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Insulin Receptor Substrate-1 Is Present in Hepatocyte Nuclei from Intact Rats

Division of Pediatric Endocrinology and Metabolism, Rhode Island Hospital and Brown University, Providence, Rhode Island 02903

Address all correspondence and requests for reprints to: Dr. Philip A. Gruppuso, Department of Pediatrics, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02903. E-mail: Philip_Gruppuso{at}brown.edu.

	Abstract

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Insulin receptor substrate-1 (IRS-1), the primary substrate for the insulin receptor tyrosine kinase in most cells and tissues, is a key component of the insulin signaling network. Numerous studies have documented the trafficking of IRS-1 from the cell membrane to intracellular, extranuclear compartments. During the course of our previous studies aimed at defining the ontogeny of insulin signaling in the rat, Western immunoblotting showed minimal IRS-1 content in late gestation fetal liver. Immunohistochemical analyses, aimed at corroborating these Western immunoblotting results, showed hepatocyte nuclear staining for IRS-1 in adult liver but not fetal liver. Further analysis of fixed tissue as well as immunofluorescent staining of liver cryosections confirmed the localization of IRS-1 to the nuclear matrix and nucleoli of adult hepatocytes within intact liver. Tissue fractionation and Western immunoblotting also showed nuclear localization of IRS-1, with this fraction accounting for approximately 25% of total liver IRS-1. Administration of insulin to intact animals did not stimulate nuclear translocation of hepatic IRS-1 or the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, insulin did activate IRS-1-associated phosphatidylinositol-3 kinase in nuclear extracts. Our results indicate that insulin signaling, which terminates in an array of nuclear events, may originate at the immediate postreceptor level with IRS-1 activation within the nucleus of normal hepatocytes.

	Introduction

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THE ROLE OF insulin in the regulation of hepatic growth and metabolism in many species is well established. Studies on intact animals, including those that have employed the model of liver regeneration after partial hepatectomy in the rat, have demonstrated a role for insulin as an adjunct factor in promoting hepatocyte proliferation (1). Insulin is a key regulator of mechanisms that terminate at the ribosome, thereby playing a direct role in the control of protein synthesis (2). Progress in elucidating the mechanisms for insulin-mediated transcriptional regulation supports a direct role for insulin in the control of hepatic glucose production (3). More compelling evidence for the direct hepatic effects of insulin comes from studies on a recently developed mouse model in which hepatic insulin signaling is abolished through tissue-specific, targeted deletion of hepatocyte insulin receptors (4). These animals exhibit systemic insulin resistance, glucose intolerance, and a failure of insulin to suppress hepatic glucose production and to regulate hepatic gene expressions.

The current model for insulin signal transduction, which is supported by a voluminous scientific literature, states that the binding of insulin to its receptor activates the insulin receptor tyrosine kinase. While the receptor kinase can phosphorylate a spectrum of cellular proteins, members of the insulin receptor substrate (IRS) family are critical to the propagation of the insulin signal through multiple downstream pathways (5). These proteins function as docking sites for numerous regulatory and adapter proteins. The first IRS protein that was identified and characterized, IRS-1 (6), is a cytosolic protein that contains 21 putative tyrosine phosphorylation sites. Tyrosine phosphorylation of IRS-1 can mediate protein-protein interactions with a spectrum of Src homology-2 domain-containing proteins. These interactions account for signaling though the phosphatidylinositol 3kinase (PI3K) pathway, the ERK pathway, and others (2).

A variety of approaches have been used to characterize the intracellular trafficking of IRS-1 following activation of the insulin receptor kinase. Elegant studies by Bergeron, Posner, and co-workers (7) showed that insulin administration to intact rats promotes the internalization of insulin receptor that leads to receptor-mediated IRS-1 phosphorylation in the endosomal compartment. These investigators showed that a number of downstream signaling events, including the activation of PI3K and Akt, also take place in endosomes (8), but they did not disclose evidence for the nuclear localization of IRS-1-mediated signaling events. Other studies have localized activated IRS-1 to intracellular membranes in primary rat adipocytes (9) and 3T3-L1 adipocytes (10) but not to the nuclei of these cells. CHO cells that overexpress rat IRS-1 do not show nuclear localization of the transfected protein (11). Several years ago, it was reported that insulin could induce the nuclear translocation of PI3K in HepG2 cells and that the association of PI3K with nuclear IRS-1 mediated this translocation (12). This appears to represent the only previous report of IRS-1 localizing to the nucleus.

Our laboratory has been focused on the ontogeny of the insulin signaling network in developing liver. We recently reported the unexpected observation that insulin-mediated activation of the ribosomal protein S6 kinases, S6K1, and S6K2, is markedly attenuated in the fetal liver (13). This finding was consistent with our previous observation that fetal liver contains a very low level of immunoreactive IRS-1 (14). When we proceeded to use immunohistochemical analysis to assess differences in IRS-1 cellular and subcellular localization during liver development, we made an unexpected observation that IRS-1 is present in nuclei of hepatocytes in the intact rat.

	Materials and Methods

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Animals
Studies were carried out using Sprague Dawley rats obtained from Charles River Laboratories, Inc. (Wilmington, MA). Fetal rats were obtained on embryonic d 19 (E19) by performing cesarean section on timed pregnant rats under pentobarbital anesthesia (50 mg/kg by ip injection). Adult male rats weighing between 125 and 175 g were similarly anesthetized before they were killed. Where noted, insulin signaling was activated in vivo by the ip administration of porcine insulin (Elanco, Indianapolis, IN) at a dose of 2.5 µg/g body weight.

Immunohistochemistry and immunofluorescence
For immunostaining analyses, as well as for the immunoprecipitation and Western immunoblotting described below, we used rabbit polyclonal antibodies directed toward either the carboxy terminus or the pleckstrin homology domains of rat IRS-1, and the p85 subunit of PI3K (Upstate Biotechnology, Inc., Lake Placid, NY).

For immunohistochemistry, liver was fixed in 10% formalin. Sections (5 µm) were stained by incubating with the above primary antibodies overnight at 4 C. After washing, secondary antibody (goat antirabbit IgG; Pierce Chemical Co., Rockford, IL) was applied for 30 min at room temperature. Staining was accomplished using the Vectastain ABC method and DAB substrate kit (both from Vector Laboratories, Burlingame, CA) following an avidin/biotin blocking step. Where noted, peroxidase-conjugated secondary antibody was used as an alternative, in which case endogenous peroxidase activity was quenched before staining.

Cryosections (7 µm) were analyzed by immunofluorescence. Following methanol fixation, sections were incubated with the primary antibodies described above for 75 min at room temperature. Detection employed fluorescein-conjugated donkey antirabbit antibodies (Pierce Chemical Co.). For both immunohistochemistry and immunofluorescence, omission of primary antibody was used as a negative control.

Preparation of nuclear and soluble fractions
Between 0.5 and 1 g of frozen liver was placed in a Teflon/glass homogenizer containing 5 ml of ice-cold buffer A1 [15 mM HEPES, pH 7.5; 300 mM sucrose; 60 mM KCl; 15 mM NaCl; 2 mM EDTA; 0.5 mM EGTA; 14 mM 2-mercaptoethanol; 10 µg/ml aprotinin; 10 µg/ml leupeptin; and 34.4 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF)]. The tissue was homogenized with 5 strokes at 800 rpm then transferred to a 15-ml conical tube and allowed to settle for 5 min on ice. The top 4 ml were transferred to an empty 15-ml tube and centrifuged at 1000 x g for 5 min. The lower 1 ml was added to the supernatant from this centrifugation. This was then homogenized with an SDT Tissumizer (Tekmar Co., Cincinnati, OH), 10 sec x 2 on ice, then 10% Nonidet P-40 (NP-40) was added to a final concentration of 1%. After 20 min on ice, the homogenate was centrifuged at 40,000 x g for 20 min. The supernatant from this centrifugation was collected as the detergent-extracted, soluble fraction. The nuclear pellet from the first slow speed centrifugation was resuspended in 5 ml of buffer A2 (buffer A1 plus 0.5% NP-40) by trituration with a Pasteur pipette. The resuspended nuclei were then layered onto 5 ml of buffer B (15 mM HEPES, pH 7.5; 30% sucrose; 60 mM KCl; 15 mM NaCl; 2 mM EDTA; 0.5 mM EGTA; 14 mM 2-mercaptoethanol; 10 µg/ml aprotinin; 10 µg/ml leupeptin; and 34.4 µg/ml AEBSF) in a 15-ml conical tube, and centrifuged for 5 min at 1600 x g. The resulting pellet contained discreet nuclei, which could be counted using a hemocytometer. To prepare a nuclear extract, this pellet was resuspended in 1 ml of PI3K lysis buffer (50 mM HEPES, pH 7.5; 137 mM NaCl; 1 mM MgCl₂; 1 mM Ca₂Cl; 2 mM EDTA; 10 mM NaF; 10 mM Na pyrophosphate; 2 mM Na₃VO₄; 1% NP-40; 10% glycerol; 10 µg/ml aprotinin; 10 µM leupeptin; and 34.4 µg/ml AEBSF), sonicated 3 x 10 sec on ice, and centrifuged at 16,000 x g for 10 min at 4 C. The resulting supernatant represented nuclear extract. Both the soluble and nuclear fractions were separated into aliquots and frozen at -70 C until use. The protein concentration of the soluble and nuclear components was determined using the bicinchoninic acid method (Pierce Chemical Co.).

The purity of our nuclei preparations was confirmed by analyzing nuclear and detergent-extracted soluble fractions for two enzyme activities. Glucose-6-phosphate dehydrogenase was employed as a marker for the cytosolic fraction, whereas 5'-nucleotidase was used as a marker for contamination with membrane fraction. Both assays were performed using reagents obtained from Sigma (St. Louis, MO). The postnuclear soluble fraction showed a 5'-nucleotidase activity of 19 mU/mg protein, whereas the activity of the nuclear extract was at the lower limit of detection of the assay (2.5 mU/mg). Glucose-6-phosphate dehydrogenase activity on the soluble fraction was 65 mU/mg, whereas no activity was detected in the nuclear extract. These results were interpreted as indicating minimal contamination of our nuclei preparations with either cytosolic or membrane-associated components.

Western immunoblotting
Nuclear and soluble fractions were boiled in Laemmli sample buffer for 10 min. Eighty micrograms of protein were separated using a 7.5% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and analyzed by Western immunoblotting (15). These analyses employed the IRS-1 carboxy terminus antibodies and the p85 antibodies that were used for immunohistochemistry and immunofluorescence. Detection employed an enhanced chemiluminescent method (Amersham Biosciences, Piscataway, NJ).

Where noted, immunoblotting was performed on IRS-1 immunoprecipitates. For these studies, 200 µg of nuclear extract protein were combined with 2 µg of the carboxy terminus IRS-1 antibody. Following overnight incubation at 4 C, immunocomplexes were recovered using protein A-Sepharose beads (Amersham Biosciences). The immunoprecipitates were analyzed by Western immunoblotting with 4G10 anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology, Inc.) as well as the carboxy terminus IRS-1 and p85 antibodies noted above.

Western immunoblotting for total and phosphorylated (serine 235/236) ribosomal protein S6 was carried out as described previously (13).

Determination of PI3K activity
Nuclear fractions containing 200 µg protein were immunoprecipitated with anti-IRS-1 antibodies as described above. The next day, protein A-Sepharose was added to each immunoprecipitate, incubated for 3 h at 4 C and pelleted by centrifugation at 16,000 x g for 10 sec. Immune complexes were washed two times with wash buffer 1 (100 mM Tris-HCl, pH 7.5; and 500 mM LiCl), one time with wash buffer 2 (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 1 mM EDTA), and one time with reaction buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; 0.5 mM EGTA; 10 mM MgCl₂; and 300 µM adenosine). Sepharose-bound immune complexes were then incubated for 10 min at 30 C with 50 µl of reaction buffer containing 5 µg phosphatidylinositol, 5 µg phosphatidylserine, and 10 µM [-³²P]ATP (8 Ci/mmol). Reactions were quenched with HCl and extracted with methanol:chloroform (1:1). Labeled phospholipids were spotted onto aluminum-backed silica gel plates (Whatman Ltd., Maidstone, Kent, UK) to separate phosphatidylinositol monophosphates.

	Results

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In our initial studies, we performed side-by-side immunohistochemical analyses of E19 and adult liver sections for IRS-1 (Fig. 1, A and B). Consistent with our prior Western immunoblotting results (14), we observed minimal immunoreactivity above background in the E19 liver. In contrast, adult liver showed significant immunostaining. Throughout multiple adult liver sections, the most intense staining was nuclear. This finding was observed with both of the IRS-1 antibodies we used, which as noted above are directed toward separate regions of IRS-1. Nuclear staining was also present when detection employed either an avidin/biotin system (Fig. 1B) or peroxidase-conjugated secondary antibody (Fig. 1C). Finally, we were able to readily detect nuclear IRS-1 in immunofluorescent stains of adult liver cryosections (Fig. 1D).

View larger version (177K): [in this window] [in a new window]   Figure 1. Immunostaining for IRS-1. Sections of formalin-fixed liver derived from an E19 fetal rat (A) and from an adult rat (B) were stained using an antibody directed toward the carboxy terminus of rat IRS-1. The inset in panel B shows a portion of the same sample stained with IRS-1 antibody directed toward the pleckstrin homology domain. These analyses used an avidin/biotin system for detection. Immunohistochemistry of adult liver using primary antibody directed toward the IRS-1 carboxy terminus and peroxidase-conjugated secondary antibody is shown in panel C. Immunofluorescent staining of an adult liver cryosection with anti-IRS-1 carboxy terminus is shown in panel D. In all cases, staining was reproduced in multiple experiments and was dependent on the inclusion of primary antibody.

View larger version (96K): [in this window] [in a new window]   Figure 2. Immunohistochemistry for the p85 subunit of PI3K. Representative results are shown for E19 and adult liver.

View larger version (87K): [in this window] [in a new window]   Figure 3. Western immunoblotting for IRS-1 and the p85 subunit of PI3K in nuclear extracts and postnuclear soluble fractions prepared from E19 fetal (F) and adult (A) livers.

View larger version (30K): [in this window] [in a new window]   Figure 4. The effect of in vivo insulin administration on hepatic nuclear IRS-1 and p85 abundance. Results are shown for liver samples taken 0–30 min after administration of insulin by ip injection. The upper panel shows the results of direct immunoblotting of nuclear extracts for IRS-1. The lower panel shows parallel results for p85. The graphs represent densitometric analysis of three separate experiments. Data, expressed as percent of maximum level for each experiment, are shown as mean + SD. The insets show representative Western blots.

View larger version (34K): [in this window] [in a new window]   Figure 5. The effect of in vivo insulin administration on hepatic nuclear IRS-1 and PI3K activation. A, Liver nuclear extracts from animals killed 0–30 min after ip insulin injection were subjected to immunoprecipitation with anti-IRS-1. The immunoprecipitates were analyzed by immunoblotting for total IRS-1, antiphosphotyrosine (PY), and the p85 subunit of PI3K. Similar results were obtained in two additional experiments. B, Triplicate samples per time point were used to prepare nuclear extracts for IRS-1-associated PI3K activity. Data are shown as mean + 1 SD. The inset shows a representative autoradiogram from a PI3K assay.

View larger version (83K): [in this window] [in a new window]   Figure 6. Immunofluorescence for IRS-1 and p85. Cryosections were prepared from liver obtained before (Control) and after insulin administration. Animals were studied from 0.5–30 min after insulin. The 20-min result shown in this figure was indistinguishable from results obtained at all other time points. IRS-1 was detected using antibodies directed toward the carboxy terminus.

View larger version (47K): [in this window] [in a new window]   Figure 7. Hepatic postnuclear (soluble) and nuclear S6 phosphorylation and abundance following insulin administration to intact animals. Samples were subjected to direct immunoblotting using antibodies directed toward phosphorylated and total ribosomal protein S6.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The generally accepted model for insulin action places initial insulin signaling events that involve IRS-1 outside the nucleus. Receptors localized to the endosomal compartment have been implicated as being involved in key signaling events in the liver of intact rats (8). However, a substantial body of work supports the possibility that insulin signaling is initiated at or within the nucleus. It is well established that insulin can translocate to the nucleus of a variety of cells, including cells of hepatic origin (17). Moreover, direct introduction of insulin into the nuclei of H35 hepatoma cells can stimulate the induction of immediate-early genes in a manner that is independent of cell surface insulin receptors (18). Another potential mechanism for propagation of the insulin signal to the nucleus involves the translocation of insulin receptors from the cell surface (19). Finally, a recent report by Ni et al. (20) defines a mechanism by which the intracellular tyrosine kinase domain of the ErbB-4 receptor undergoes regulated intramembrane proteolysis and translocation to the nucleus. Such a mechanism might provide an alternative pathway for insulin signaling to reach the nucleus.

To assess the function of nuclear IRS-1, we examined the activation of PI3K in nuclear extracts. Upon finding that insulin does indeed activate nuclear IRS-1-associated PI3K, we attempted to examine downstream signaling by using phosphorylation of ribosomal protein S6 as an outcome measure. Our results showed insulin-mediated nuclear S6 phosphorylation, but they also disclosed a difficulty in interpreting such data. In addition to inducing the appearance of phosphorylated S6 in liver nuclear extracts, insulin also promoted an increase in total nuclear S6. This raises the possibility that any or all insulin-mediated signaling events in liver nuclei result from translocation of signaling molecules into the nucleus. This is a well-established phenomenon for a number of signaling kinases that are activated in response to insulin, including the mammalian target of rapamycin, mTOR (21), Akt (22), and ribosomal protein S6 kinases (23, 24). In fact, our findings point out the possibility that the appearance of tyrosine phosphorylated IRS-1 in the nucleus after insulin stimulation may be a result of the selective translocation of phosphorylated IRS-1 rather than the phosphorylation of IRS-1 that already resides in the nucleus. Such a mechanism is not ruled out by the apparent absence of a detectable shift in the total IRS-1 pool. These caveats notwithstanding, our data indicate that the most proximal postreceptor step, tyrosine phosphorylation of IRS-1, can be activated in the liver nuclei of intact rats by the administration of insulin.

	Acknowledgments

 
We thank Ms. Theresa Bienieki for providing assistance with the animal portion of the studies and with the immunohistochemical and immunofluorescent analyses.

	Footnotes

 
This work was supported by NIH Grants HD-24455, HD-11343, and HD-35831; and by the Rhode Island Hospital Department of Pediatrics Research Endowment.

Abbreviations: AEBSF, 4-(2-Aminoethyl)-benzenesulfonyl fluoride; E19, embryonic d 19; IRS, insulin receptor substrate; NP-40, Nonidet P-40; PI3K, phosphatidylinositol 3-kinase; S6K, ribosomal protein S6 kinase.

Received March 19, 2002.

Accepted for publication July 3, 2002.

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