Intranuclear localization of insulin in rat hepatoma cells: insulin/matrix association.
Previous studies have documented nuclear insulin accumulation in a variety of cell types. The present investigation extends these observations by demonstrating that insulin associates with the matrix fraction of H35 rat hepatoma cell nuclei. Nuclei were isolated from [125I]insulin-loaded cells and extracted with DNase I, RNase A and high salt. The resulting matrix fraction was found to contain greater than 75% of the radiolabel initially present. Ultrastructural studies to confirm these findings were carried out using an agarose-encapsulated nuclear matrix preparation. Electron microscopic immunocytochemistry specifically detected insulin in matrices prepared from insulin-treated cells. No reaction was observed in matrices obtained from non-insulin-treated (control) cells. Further biochemical analysis revealed that matrix-associated insulin could be solubilized with 1% sodium dodecyl sulfate (SDS) or in the presence of high urea concentrations. Gel filtration analysis of urea-solubilized matrix material revealed the presence of apparently intact [125I]insulin and a higher molecular weight peak. It is hypothesized that the latter may represent a tightly associated complex of insulin with some matrix protein(s).
Thompson
K A
KA
Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Soler
A P
AP
Smith
R M
RM
Jarett
L
L
D000818:Animals; D002850:Chromatography, Gel; D007150:Immunohistochemistry; D007328:Insulin / Q000032:analysis* / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000032:analysis*; D008854:Microscopy, Electron; D015530:Nuclear Matrix / Q000032:analysis* / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D014407:Tumor Cells, Cultured
D007328:Insulin