Nuclear and cytosolic Ca2+ signals were monitored during insulin (10-nM) stimulation. InsP3-Buffer-NLS and InsP3-Buffer-NES were correctly localized in the nucleus and in the cytosol, respectively (Fig. 2A). In control cells, insulin-induced Ca2+ signals occurred in the nucleus and in the cytosol. However, the Ca2+ increase occurred first in the nucleus (Fig. 2A,B). Both nuclear and cytosolic Ca2+ signals were nearly eliminated by buffering InsP3 in the nucleus (Fig. 2A,C,E); nuclear Ca2+ signals were not affected in

the presence of the cytosolic InsP3 buffer, whereas cytosolic Ca2+ signals had a minimal decrease (Fig. 2A,D,E). These Sirolimus results are similar to previous findings in SkHep-1 cells.[11] Collectively, these observations demonstrate that insulin promotes IR translocation to the nucleus and initiation of Ca2+ signals dependent on nuclear InsP3. Insulin regulates viability, growth, and Bortezomib concentration proliferation of primary hepatocytes and hepatoma cell lines,[4, 27] and nuclear, rather than cytosolic,

Ca2+ is required for cell proliferation.[16] To verify whether nuclear InsP3 is the upstream regulator of insulin-induced cell proliferation, SkHep-1 cells were synchronized in G0 by serum withdrawal, transfected with InsP3-Buffer-NLS, and assayed for BrdU incorporation. Insulin, 10% FBS, and HGF each induced significant increases in BrdU uptake, when compared to unstimulated control cells, as expected. However, BrdU uptake was reduced in cells expressing InsP3-Buffer-NLS, relative to control cells treated with insulin. Nuclear InsP3-buffered cells treated with insulin also had significantly many smaller BrdU uptake than control cells stimulated with insulin. BrdU uptake in InsP3-Buffer-NLS cells stimulated with insulin was not significantly higher than in untreated InsP3-Buffer-NLS cells (Fig. 2F). Together, these results indicate that formation of InsP3 in the nucleus is required for insulin-induced cell proliferation.

Upon insulin stimulation, the IR undergoes endocytosis through the classic clathrin (cla)-dependent pathway, such as does other RTKs.[28] However, a subpopulation of IRs on the PM is associated with caveolin (cav)-enriched membrane domains.[29] To determine whether cla and/or cav are necessary to mediate IR translocation from the plasma membrane to the nucleus, we used specific siRNAs that allowed a knockdown of 97% in both cla and cav expression, compared to scrambled siRNA-transfected cells (Fig. 3A-D). Immunoblottings of non-nuclear and nuclear fractions showed that silencing of cav caused a decrease in nuclear IR by 46.5%, when compared to scrambled siRNA-transfected cells stimulated with 10 nM of insulin. Silencing of cla caused a 24.7% decrease in nuclear IR, as compared to scrambled siRNA-transfected cells stimulated with insulin (10 nM), which was marginally significant (P = 0.08). Furthermore, simultaneous silencing of both proteins had an additive effect, causing a decrease in nuclear IR by 65.