A microtiter well assay system to measure insulin activation of insulin receptor kinase in intact human mononuclear cells. Decreased insulin effect in cells from patients with NIDDM.
A sensitive microtiter well-based assay for the measurement of insulin activation of insulin receptor kinase in intact human circulating mononuclear cells has been developed and characterized. Mononuclear cells from 100-150 ml blood were incubated with various insulin concentrations to activate the receptor kinase. The cells were then solubilized in the presence of phosphatase and kinase inhibitors and the receptors immobilized to microwells coated with anti-insulin receptor antibody (efficiency of receptor immobilization > 85%). Receptor kinase activity and binding activity were then consecutively measured in the same wells. Insulin incubation of the cells increased the kinase activity three- to fourfold with a half-maximal effect at 5 nM and a maximal effect at 87 nM. In mononuclear cells from 16 subjects with NIDDM, the insulin effect on receptor kinase activation was significantly reduced compared with 16 nondiabetic control subjects (0.135 +/- 0.016 vs. 0.195 +/- 0.024 fmol P.fmol binding activity-1 x min-1, respectively; P < 0.05). We conclude that; 1) it is possible to determine insulin activation of receptor kinase in intact cells in this easily accessible human tissue; 2) insulin activation of insulin receptor kinase is impaired in intact mononuclear cells from patients with NIDDM; and 3) the finding that kinase activation in NIDDM is reduced in a tissue that, according to the literature, contains only the A isoform of the insulin receptor, suggests that mechanisms other than a different abundance of the A and B insulin receptor isoforms must exist that contribute to the decreased kinase activity in NIDDM.
Klein
H H
HH
Department of Internal Medicine, Medical University of Lübeck, Germany.
Kowalewski
B
B
Drenckhan
M
M
Neugebauer
S
S
Matthaei
S
S
Kotzke
G
G
D000368:Aged; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D010761:Phosphorus Radioisotopes; D011505:Protein-Tyrosine Kinases / Q000097:blood* / Q000187:drug effects; D011972:Receptor, Insulin
D007328:Insulin; D010761:Phosphorus Radioisotopes; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin