Cation-induced restoration of insulin action in insulin-desensitized HTC cells.
Insulin desensitization of amino acid uptake in HTC cells was induced by preincubation with 4 or 10 micrograms/ml insulin. Insulin binding after desensitisation was decreased by both insulin concentrations due to a 45-49% decrease in insulin receptor numbers. Desensitization with 4 micrograms/ml insulin increased the ED50 for half-maximal stimulation of amino acid uptake from 19.5 +/- 9.2 ng/ml in control cells to 84.0 +/- 8.3 ng/ml (P less than 0.0001). It also decreased the maximal insulin response of amino acid uptake from 1.40 +/- 0.10 to 1.14 +/- 0.10 nmol/mg protein, indicating the production of a mild postreceptor defect. Desensitization with 10 micrograms/ml insulin completely abolished this insulin response. When cellular receptors were down-regulated with 4 micrograms/ml insulin and restimulated with insulin in the presence of 0.03 mM ruthenium red (RR) or 10 mM Ca2+, both the insulin response and insulin binding were increased. Insulin binding was restored to levels comparable to those observed in control cells by an increase in receptor affinity. The ED50 of amino acid uptake decreased to 20.5 +/- 7.3 ng/ml insulin in the presence of RR and to 42.2 +/- 8.9 ng/ml in the presence of 10 mM Ca2+ (both P less than 0.0001 from down-regulated cells). In addition, the maximal insulin response increased from 1.14 +/- 0.10 to 1.40 +/- 0.10 and 1.45 +/- 0.10 nmol/mg protein, respectively. Preincubation with 10 micrograms/ml insulin prevented the effect of RR and Ca2+ on the recovery of insulin responses. These experiments suggest that insulin-desensitized cells undergo a progressive loss of their insulin response and that RR and Ca2+ provide useful reagents to investigate the mechanisms of this process because they can counteract the decrease in insulin response by increasing receptor affinity and receptor-effector coupling.
Williams
P F
PF
Department of Endocrinology, Royal Prince Alfred Hospital, Camperdown, Australia.
Zilkens
R R
RR
Caterson
I D
ID
Cooney
G J
GJ
D000596:Amino Acids / Q000378:metabolism; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular; D002412:Cations; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D007333:Insulin Resistance; D008113:Liver Neoplasms; D011972:Receptor, Insulin / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D002412:Cations; D007328:Insulin; D012430:Ruthenium Red; D011972:Receptor, Insulin; D002118:Calcium