The insulin receptor translocates to the nucleus to regulate cell proliferation in liver

Abstract

Insulin's metabolic effects in the liver are widely appreciated, but insulin's ability to act as a hepatic mitogen is less well understood. Because the insulin receptor (IR) can traffic to the nucleus, and Ca(2+) signals within the nucleus regulate cell proliferation, we investigated whether insulin's mitogenic effects result from activation of Ca(2+)-signaling pathways by IRs within the nucleus. Insulin-induced increases in Ca(2+) and cell proliferation depended upon clathrin- and caveolin-dependent translocation of the IR to the nucleus, as well as upon formation of inositol 1,4,5,-trisphosphate (InsP3) in the nucleus, whereas insulin's metabolic effects did not depend on either of these events. Moreover, liver regeneration after partial hepatectomy also depended upon the formation of InsP3 in the nucleus, but not the cytosol, whereas hepatic glucose metabolism was not affected by buffering InsP3 in the nucleus.

Conclusion: These findings provide evidence that insulin's mitogenic effects are mediated by a subpopulation of IRs that traffic to the nucleus to locally activate InsP3 -dependent Ca(2+)-signaling pathways. The steps along this signaling pathway reveal a number of potential targets for therapeutic modulation of liver growth in health and disease.

Figure 1. The insulin receptor translocates from the plasma membrane to the nucleus upon stimulation with insulin in SkHep-1 cells

A. Confocal immunofluorescence images of the insulin receptor before and after insulin stimulation (10 nM) for the indicated times in SkHep-1 cells. The insulin receptor was labeled in green and the nucleus was stained with dapi (blue). B. Quantification of nuclear fluorescence intensity in images shown in (A) (**p<0.001, n=3 experiments). Scale bar=10 μm. Values are mean ± S.D. Data were analyzed by one-way ANOVA followed by Bonferroni post-tests. C. Immunoblots of the insulin receptor (IR) in non-nuclear and nuclear fractions of SkHep-1 cells before and after insulin stimulation (10 nM). Histone H3 and Lamin B1were used as purity controls for the nuclear fractions and Na⁺/K⁺ ATPase, GAPDH and α-tubulin for the non-nuclear fractions(n=3 experiments). D. Immunoblots of IR in biotinylated and fractioned SkHep-1 cells subjected to streptavidin pull-down before and after insulin (10 nM) stimulation. Na⁺/K⁺ ATPase was used to verify the purity of the non-nuclear and nuclear fractions as well as the biotinylated fractions; n=3 experiments.

Figure 2. Insulin-induced Ca²⁺ signals and cell proliferation depend on nuclear InsP₃

A. Confocal images of SkHep-1 cells loaded with Fluo-4/AM (6 µM) and stimulated with insulin (10 nM). Control, InsP₃-Buffer-NLS and InsP₃-Buffer-NES infected cells were analyzed. Right bottom panels show correct subcellular localization (red) of each adenoviral construct. Images were pseudocolored according to the scale shown at the bottom. Scale bar=10 μm. Graphical representation of the fluorescence increase in the nucleus (red traces) and cytosol (blue traces) of control (B), InsP₃-Buffer-NLS (C) and InsP₃-Buffer-NES (D) cells. E. Summary of InsP₃ buffer studies. Values are mean ± S.D of the peak Fluo-4 fluorescence acquired during the observation period (expressed as % of baseline) and include the response from 55 nontransfected cells, 55 cells expressing InsP₃-Buffer-NLS, and 44 cells expressing InsP₃-Buffer-NES (*p < 0.05, ***p<0.0001). F. BrdU uptake in control and InsP₃-Buffer-NLS infected cells before and after insulin (10 nM, 10 min) stimulation. 10% serum and HGF were used as additional positive controls for cell proliferation. (*p<0.05, **p<0.01, ***p<0.0001, n=3 experiments). Values are mean ± S.D Data were analyzed by one-way ANOVA followed by Bonferroni post-tests.

Figure 3. IR translocates to the nucleus in a clathrin heavy chain and caveolin-1-dependent manner to regulate insulin-induced cell proliferation

Immunoblots of total SkHep-1 cell lysates (A,C) and densitometric analysis (B,D) after treatment with 5 nM scrambled, clathrin heavy chain (A,B) or caveolin-1 (C,D) siRNAs. (**p<0.001, ***p<0.0001; n=3 experiments). Values are mean ± S.D. E. Immunoblots of non-nuclear and nuclear SkHep-1 fractions of cells stimulated with insulin (10 nM) under scrambled, clathrin heavy chain (cla), caveolin-1 (cav), and cla/cav silencing conditions. F. Densitometric analysis of (E). (**p<0.001, ***p<0.0001, n=5 experiments). G. BrdU uptake in cells subjected to experimental conditions in (E). (*p<0.05, ***p<0.0001; n=3 experiments). Values are mean ± S.D. Data were analyzed by one-way ANOVA followed by Bonferroni post-tests.

Figure 3. IR translocates to the nucleus in a clathrin heavy chain and caveolin-1-dependent manner to regulate insulin-induced cell proliferation

Immunoblots of total SkHep-1 cell lysates (A,C) and densitometric analysis (B,D) after treatment with 5 nM scrambled, clathrin heavy chain (A,B) or caveolin-1 (C,D) siRNAs. (**p<0.001, ***p<0.0001; n=3 experiments). Values are mean ± S.D. E. Immunoblots of non-nuclear and nuclear SkHep-1 fractions of cells stimulated with insulin (10 nM) under scrambled, clathrin heavy chain (cla), caveolin-1 (cav), and cla/cav silencing conditions. F. Densitometric analysis of (E). (**p<0.001, ***p<0.0001, n=5 experiments). G. BrdU uptake in cells subjected to experimental conditions in (E). (*p<0.05, ***p<0.0001; n=3 experiments). Values are mean ± S.D. Data were analyzed by one-way ANOVA followed by Bonferroni post-tests.

Figure 4. IR translocation to the nucleus mediates insulin-induced Ca²⁺ signals but not Akt activation

A. Confocal images of SkHep-1 cells loaded with Fluo-4/AM (6 µM) and stimulated with insulin (10 nM) under scrambled (sc), clathrin heavy chain (cla), caveolin-1 (cav) and cla/cav silencing conditions. Images were pseudocolored according to the scale shown at the bottom. Scale bar=10 μm. B. Graphical representation of the fluorescence increase in the nucleus (red traces) and cytosol (blue traces). C. Summary of siRNA studies. Values are mean ± S.D of the peak Fluo-4 fluorescence acquired during the observation period (expressed as % of baseline) and include the response from 12 scrambled siRNA cells, 16 cla siRNA cells, 16 cav siRNA cells and 30 cla/cav siRNA cells (***p<0.0001). (D) Immunoblots of total SkHep-1 cell lysates. (E) Densitometric analysis of Akt phosphorylation in cells stimulated with insulin (10 nM) under scrambled, clathrin heavy chain (cla), caveolin-1 (cav), and cla/cav silencing conditions. (p<0.0001; n=3 experiments). Values are mean ± S.D. Data were analyzed by one-way ANOVA followed by Bonferroni post-tests.

Figure 5. Liver regeneration but not insulin’s metabolic effects depend on nuclear InsP₃

A. Immunohistochemistry images of liver sections from sham, PH, InsP₃-Buffer-NLS and InsP₃-Buffer-NES animals 48 hrs after PH and 72 hrs after infection and treatment with BrdU. BrdU staining in the nucleus allows the identification of proliferating cells in each group. Inset in sham panel represents the negative control of the technique. The near-100% efficiency of infection is highlighted in the insert in the InsP3-Buffer-NLS panel, in which a red nuclear stain is observed due to the mRFP tag. Scale bar=50 μm. Objective lens: 10x. B. BrdU uptake in control non-hepatectomized (sham), control hepatectomized (PH) animals and PH animals (2 days after PH) infected with adenoviral forms of either InsP₃-Buffer-NLS or InsP₃-Buffer-NES for 72 hrs (*p<0.05, **p<0.01; n=4 animals per condition).C. Liver/body weight ratio in animals subjected to experimental conditions in A (**p<0.001, ***p<0.0001; n=4 animals per condition). Values are mean ± S.D. D. Immunohistochemistry images rarely detect the IR in the hepatocyte nucleus in sham (top row) but frequently detect it in PH animals (bottom row) 24 hrs after PH. Arrows: negatively stained nuclei; arrowheads: IR positive nuclei; insert: negative control; Scale Bar=40 µm. Objective lens: 100x. (E) Quantification of blood glucose levels and (F) liver glycogen content in control animals, and in animals infected with either InsP₃-Buffer-NLS or InsP₃-Buffer-NES. (*p<0.05, **p<0.001; n=4 animals per condition). Values are mean ± S.D. Data were analyzed by one-way ANOVA followed by Bonferroni post-tests.