulin-treated cells revealed that accumulation of G-6-P reached a plateau when extracellular glucose was increased >2 mM. At this point the estimated intracellular glucose concentration was 300 μM, or approximately 10 times the Michaelis constant of hexokinase for glucose. These results indicate that in the presence of insulin and physiological concentrations of glucose, hexokinase is saturated with glucose. Consequently, the rate- limiting step for insulin-stimulated glucose utilization is glucose phosphorylation rather than glucose transport.
KW - glucose phosphorylation
KW - hexokinase
KW - microanalytic methods
UR - http://www.scopus.com/inward/record.url?scp=0028347762&partnerID=8YFLogxK
U2 - 10.1152/ajpendo.1994.266.3.e326
DO - 10.1152/ajpendo.1994.266.3.e326
M3 - Article
C2 - 8166252
AN - SCOPUS:0028347762
SN - 0002-9513
VL - 266
SP - E326-E333
JO - American Journal of Physiology - Endocrinology and Metabolism
JF - American Journal of Physiology - Endocrinology and Metabolism
IS - 3 29-3
ER -