Investigations on glucose uptake in isolated human leucocytes from normal and diabetic subjects

Summary 1. Leucocyte preparations have been obtained from the blood of 135 healthy control persons and 10 poorly-controlled insulin-dependent diabetics according to a procedure described earlier. Cells were suspended in a Krebs-Ringer-Bicarbonate-Buffer. Glucose uptake, lactate production and cell glucose space were determined after incubating the cells for 1 h at 37° C in an atmosphere of O₂/CO₂ (95/5). Extracellular water-space of the cell sediment was corrected by measuring U-¹⁴C-sucrose levels in the medium before and after addition to the cell sediment. — 2. The cells showed intact structures and amoeboid motility under the light-microscope as well as under the phase-contrast-microscope. During an incubation lasting 1 h, the ATP/ADP quotient diminished by about 20%; the following metabolites: glucose-6-phosphate, fructose-1,6-diphosphate, 1,3-phosphoglycerate, pyruvate and lactate increased slightly up to markedly, especially glucose-6-phosphate and lactate. — 3. Sources of methodological errors were investigated in preliminary experiments. Disregarding corrections for the extracellular space of the cell sediments led to a dilution effect which imitates an apparent glucose uptake. For measurements of intracellular glucose and cell glucose space the procedure of Crofford and Renold provided the best results. Falsely high values for cell glucose have been found using the glucose oxidase reaction. — 4. Glucose uptake of healthy leucocytes increases at first steeply, later on less with rising medium glucose concentrations. Addition of insulin (50–500 mU/ml) gave no consistent effects. At medium glucose concentrations of 400 and 600 mg/100 ml intracellular glucose could be demonstrated with statistical significance. Intracellular glucose and cell glucose space were not affected by insulin to any marked degree. Determinations of lactate production indicated that human leucocytes utilize the largest portion of glucose via glycolysis. — 5. At medium glucose concentrations of 600 mg/100 ml leucocytes from diabetics who had received no insulin for 14 h prior to incubation showed a significant diminution of glucose uptake. — 6. Theoretical curves for inward transport and phosphorylation have been calculated from the data for glucose uptake and intracellular glucose concentration.The authors are greatly indepted to Prof. Wilbrandt, Department of Pharmacology, University Bern, for advice and discussion.