Insulin receptors and insulin action in dissociated brain cells.
The present study was conducted to characterize insulin receptors and insulin action in rat brain cells. Binding of [125I]insulin to cells obtained by mechanically dissociating rat brains was 86% specific, time-dependent and reached equilibrium within 90 min. The t1/2 of association was 14 min and t1/2 of dissociation was 8 min. Scatchard analysis demonstrated the typical curvilinear plot providing high affinity (0.03 nM) and low affinity (6.6 nM) binding sites. The total number of binding sites were 0.15 pmol/mg protein. Crosslinking of [125I]insulin to its receptors on dissociated brain cells followed by SDS-PAGE and autoradiography showed that the alpha-subunit of the receptor had a molecular weight of 122,000. This was in contrast with a molecular weight of 134,000 for the liver alpha-subunit. Incubation of dissociated brain cells with insulin resulted in a concentration-dependent inhibition of total [3H]norepinephrine (NE) uptake. This inhibitory effect of insulin on [3H]NE uptake was sodium ion-dependent suggesting that 80-90% of the sodium ion-dependent uptake was insulin-sensitive. Incubation of lectin-purified insulin receptors with insulin resulted in a time- and concentration-dependent stimulation of phosphorylation of the tyrosine residue of an exogenous substrate poly (Glu, Tyr) (4:1). In addition, insulin also stimulated the autophosphorylation of the beta-subunit of the insulin receptors. These observations corroborate our contention that insulin exerts neuromodulatory effects mediated by the specific insulin receptors in the brain.
Masters
B A
BA
Department of Physiology, University of Florida, Gainesville 32610.
Shemer
J
J
Judkins
J H
JH
Clarke
D W
DW
Le Roith
D
D
Raizada
M K
MK
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D009638:Norepinephrine / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000378:metabolism*; D013997:Time Factors
D007328:Insulin; D011972:Receptor, Insulin; D009638:Norepinephrine