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Endocrinology Vol. 142, No. 3 1041-1049
Copyright © 2001 by The Endocrine Society

ARTICLES

Physiological Concentrations of Insulin Promote Binding of Nuclear Proteins to the Insulin-Like Growth Factor I Gene1

Elizabeth N. Kaytor, Juan Li Zhu, Ching-I Pao and Lawrence S. Phillips

Emory University School of Medicine, Atlanta, Georgia 30322

Address all correspondence and requests for reprints to: Lawrence S. Phillips, M.D., Division of Endocrinology, Emory University School of Medicine, 1639 Pierce Drive, 1301 WMRB, Atlanta, Georgia 30322. E-mail: medlsp{at}emory.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Limitations in understanding the mechanism of transcriptional regulation by insulin are due in part to lack of models in which there is insulin-responsive binding of nuclear factors to critical promoter regions. The insulin-like growth factor I (IGF-I) gene responds to diabetes status via a footprinted sequence, region V, which contains an AT-rich element and a GC-rich site. We tested the hypothesis that insulin regulates nuclear factor binding to the AT-rich site. Gel shift analysis with liver nuclear extracts and a region V probe showed binding of Sp1, Sp3, and B1, which persisted despite the presence of antibodies against Sp1 and Sp3. B1 was detected by a probe mutated in the GC-rich site (VmSp1), but not by a probe mutated at the AT-rich site (VmAT). We then asked whether B1 was responsive to insulin. For both region V and VmSp1 probes, nuclear extracts from normal rat hepatocytes, H4IIE cells, and CHO-IR cells exposed to 10-6 M insulin exhibited an increase in binding, designated insulin-responsive binding protein (IRBP); IRBP comigrated with B1 from liver extracts. IRBP binding to region V was competed by VmSp1, but not by VmAT, indicating specific interactions with the AT-rich sequence; insulin response elements from other genes also failed to compete. After addition of insulin, IRBP began to increase by 1 h and rose further at 24 h, suggesting involvement of both posttranslational and transcriptional mechanisms. IRBP responded to as little as 10-10 M insulin, indicating physiological relevance. Induction of IRBP was blunted by the phosphatidylinositol 3'-kinase inhibitor LY294002, whereas other signal transduction inhibitors had little effect. IRBP interacts with an important sequence in the IGF-I gene and may participate in the metabolic regulation of IGF-I expression. As most insulin-responsive genes do not exhibit insulin-responsive nuclear factor binding, further studies of IRBP may also contribute to understanding of the mechanism of insulin action on gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AS THE MAJOR anabolic hormone of the fed state, insulin alters protein, fat, and carbohydrate metabolism to limit tissue breakdown and promote utilization and storage of exogenous nutrients that are in excess of energy needs. The actions of insulin include both rapid effects related to glucose disposition and slower effects on a wide variety of body processes. The signal transduction pathways activated by insulin lead to regulated transcription of many genes, and insulin-responsive sequences have been identified for glucokinase (GK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fatty acid synthase (FAS), insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), IGFBP-3, and phosphoenolpyruvate carboxykinase (PEPCK), among others (1, 2, 3, 4, 5, 6, 7). However, although such sequences are binding sites for transcription factors such as hepatocyte nuclear factor-3 (HNF-3), high mobility group factor I (HMGI), Forkhead/winged helix protein, and sterol regulatory element binding protein (SREBP) (8, 9, 10, 11, 12), understanding of the transcriptional effects of insulin has been limited by the paucity of models in which binding of these or other nuclear factors is insulin responsive.

IGF-I is similar to insulin both structurally and functionally (13), with broad effects on growth and development and an anabolic role in metabolic regulation (reviewed in Ref. 14). The IGF-I gene is expressed in diverse tissues, but the major source of circulating (endocrine) IGF-I is the liver (15). The physiological importance of endocrine IGF-I has been shown by liver-specific IGF-I knockout studies in which circulating IGF-I fell by 75%, and growth was maintained only by a 6-fold increase in GH production (16). Circulating IGF-I levels are reduced in rats with diabetes mellitus, but are restored by insulin treatment (17, 18). Much of the control of IGF-I occurs at the messenger RNA level, and IGF-I expression levels and rates of gene transcription in nuclear run-on assays are diabetes responsive in animals (18) and insulin responsive in cultured hepatocytes (19).

The IGF-I gene contains six exons (20, 21), and most transcripts initiate in exon 1, which includes two major and two minor start sites (22, 23). In the liver, transcripts are generally initiated at sites two and three (IS2 and IS3; Fig. 1AGo). A number of transcription factors are known to activate IGF-I expression through binding sites in exon 1, including CCAAT enhancer-binding protein {alpha} (C/EBP{alpha}), HNF-1{alpha}, HNF-3{beta}, and C/EBP{delta} (24, 25, 26, 27, 28). However, such factors are not known to play a role in IGF-I regulation by diabetes and insulin status. In vitro transcription studies have shown that although sequences upstream from IS3 are necessary for maximal expression, elements within several hundred bases downstream from IS3 are required for diabetes-mediated regulation (29). Deoxyribonuclease I footprinting experiments have identified six protected regions within approximately 200 bp downstream of IS3 (Fig. 1AGo) (29). In vitro transcription studies using IGF-I genomic templates demonstrated that the presence of region V is necessary for regulation by diabetes status, as deletion of this region abolished the differential expression previously observed with liver nuclear extracts from normal and diabetic animals (29). Region V contains a GC-rich motif, and we have found that Sp1 may play a role in IGF-I gene expression (30). However, Sp1 is ubiquitous and contributes to the expression of numerous genes that are regulated by a diverse range of stimuli (31, 32, 33), and Sp1 abundance is not known to be responsive to diabetes or insulin status. Thus, additional transcription factors must be involved in regulating IGF-I expression in response to insulin.



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  		Figure 1. Location of footprinted sequences and transcription initiation sites in IGF-I exon 1. A, The first exon of the IGF-I gene contains two minor and two major transcription initiation sites; the major sites (IS2 and IS3) are indicated by the larger arrows. Footprinted regions downstream from IS3 are shown by Roman numerals. B, The sequence of the region V footprint, with the AT-rich and GC-rich elements boxed, and the sequences of the VmAT and VmSp1 oligonucleotides with the 4 bp mutations underlined and in lower case.

 
The IGF-I region V sequence also contains an AT-rich element, and uracil interference studies have demonstrated interactions between nuclear proteins and bases within the AT-rich sequence (34). Additionally, gel shift experiments have shown that oligonucleotides mutated in the AT-rich site compete poorly for nuclear factor binding to wild-type region V probes, indicating the involvement of bases within the AT-rich element (34). In view of the importance of the region V sequence for control of IGF-I expression by diabetes status, we hypothesized that an insulin-responsive nuclear factor(s) interacts with region V through the AT-rich motif. We now describe the recognition by region V of an insulin-responsive binding protein, designated IRBP, that interacts specifically through the AT-rich element.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
PD98059 and rapamycin were purchased from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma (St. Louis, MO). Mediatech Cellgro cell culture medium was obtained through Fisher (Pittsburgh, PA), and FBS and antibiotics were purchased from Life Technologies, Inc. (Gaithersburg, MD). [{gamma}-32P]ATP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Oligonucleotides corresponding to IGF-I region V and insulin response element (IRE) sequences from other genes were obtained from Life Technologies, Inc. Antibodies against Sp1 and Sp3 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Cell culture
Rat hepatocytes were prepared as previously described (35) and maintained in DMEM/Ham’s F-12 medium containing 10% FBS. Chinese hamster ovary cells stably transfected with the insulin receptor (CHO-IR) were provided by Drs. Barry Goldstein and Morris White and cultured in Ham’s F-12 medium supplemented with 10% FBS. Rat hepatoma cells (H4IIE) were obtained from American Type Culture Collection (Manassas, VA) and were maintained in DMEM containing 10% FBS. Recombinant human insulin was purchased from Life Technologies, Inc., and was dissolved in 10-4 M HCl before addition to cell cultures in serum-free medium. When included, LY294002 was used at 50 µM, PD98059 was used at 50 µM, and rapamycin was used at 200 nM.

Liver nuclear extract preparation
Male Sprague Dawley rats were obtained from Charles River Laboratories, Inc. (Raleigh, NC). Nuclear extracts from livers of normal rats were prepared using a modification of the method previously described (29, 36). Briefly, livers were homogenized in an anaerobic tissue processor in 2 M sucrose with 1% dry milk and filtered through cheesecloth, and nuclei were pelleted by centrifugation through a 2-M sucrose cushion. Nuclei were resuspended in storage buffer [20 mM Tris-Cl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 0.85 mM dithiothreitol (DTT), 0.125 mM phenylmethylsulfonylfluoride (PMSF), and 50% glycerol] and repelleted, followed by lysis in NUN buffer [1.1 M urea, 0.33 M NaCl, 1.1% Nonidet P-40, 27.5 mM HEPES (pH 7.6), 11% glycerol, 1 mM DTT, 1.1% aprotinin, 0.77 µg/ml leupeptin, and 0.77 µg/ml pepstatin A]. The Emory University animal care and use committee approved all animal use.

Nuclear extract preparation from cultured cells
Nuclear extracts were prepared from CHO-IR cells, H4IIE cells, and rat hepatocytes in primary culture using the method of Schreiber et al. (37). Briefly, cells were washed with PBS, scraped, pelleted, and resuspended in buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml aprotinin]. After swelling on ice, cells were lysed by the addition of Nonidet P-40. Nuclei were pelleted, resuspended in buffer B [20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml aprotinin], and lysed by vigorous shaking on a vortex mixer.

Gel mobility shift assays
Radiolabeled double stranded oligonucleotides (50,000 cpm/reaction) were incubated with 3–5 µg nuclear extract in 20 µl binding buffer containing 5% glycerol, 1 mM EDTA, 50 mM NaCl, 10 mM Tris-Cl (pH 7.5), 1 mM DTT, and 1 µg poly(dI-dC) (Roche Molecular Biochemicals, Indianapolis, IN) for 30 min at 25 C. For competition studies, unlabeled competitor oligonucleotides were added to the reaction mixture 30 min before the addition of radiolabeled probe. In antibody supershift analyses, Sp1 or Sp3 antibodies (1 µg) were added to the binding mixture and incubated on ice for 2 h before the addition of probe, at which point the incubation was carried out for 30 min at 25 C as usual. Reactions were subjected to electrophoresis on 4.5% polyacrylamide gels in TGE buffer (25 mM Tris, 190 mM glycine, and 1 mM EDTA) at 4 C at 150 V for 3 h, followed by autoradiography and quantitation by densitometry (Molecular Dynamics, Inc., Sunnyvale, CA). All experiments were repeated at least three times.

Statistics
Where appropriate, results are expressed as the mean ± SEM. Statistical significance was assessed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Interaction of region V with nuclear factors in rat liver extracts
IGF-I downstream footprint region V is located approximately 140 bp from IS3 and is a 24-bp sequence that contains a GC-rich element adjacent to an AT-rich site (Fig. 1AGo). We first conducted in vitro gel mobility shift experiments using normal rat liver nuclear extracts and a radiolabeled region V probe. As previously described (30), we observed three major complexes, designated B1, B2, and B3 (Fig. 2AGo, lane 2). Addition of antibodies against Sp1 and the closely related protein, Sp3, demonstrated that B2 was supershifted with anti-Sp1, whereas both B2 and B3 disappeared with anti-Sp3. However, B1 was largely unaffected by either antibody (Fig. 2AGo, lanes 3–5). These results indicate that a nuclear factor(s) distinct from Sp1 and Sp3 can bind to region V.



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  		Figure 2. Nuclear factor B1 interacts with region V through the AT-rich element. Normal rat liver nuclear extracts were used in gel shift assays with region V probes. A, Supershift analysis, in which extracts were preincubated with antibodies against Sp1 (lane 3), Sp3 (lane 4), or both (lane 5) before addition of wtV probe. Complexes B1, B2, and B3 are indicated, as are the supershifted and nonspecific complexes (SS and NS). B, Competition gel shift study in which extracts were incubated with a 100-fold molar excess of wtV (lane 2), VmAT (lane 3), or VmSp1 (lane 4) competitor DNA before addition of wtV probe. C, Extracts were incubated with either wtV or VmSp1 probe, as indicated.

 
To determine which portion of region V is recognized by B1, gel shift competition studies were carried out. Unlabeled oligonucleotide competitors corresponding to region V sequences that were mutated at either the GC-rich or AT-rich site (VmAT and VmSp1; Fig. 1BGo) were added at 100-fold molar excess to gel shift reactions containing rat liver nuclear extracts and a wild-type region V probe (wtV). As shown in Fig. 2BGo, the wtV sequence competed for binding of all three complexes, whereas VmSp1 strongly competed for B1, marginally competed for Sp3, which appears to have broader region V binding recognition than Sp1 (30), and did not affect Sp1 binding. Conversely, the VmAT sequence competed for B2 and B3 and failed to compete for B1, indicating that the B1 complex interacts with region V through the AT-rich sequence.

This specificity of B1 binding was supported by gel shift experiments using radiolabeled probes corresponding to wtV, VmAT, and VmSp1. Although nuclear extracts incubated with the wtV probe clearly resulted in three complexes, only B1 was detected with the VmSp1 probe (Fig. 2CGo). However, the VmAT probe completely prohibited formation of B1 (data not shown). Taken together, our data support the presence of a nuclear factor, B1, which can bind to IGF-I region V, is distinct from Sp1 and Sp3, and binds through the AT-rich element. We then attempted to determine whether B1 might contain insulin-responsive factor(s).

B1 comigrates with an insulin-responsive factor
The metabolic alterations that occur in diabetic compared with normal animals are complex, involving the integration of numerous physiological signals. To test the impact of insulin more specifically, we treated normal rat hepatocytes in primary culture with 10-11 or 10-6 M insulin for 48 h, at which point nuclear extracts were prepared. Gel shift experiments with the wtV probe revealed insulin-induced binding of one major complex (Fig. 3AGo). An insulin-responsive complex bound to region V was also observed with nuclear extracts from H4IIE hepatoma cells and CHO-IR cells overexpressing the insulin receptor (Fig. 3BGo). Although Western blots demonstrated the presence of Sp1 in nuclear extracts from each of these cell types (data not shown), the insulin-responsive complex is typically the only one observed with the experimental conditions used in the present studies. In control experiments, we failed to observe insulin-responsive binding to probes corresponding to footprint regions III and IV from the IGF-I gene (data not shown).



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  		Figure 3. Insulin treatment enhances binding to region V in nuclear extracts from cultured cells. A, Normal rat hepatocytes were placed in primary culture overnight and then treated with medium containing 10-11 or 10-6 M insulin for 48 h. Nuclear extracts were prepared and used in gel shift assays with the wtV probe. Insulin-responsive binding is indicated by the arrow. B, Nuclear extracts were prepared from H4IIE and CHO-IR cells treated with or without 10-6 M insulin for 24 h and used in gel shift studies as described above. C, Nuclear extracts from liver and CHO-IR cells were incubated with the wtV probe (lanes 1–3) or the VmSp1 probe (lanes 4–6). Locations of B1, B2, B3, and the insulin-responsive complex are indicated.

 
To determine whether B1 and the insulin-responsive complex detected in cellular extracts might contain the same protein components, they were compared directly in adjacent lanes of a gel shift experiment. Using region V probes, the insulin-responsive complex in CHO-IR extracts exhibited the same mobility as the B1 complex in liver nuclear extracts that binds to the wtV and VmSp1 probes (Fig. 3CGo). This result suggests that the B1 complex observed with liver nuclear extracts may be the insulin-responsive factor found in extracts from cultured cells, which we designate insulin-responsive binding protein (IRBP).

Binding specificity of IRBP
To further compare B1 and IRBP, gel shift competition studies were conducted with the wtV probe, CHO-IR cell nuclear extracts, and the three region V oligonucleotides (Fig. 1BGo). As we observed with liver extract complex B1, the wtV and VmSp1 competitors were able to compete for IRBP binding in both CHO-IR and hepatocyte nuclear extracts, whereas the VmAT oligonucleotide failed to compete (Fig. 4Go, A and B). This result is consistent with the observation that IRBP binding to region V persisted when the VmSp1 sequence was used as a probe, as in Fig. 3CGo. Therefore, the interaction of both B1 and IRBP with region V is independent of the Sp1 site and occurs specifically through the AT-rich element.



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  		Figure 4. IRBP binds to region V through the AT-rich element. A, Nuclear extracts from CHO-IR cells treated with or without 10-6 M insulin for 24 h, as indicated, were preincubated with a 100-fold molar excess of the wtV (lanes 3 and 4), VmAT (lanes 5 and 6), or VmSp1 (lanes 7 and 8) competitor before addition of wtV probe. B, Nuclear extracts from rat hepatocytes were prepared after 48 h of treatment with 10-11 or 10-6 M insulin, and used in competition gel shift studies as described above.

 
IRBP does not bind to other IREs
IREs have been identified for a number of insulin-responsive genes (Table 1Go). However, the sequences of these IREs differ significantly from the AT-rich element in IGF-I region V, and these IREs have generally not exhibited insulin-responsive nuclear factor binding (5, 7). We determined whether IRBP from CHO-IR nuclear extracts was recognized by other IREs in gel shift assays. Competition studies using the IREs from IGFBP-1, IGFBP-3, PEPCK, and FAS at 100-fold molar excess showed no effect on IRBP binding to the VmSp1 probe (Fig. 5AGo). Lack of competition was also found with oligonucleotides containing IREs from the GK, tyrosine aminotransferase, amylase, GAPDH, and PRL genes and several other upstream and downstream footprinted regions from the IGF-I gene (data not shown).


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  		Table 1. Insulin-responsive sequences and associated transcription factors

 


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  		Figure 5. IRBP does not interact with IREs from other genes. Nuclear extracts were prepared from CHO-IR cells treated with or without 10-6 M insulin for 24 h and used in gel shift studies. A, Extracts were preincubated with 100-fold molar excesses of unlabeled oligonucleotides corresponding to the IREs of the IGFBP-1, IGFBP-3, PEPCK, or FAS genes (lanes 5–8) or region V sequence (lanes 3 and 4) before addition of VmSp1 probe. B, The VmSp1, IGFBP-1, IGFBP-3, and PEPCK IRE sequences were used as probes for gel shift assays with CHO-IR extracts.

 
To further test the specificity of IRBP binding, IREs from the IGFBP-1, IGFBP-3, and PEPCK genes were radiolabeled to similar specific activities and used as probes in gel shift assays. As demonstrated in Fig. 5BGo, the IGF-I VmSp1 probe exhibited recognition of insulin-responsive IRBP binding with CHO-IR cell nuclear extracts, whereas the IGFBP-1, IGFBP-3, and PEPCK IRE probes showed no band similar to IRBP. Taken together, these data indicate that the IGF-I IRBP is distinct from the factors that bind to these other IREs.

Regulation of IRBP
We then undertook additional studies to characterize the regulation of IRBP interactions with region V. For time-course experiments, CHO-IR cells were exposed to 10-6 M insulin for 1–48 h before preparation of nuclear extracts. Gel shift assays were conducted with the VmSp1 probe, and IRBP binding was quantitated by densitometry. Binding was increased 2-fold after 1 h of insulin treatment and rose further to approximately 3-fold after 2 and 4 h, before decreasing to 2-fold after 6 h (Fig. 6Go). However, binding was increased 5-fold after 24 and 48 h of insulin treatment (P < 0.01 and P < 0.03, respectively).



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  		Figure 6. Time course of insulin induction of IRBP binding to region V. CHO-IR cells were treated with or without 10-6 M insulin for 1, 2, 4, 6, 24, and 48 h, and nuclear extracts were prepared at each time point. Gel shift studies were conducted with the VmSp1 probe. Autoradiography results were quantitated by densitometry; data shown are the mean ± SEM from three experiments.

 
Dose-response experiments were also conducted, using nuclear extracts from CHO-IR cells incubated for 24 h with insulin concentrations from 10-10–10-7 M. The lowest dose of insulin resulted in a 2-fold increase in binding (P < 0.02), which increased to 6-fold at 10-7 M (Fig. 7Go). Therefore, the induction of IRBP binding to IGF-I region V occurs at physiological concentrations of insulin (38).



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  		Figure 7. Physiological concentrations of insulin stimulate IRBP interactions with region V. CHO-IR cells were incubated with medium containing 10-10, 10-9, 10-8, or 10-7 M insulin for 24 h, at which point nuclear extracts were prepared for gel shift studies with the VmSp1 probe. Autoradiograms were quantitated by densitometry; data are the mean ± SEM from three experiments.

 
Finally, we explored the signal transduction pathways that might be responsible for the induction of IRBP binding. CHO-IR cells were treated with 10-6 M insulin for 24 h in the presence of LY294002, which inhibits phosphatidylinositol 3'-kinase (PI3-kinase) (39), PD98059, which inhibits mitogen-activated protein kinase kinase (40), and rapamycin, which inhibits p70S6 kinase (41). Control cells were treated with the dimethylsulfoxide solvent and demonstrated an average 2.5-fold increase in IRBP with addition of insulin. LY294002 treatment had no effect on IRBP under basal conditions, but blunted the increase in binding with insulin by 44% (P < 0.04; Fig. 8Go). In contrast, PD98059 and rapamycin had no significant effect on the ability of insulin to induce IRBP binding. These data suggest that the PI3-kinase pathway may be involved in the effect of insulin on IRBP.



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  		Figure 8. The PI3-kinase inhibitor LY294002 blocks insulin-mediated induction of IRBP. CHO-IR cells were treated with or without 10-6 M insulin for 24 h in the presence of 0.1% dimethylsulfoxide (control), 50 µM LY294002, 50 µM PD98059, or 200 nM rapamycin. Nuclear extracts were prepared and used in gel shift assays with VmSp1 probe. Autoradiography results were quantitated by densitometry. Data are the mean ± SEM from four experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate the presence of an insulin-responsive nuclear complex in cultured cells that interacts with the IGF-I region V sequence that is critical for transcriptional regulation by diabetes status (29). This complex, designated IRBP, comigrates with the B1 complex that is observed in gel shift assays using liver nuclear extracts and displays a similar pattern of binding site recognition. Additionally, IRBP interactions with region V are increased by physiological concentrations of insulin, indicating probable biological relevance.

Time-course studies revealed that IRBP interactions with region V were increased 2-fold after only 1 h, rose to 3-fold after 2 h, but fell to 2-fold at 6 h. We attempted to examine the effect of cycloheximide on IRBP induction over the shorter time courses (up to 2 h), but these studies were inconclusive due to variability of the responses at early time points. Interestingly, IRBP binding at 24 h was increased 5-fold, and despite the replacement of medium and insulin at the 24 h point did not rise further at 48 h. These findings suggest that control of IRBP may be biphasic, with posttranslational mechanisms at early time points and control at the messenger RNA level at later times. Other biological processes, such as insulin secretion (reviewed in Ref. 42), are also controlled at dual levels.

The addition of signal transduction inhibitors to cultured CHO-IR cells also provided an indication of possible mechanisms of control at the 24 h point. PD98059, which inhibits mitogen-activated protein kinase kinase, and rapamycin, which blocks p70S6 kinase, did not affect the ability of insulin to increase IRBP binding. In contrast, the PI3-kinase inhibitor LY294002 resulted in decreased binding after insulin treatment, with no effect on basal binding. This indicates that the PI3-kinase pathway may be involved in the induction of IRBP by insulin. Similar experiments conducted for the 1 and 2 h points were inconclusive due to greater variability of responses at these times. PI3-kinase has been implicated in the effects of insulin on glucose transport, glycogen accumulation, protein synthesis, and gene expression (reviewed in Ref. 43, 44, 45, 46, 47, 48). The hypothesis that PI3-kinase plays a major role in insulin action is also supported by the finding that type 2 diabetes results in reduced PI3-kinase activity in muscle (49). The action of PI3-kinase promotes the action of 3'-phosphoinositide-dependent kinase-1 (PDK1), which, in turn, activates a number of other kinases (reviewed in Refs. 50 and 51). Recent evidence supports the involvement of one such kinase, Akt/PKB, in insulin signaling (reviewed in Ref. (52), and it will be of interest to learn whether Akt or one of the other PDK1-dependent kinases mediates the effects of insulin on IRBP.

We have observed IRBP in extracts from both hepatic and nonhepatic cells, suggesting that IRBP is not likely to be a liver-specific factor. Based on the similar mobility shifts and parallel competition profiles with extracts of different origins, it seems likely that the same IRBP is present in different tissues. If this is the case, then IRBP could participate in insulin-mediated IGF-I responsiveness in both the liver and other tissues, whereas factors other than IRBP (e.g. HNF-1 and C/EBP (24, 25) could contribute to the high level of hepatic IGF-I expression.

The nature of the region V binding site may provide a clue to the structure of the IRBP protein(s). IGF-I genomic templates containing region V demonstrate increased transcriptional activity with nuclear extracts from normal compared with diabetic animals (29), indicating that factors binding to region V, such as IRBP, might be transcriptional activators. Furthermore, IRBP binding requires the presence of the AT-rich element, suggesting that IRBP may be a member of the homeodomain family of transcription factors. Homeodomain proteins contain a 60-amino acid element that includes a DNA-binding region, which recognizes a core 5'-ATTA-3' sequence such as that present in IGF-I region V. This group of transcription factors is involved in the control of development in higher organisms, and includes Drosophila factors such as Antennapedia, engrailed, and bicoid, and the mammalian Hox proteins (reviewed in Ref. 53). The cloning or purification of IRBP will be necessary to establish whether it truly is a homeodomain protein.

The AT-rich site in region V is immediately adjacent to a GC-rich site that closely resembles an Sp1 binding site. We recently demonstrated that this GC-rich element is capable of interacting with Sp1 in vitro and is necessary for activation of region V reporter constructs by Sp1 in cotransfection studies (30). As Sp1 acts as a general accessory factor in the expression of a number of genes, facilitating the action of other transcription factors that confer specificity, it is possible that IRBP and Sp1 act in concert to control IGF-I gene expression. The IGF-I gene might therefore be similar to the low density lipoprotein, 3-hydroxy-3-methylglutaryl coenzyme A synthase, ACC, and FAS genes, where Sp1 acts with SREBP to stimulate expression in response to cholesterol (54, 55, 56, 57); the HIV-1 gene, which requires both Sp1 and NFKB for expression in response to mitogens (58); and the CYP2D5 P-450 gene, which is dependent on Sp1 and C/EBP for promoter activation (59). We are developing studies to ascertain whether Sp1 and IRBP act in a cooperative manner to stimulate IGF-I expression.

IREs have been identified for a number of genes, and several of the associated binding factors have also been identified. For example, the Forkhead/winged helix protein has been shown to interact with the negatively regulated IREs from the IGFBP-1, PEPCK, and tyrosine aminotransferase genes (10), and the basic/helix-loop-helix/leucine zipper protein SREBP can bind to the FAS IRE and is thought to control expression of GK by insulin (12). However, none of these IREs exhibits insulin-responsive binding with unfractionated nuclear extracts (5, 7), none contains an AT-rich element such as that required for binding of IRBP, and none competes for IRBP binding to region V. Furthermore, use of the IGFBP-1, IGFBP-3, and PEPCK IREs as probes in gel shift studies revealed that only region V provided recognition of IRBP (Fig. 5BGo). Accordingly, the protein(s) contained in the IRBP complex may be distinct from other IRE-binding factors.

Our data indicate that IRBP may be involved in insulin-regulated expression of the IGF-I gene, but it is also possible that IRBP plays a role in the insulin-responsive regulation of other genes. However, as the IGF-I region V sequence is different from other IREs, and we were unable to detect IRBP interaction with other IRE sequences, a role for IRBP in the effects of insulin on other genes would probably involve protein-protein interactions rather than direct DNA binding. Testing this hypothesis will require identification of the protein(s) that comprises IRBP, and efforts toward that objective are currently underway in our laboratory.


   Footnotes
 
1 This work was supported in part by research and training awards from the NIH [DK-07298 and DK-09922 (to E.N.K.) and DK-33475 (to L.S.P.)]. This research was previously presented in part at meetings of the American Society of Biochemistry and Molecular Biology and The Endocrine Society. Back

Received August 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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