Insulin induces rapid accumulation of insulin receptors and increases tyrosine kinase activity in the nucleus of cultured adipocytes.
To better understand the mechanism by which insulin exerts effects on events at the cell nucleus, we have studied insulin receptors and tyrosine kinase activity in nuclei isolated by sucrose density gradient centrifugation following insulin treatment of differentiated 3T3-F442A cells. Insulin stimulated nuclear accumulation of insulin receptors by approximately threefold at 5 min. The half-maximal effect was observed with 1-10 nM insulin. Following insulin treatment, phosphotyrosine content associated with the nuclear insulin receptor was also increased by twofold at 5 min with a similar insulin concentration dependency. These nuclear insulin receptors differ from the membrane-associated insulin receptors in that they were not efficiently solubilized with 1% Triton X-100. During the same period of time, insulin stimulated nuclear tyrosine kinase activity toward the exogenous substrate poly Glu4:Tyr1 tenfold in a time-dependent manner reaching a maximum at 30 min. The insulin receptor substrate protein 1 (IRS-1) could not be detected in the nucleus by immunoblotting. However, a nuclear protein with M(r) approximately 220 kDa was tyrosine phosphorylated, and insulin further stimulated this process threefold > 30 mins. Surface labeling was performed to determine if the nuclear insulin receptors would emerge from the plasma membrane fraction. Using 125I-BPA-insulin with intact cells, the intensity of nuclear insulin receptor labeling was negligible and not increased throughout 30 min incubation at 37 degrees C. In contrast, there was an increase in labeled receptors in the microsomal fraction following insulin treatment. Taken together, these results indicate that insulin rapidly increases nuclear insulin receptor appearance and activates nuclear tyrosine kinase activity. The insulin-induced accumulation of nuclear insulin receptors cannot be accounted for by internalization of surface membrane receptors. These effects of insulin may play an important role in action of the hormone at the nuclear level.
Kim
S J
SJ
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
Kahn
C R
CR
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000648:ultrastructure; D002467:Cell Nucleus / Q000737:chemistry* / Q000201:enzymology* / Q000648:ultrastructure; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D011972:Receptor, Insulin / Q000032:analysis* / Q000378:metabolism*; D013997:Time Factors
D007328:Insulin; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin