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Endocrinology Vol. 142, No. 4 1635-1642
Copyright © 2001 by The Endocrine Society

ARTICLES

Insulin Deprivation Leads to Deficiency of Sp1 Transcription Factor in H-411E Hepatoma Cells and in Streptozotocin-Induced Diabetic Ketoacidosis in the Rat1

Xiaolei Pan, Solomon S. Solomon, Dawn M. Borromeo2, Antonio Martinez-Hernandez and Rajendra Raghow3

Research, Medical, and Pathology Services, Veterans Affairs Medical Center of Memphis, and Departments of Medicine, Pharmacology, and Pathology, University of Tennessee, Memphis, Tennessee 38104

Address all correspondence and requests for reprints to: Solomon S. Solomon, M.D., Veterans Affairs Medical Center, Research Service (151), 1030 Jefferson Avenue, Memphis, Tennessee 38104.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Members of the family of Sp transcription factors include Sp1, Sp3, and Sp4 and are important regulators of eukaryotic gene expression. We previously reported that Sp1 mediated stimulation of rat calmodulin I gene expression in response to insulin. To test whether other members of the Sp family are direct targets of insulin action, we compared the levels of Sp1 and Sp3 proteins from nuclear extracts obtained from both insulin-treated and untreated rat hepatoma (H-411E) cells. We demonstrated by Western blot analysis that levels of Sp1 and Sp3 proteins were increased more than 2-fold in the insulin-treated group. Additionally, the up-regulation of both Sp1 and Sp3 transcription factors by insulin was antagonized by tumor necrosis factor-{alpha}, a known inhibitor of insulin action. Immunohistochemical analysis demonstrated that H-411E cells treated with insulin (10,000 µU/ml) had a marked increase in demonstrable Sp1 in the nucleus compared with cells incubated in insulin-free medium. We extended these in vitro observations to in vivo studies in the streptozotocin-diabetic rat model. We demonstrated in rat liver tissue by both Western blot and immunohistochemical staining with anti-Sp1 antibody that 1) livers of fully diabetic streptozotocin rats have low levels of Sp1 transcription factor; and 2) insulin treatment of the diabetic rat rapidly reversed this process by markedly stimulating accumulation of Sp1 in rat liver. Studies of the signal transduction mechanisms involved in insulin’s effect on Sp1 demonstrate a facilitating role for phosphoinositol 3-kinase and an inhibitory role for cyclic nucleotides. In summary, insulin stimulates Sp1 protein, a transcription factor that is shown to regulate calmodulin gene expression and most likely other, as yet untested, genes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOSE UTILIZATION and its storage as glycogen, key events in maintaining cellular energy homeostasis, are primarily regulated by the hormone insulin. Insulin’s ability to orchestrate these functions is critically dependent on its binding to a unique receptor, followed by activation of tyrosine kinase and multiple other signal transduction pathways, including phosphoinositol 3-kinase (1). Calmodulin (CaM) is also an important intracellular regulator of metabolic function and cell growth both through direct effects and indirectly by its ability to bind cytosolic Ca2+ and influence its intracellular Ca2+ flux (2). Insulin has been shown to reverse diabetic coma in animals and humans in part by stimulating the activity of the low Km cAMP phosphodiesterase (PDE) (3, 4, 5). Therefore, we and others have postulated that in diabetes mellitus the absence of insulin’s stimulatory effect on low Km cAMP PDE would result in acute diabetic ketoacidosis (3, 4, 5, 6, 7, 8). In conditions of insulin deprivation and acute diabetes, both insulin-sensitive low Km cAMP PDE activity and CaM activity are decreased synchronously (3, 4, 5, 6, 9, 10, 11, 12). Our laboratory has previously reported the existence of an insulin-sensitive CaM-dependent isozyme of PDE (9, 13) and the coregulation of PDE and CaM by insulin in both streptozotocin-induced and spontaneous diabetic BB Wistar rats (5, 10, 11, 13). Insulin has been shown to regulate transcription of the CaM gene, as demonstrated by both nuclear transcription runoff assays (11) and experiments using antisense oligonucleotides (13). Binding of Sp1 to three putative Sp1 sites in the rat CaM I (rCaM I) gene promoter appears to be a critical determinant of both basal and insulin-stimulated CaM gene expression (14, 15).

The family of Sp transcription factors includes not only Sp1, but two other, less well understood, factors, Sp3 and Sp4 (16, 17). Using Drosophila SL2 cells (devoid of Sp proteins) cotransfected with Sp expression vectors and rCaM I-392 promoter-luciferase DNA, we demonstrated that both basal and insulin-stimulated CaM gene expressions were stimulated by Sp1, but were inhibited by Sp3 and Sp4 (18). This ability to both stimulate and inhibit gene expression by closely related Sp family members creates optimal flexibility for transcriptional regulation of the target genes. As transcription of CaM gene expression is intimately related to synthesis of Sp1, we quantitated Sp1 by Western blot and immunohistochemical staining in H-411E hepatoma cells in culture in the presence or absence of insulin and tumor necrosis factor-{alpha} (TNF{alpha}), a cytokine known to antagonize key insulin-dependent cellular events (19, 20, 21). We extended these observations to livers of animals rendered diabetic with streptozotocin (STZ-DM) in diabetic ketoacidosis, untreated and after treatment with insulin. We report that insulin has profound regulatory influence on the biosynthesis and/or accumulation of Sp1 both in vitro in hepatocytes in culture and in vivo in the livers of diabetic rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Male Holtzman rats were rendered diabetic (STZ-DM) by injection of 65 mg/kg streptozotocin via the tail vein (4). After injection the rats were maintained on Purina chow (Ralston Purina Co., St. Louis, MO) in metabolic cages, with daily measurements of weight, urine volume, glucose, and ketones. At the time of death, blood glucose and plasma insulin were measured (4, 5, 22). Untreated STZ-DM rats were killed on day 3, insulin-treated STZ-DM rats (6 U 70N/30R insulin/day) on day 5, and control rats (saline) on day 6. The clinical parameters of diabetes were assessed in the different groups of rats and are shown in Table 1Go.


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  		Table 1. Clinical laboratory data from streptozotocin-diabetic (STZ-DM) rats with and without insulin (INS) treatment

 
Cell cultures
A minimal deviant rat hepatoma cell line, H-411E, was grown in Eagle’s MEM enriched with glutamine, nonessential amino acids, and antibiotics. Cells were grown in the above medium containing 10% calf serum and 5% FCS at 37 C in 5% CO2-95% air. After 48 h, serum-enriched medium was replaced by serum-free medium, and subconfluent cells were treated with 10,000 µU/ml insulin overnight. In some experiments cells were treated (4–6 h) with 5 ng/ml TNF{alpha} before and again during treatment with insulin (13, 14, 19). In a similar fashion, confluent H-411E cells were treated for 1 h with a specific additive, e.g. forskolin, wortmannin, 3-isobutyl-1-methylxanthine (IBMX), okadaic acid, cAMP, or (Bu)2cAMP, and then for 4 additional h with the selected additive and insulin. After these treatments, cells were harvested and processed for immunocytochemistry and Western blot analyses.

Western immunoblot analysis of Sp1 and Sp3
Nuclear proteins were prepared from H-411E cells as presented previously (23). Whole cellular protein was prepared from rat livers using RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM phenylmethylsulfonylfluoride, 0.5 mM dithiothreitol, 1.0 mM sodium orthovanadate, and protease inhibitors; Roche Molecular Biochemicals, Mannheim, Germany). Nuclear extracts from untreated or insulin- and/or TNF{alpha}-treated H-411E cells and cellular protein from rat livers were electrophoresed on 7.5% SDS-PAGE and transferred to Immobilon-P transfer membrane (Millipore Corp., Bedford, MA) by a Trans-Blot electrophoretic transfer cell (Bio-Rad Laboratories, Inc., Hercules, CA). Nonspecific binding was blocked with 5% nonfat milk in PBS-Tween-20 overnight at 4 C. The membrane was probed with either rabbit antirat Sp1 or Sp3 polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or actin monoclonal antibodies (Chemicon International, Temecula, CA) for 1 h at room temperature and then washed three times with PBS-Tween-20. The membranes reacted with primary antibodies were incubated with antirabbit peroxidase-conjugated IgG antibody for 1 h at room temperature, and binding was detected with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Western blots were exposed to x-ray films and subsequently scanned into a computer. Individual polypeptide bands were quantified with the Quantity-One software program (Bio-Rad Laboratories, Inc.) with a G-3 Macintosh computer (Apple Computer Inc., Cupertino, CA).

Immunohistochemistry
After brief rinses with PBS, cells grown in chamber slides were fixed in buffered 10% formalin for 1 h. At the time of death, thin slices of liver tissue (2 x 1 x 0.5 cm) were quickly fixed in 10% buffered formalin for 4 h. Tissue blocks were dehydrated and embedded in paraffin, and 5-µm-thick sections were cut by standard methods. Both cultured cells and tissue sections were reacted with the same anti-Sp1 antibody used for Western blotting. Immunohistochemical staining was performed according to previously described procedures (24, 25). Quantitative data are presented as the mean ± SEM. Statistical analysis was performed using Student’s t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of insulin on Sp1 and Sp3 protein levels
To determine how insulin affects Sp protein expression, a Western immunoblot was performed using nuclear extracts from insulin-treated and untreated H-411E cells. A polypeptide band of approximately 100 kDa was detected with anti-Sp1 antibody. Insulin treatment of the cells significantly increased Sp1 protein levels in the nuclear extracts (Fig. 1Go). Densitometric analysis yielded a 3-fold increase in Sp1 in the insulin-treated cells (P < 0.0001). Anti-Sp3 antibody detected one band of approximately 110 kDa and a doublet of about 75 kDa. Insulin significantly increased both bands (Fig. 2Go). Densitometric analysis indicated that insulin treatment resulted in a 2-fold increase in the 110 kDa band (P < 0.05) and a 3-fold increase in the 75-kDa band (P = NS). Western blot with antiactin antibody detected no change after insulin (data not shown). Although the data presented here examine levels of Sp1 and Sp3 in hepatoma cultures treated with or without insulin for 24 h, we have studied the temporal responses of both of these proteins. Insulin treatment results in enhanced accumulation of Sp1 as well as induction of CaM gene transcription after 3 h of treatment (15, 18).



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  		Figure 1. The effect of insulin (10,000 µU/ml) on Sp1 protein expression. A, Western immunoblot analysis of Sp1 in nuclear extracts from untreated and insulin-treated H-411E hepatoma cells. Nuclear extracts were electrophoresed on 7.5% SDS-PAGE, probed with an Sp1 antibody, and visualized by enhanced chemiluminescence. One representative of five replicate experiments is shown. B, Densitometrically analyzed values of Sp1 protein levels in nuclear extracts. Data are the mean ± SE obtained from five separate experiments. *, P < 0.0001.

 


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  		Figure 2. The effect of insulin (10,000 µU/ml) on Sp3 protein expression. A, Western immunoblot analysis of Sp3 in nuclear extracts from untreated and insulin-treated H-411E hepatoma cells. Nuclear extracts were electrophoresed on 7.5% SDS-PAGE, probed with an Sp3 antibody, and visualized by enhanced chemiluminescence. One representative of four replicate experiments is shown. B, Densitometrically analyzed values of different bands of Sp3 protein levels in nuclear extracts. Data are the mean ± SE obtained from four separate experiments. *, P < 0.05.

 
Effect of TNF{alpha} on insulin-stimulated Sp1 and Sp3 protein levels
TNF{alpha} is known to antagonize the action of insulin, inducing insulin resistance in tissues (12). To determine whether the effects of insulin on Sp1 and Sp3 protein levels could be antagonized by TNF{alpha}, cells were pretreated with TNF{alpha} before treatment with insulin. Our data indicate that in the presence of 10,000 µU/ml insulin, 5 ng/ml TNF{alpha} inhibited Sp1 protein levels by 54% (P < 0.05; Fig. 3Go). In the presence of 10,000 µU/ml insulin, 5 ng/ml TNF{alpha} also inhibited Sp3 protein levels, the 110-kDa Sp3 by 54% and the 75-kDa Sp3 by 63% (P < 0.02; Fig. 4Go).



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  		Figure 3. The effect of TNF{alpha} (5 ng/ml) on insulin (10,000 µU/ml)-stimulated Sp1 protein expression. A, Western immunoblot analysis of Sp1 in nuclear extracts from insulin-treated and insulin-plus TNF{alpha}-treated H-411E hepatoma cells. Nuclear extracts were electrophoresed on 7.5% SDS-PAGE, probed with an Sp1 antibody, and visualized by enhanced chemiluminescence. One representative of four replicate experiments is shown. B, Densitometrically analyzed values of Sp1 protein levels in nuclear extracts. Data are the means ± SE obtained from four separate experiments. *, P < 0.05.

 


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  		Figure 4. The effects of TNF{alpha} (5 ng/ml) on insulin (10,000 µU/ml)-stimulated Sp3 protein expression. A, Western immunoblot analysis of Sp3 in nuclear extracts from insulin-treated and insulin-plus TNF{alpha}-treated H-411E hepatoma cells. Nuclear extracts were electrophoresed on 7.5% SDS-PAGE, probed with an Sp3 antibody and visualized by enhanced chemiluminescence. One representative of five replicate experiments is shown. B, Densitometrically analyzed values of Sp3 protein levels in nuclear extracts. Data are the mean ± SE obtained from five separate experiments. The lower (75-kDa) band, INS+TNF-{alpha}, was decreased significantly (P < 0.02, by normalized paired Student’s t test). The upper (110-kDa) band was decreased, but this was not statistically significant.

 
Immunohistochemical analysis of Sp1
After we had established that the levels of Sp1 and other Sp family member proteins (Western blot) were modulated in cells incubated with insulin and/or insulin antagonists, i.e. TNF{alpha}, etc., we performed immunostaining to directly visualize the dynamics of Sp1 under insulin-deprived and insulin-treated conditions.

To examine the above effects further, we stained H-411E cells with anti-Sp1 antibody in the presence and absence of insulin (10,000 µU/ml) and/or TNF{alpha} (5 ng/ml). Immunohistochemical analysis demonstrated that H-411E cells treated with insulin showed a significant increase in peroxidase staining (brown) of the nucleus compared with control (Fig. 5Go, A and B). Furthermore, incubation with TNF{alpha} significantly inhibited this staining pattern [Fig. 5Go, C and D; controls for these experiments, i.e. nonimmune serum and irrelevant antibody (anti-{alpha}-fetal protein antibody) were negative; data not shown].



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  		Figure 5. Immunohistochemical staining of Sp1 antibodies of H-411E cultured with standard medium (A), medium supplemented with insulin (B), medium containing TNF{alpha} (C), and medium containing TNF{alpha} and insulin (D). The presence of Sp1 antigen is demonstrated by the peroxidase reaction product (brown). See Materials and Methods for details of cell culture and immunohistochemical procedures. A, Cells cultured with standard medium. Although there is some variability, all nuclei contain demonstrable Sp1 antigen. B, Cells cultured with medium supplemented with insulin. All cells contain Sp1 antigen, and in many cells there is demonstrable cytoplasmic antigen. C, Cells cultured with medium supplemented with TNF{alpha}. The nuclear staining is weaker than that in A or B, indicating decreased antigen levels. D, Cells cultured with medium supplemented with TNF{alpha} and insulin. The addition of insulin to the medium restores the nuclear staining intensity at least to that of cells cultured with standard medium (A), demonstrating insulin’s ability to prevent or correct the effects of TNF{alpha}. All cell cultures were lightly counterstained with hematoxylin. Original magnification, x160.

 
Studies in STZ-DM rats
Based on these analyses, we predicted that changes in Sp1 in cultured cells with and without insulin would reflect changes one might expect to see pathophysiologically in untreated and insulin-treated diabetes mellitus. We then moved forward to animal models of diabetes mellitus. To test this prediction, we measured steady state levels of Sp1 in the livers of STZ-DM rats.

The data shown in Table 1Go illustrate that after the Holtzman rats were rendered diabetic they lost weight compared with controls (-27.3 g; P < 0.05), had marked elevation of urine volume (84 vs. 10 ml/24 h; P < 0.01) with positive urinary glucose and acetone, had marked elevation of fasting blood glucose (436 vs. 95 mg/100 ml; P < 0.05), and had marked reduction of fasting plasma insulin (10 vs. 0.3 ng/ml; P < 0.01). Administration of insulin to the STZ-DM group significantly reduced the weight loss, urine volume, fasting plasma glucose (436 vs. 60 mg/100 ml; P < 0.05) and significantly raised the plasma insulin (0.3 to 84 ng/ml; P < 0.01). Hence, after 2 days without insulin treatment, STZ-DM animals were in marked diabetic ketoacidosis. After insulin treatment for 5 days, most clinical parameters were restored to normal, and the animals were no longer ketoacidotic.

The data presented in Fig. 6Go demonstrate by Western blot that Sp1 protein from STZ-DM animals was reduced by 85% by day 2 (P < 0.001) and was dramatically restored (3.5-fold; P < 0.02) after 7 days of insulin treatment. The data shown in Fig. 7Go represent sections of livers from these three groups of animals specifically stained for Sp1. Herein, after 3 days without insulin and in acute diabetic ketoacidosis, one easily observes marked diminution of brown (horseradish peroxidase) stain in the STZ-DM rat liver with subsequent restoration of Sp1 staining, primarily in the nucleus, after 7 days of insulin treatment.



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  		Figure 6. The effects of STZ-DM on Sp1 protein levels, determined by Western blot, in male Holtzman rat livers obtained from control (n = 3), STZ-DM untreated (n = 3), and insulin-treated STZ-DM (n = 3). See text for methods. STZ-DM different from control, P < 0.001; STZ-DM plus insulin different from STZ-DM, P < 0.05.

 


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  		Figure 7. Immunohistochemical staining with anti-Sp1 antibodies of liver tissue from normal (A), diabetic (B), and insulin-treated diabetic (C) rats. The presence of Sp1 antigen in cell nuclei is demonstrated by the peroxidase reaction product (brown). A, Normal rat. Although there is some variability in the staining intensity, most cell nuclei contain immunoreactive Sp1. B, Diabetic rat (STZ-DM). As is often the case in diabetes, many hepatocytes contain lipid (steatosis) droplets (arrowheads). The nuclear staining for Sp1 is notably less intense than that in normal animals (compare with A). C, Insulin-treated diabetic rat (STZ-DM+insulin). The nuclear staining for Sp1 is at least as intense as that in the normal animal (A) and in many cases it is even stronger. All sections were lightly counterstained with hematoxylin. Original magnification, x160.

 
Signaling mechanism(s) involved in the regulation of Sp1 by insulin
To define the mechanisms involved, we studied the effects of specific perterbators of signal transduction on insulin’s ability to stimulate Sp1. The data presented in Fig. 8Go demonstrate that the stimulatory effect of insulin on Sp1 protein is abrogated by cAMP and (Bu)2AMP. Insulin’s effect on Sp1 is also significantly blunted by IBMX, an inhibitor of cAMP PDE, and wortmannin, an inositol trisphosphate 3-kinase inhibitor. Regulation of Sp1 by insulin was affected to a lesser extent by the selective phosphatase inhibitor, okadaic acid. Forskolin, a known powerful stimulator of adenyl cyclase, did not inhibit, as would have been predicted. Forskolin’s additional stimulatory effect on insulin-stimulated Sp1 would then appear to be independent of its effects on cAMP.



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  		Figure 8. The effect of insulin (10,000 µU/ml) on Sp1 protein expression in the presence of forskolin, cAMP, (Bu)2AMP (dcAMP), wortmannin, IBMX, and okadaic acid. Both inhibitors and concentrations are shown above the Western immunoblot for Sp1. Nuclear extracts of treated H-411E hepatoma cells were electrophoresed on 7.5% SDS-PAGE and probed with an Sp1 antibody or an antiactin antibody (ACT) in the absence and presence of insulin. The data shown are representative of four replicate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Insulin exerts its cellular effects through binding to a specific receptor and activating a cascade of signal transduction mechanisms. Its effects appear to be specific and unique on different subcellular components, i.e. membrane, mitochondria, Golgi, nucleus, etc. Insulin has been shown to regulate the expression of many genes (26). Therefore, it appears almost certain that both basal and insulin-regulated gene expressions play a role in the pathophysiology of both type I insulin-dependent diabetes mellitus and type II noninsulin-dependent diabetes mellitus. We have previously shown that Sp1 is required for both basal and insulin-stimulated rCaM I gene expression (14, 15, 18). We studied other members of the Sp family of transcription factors, i.e. Sp1, Sp3, and Sp4. We found that Sp1 and USp3, a long form of Sp3, mediated insulin stimulation of rCaM gene expression in Schneider’s Drosophila SL2 cells that lack Sp1 transcription factor. Furthermore, we reported that Sp4 and a short form of Sp3 exerted inhibitory effects on Sp1-mediated rCaM I gene expression (18). Herein, we have extended our earlier studies to examine the steady state levels of Sp1 and Sp3 in H-411E hepatoma cells and STZ-DM rat livers under insulin- deprived and insulin-sufficient states. We demonstrate that insulin is a potent inducer of nuclear Sp1 protein levels. As both basal and insulin-stimulated transcriptions of the rCaM I gene are critically dependent on Sp1, this increase in Sp1 protein is most likely directly responsible for insulin’s stimulatory effect on rCaM I gene expression (15, 18). As increased levels of Sp1 and Sp3 proteins were induced by insulin and significantly blocked by TNF{alpha}, these data may well explain the known antagonism between insulin and TNF{alpha} reported previously (19, 20, 21). It has been suggested that insulin-stimulated changes in the phosphorylation-dephosphorylation of transcription factor(s) could potentially affect their nuclear translocation, DNA binding, and trans-activation potential (27, 28). Sp1 is reported to be modified by both phosphorylation and O-glycosylation (29, 30, 31). As the Sp family of transcription factors is regulated by insulin, it is possible that insulin may not only increase Sp1 protein levels, but also modify Sp1 binding by selective phosphorylation and/or O-glycosylation.

Insulin’s actions generally fall into two groups, those involving the cell membrane and those involving the nucleus of the cell (1). Membrane events are mediated by the binding of insulin to its receptor, triggering the initiation and propagation of signal transduction pathways, most likely through phosphorylation-dephosphorylation, that eventually will activate or inhibit the transcription of target genes in the nucleus. The initial events at the membrane are rapid, i.e. seconds to minutes; the nuclear-transcriptional events are much slower and long-lived, involving hours to days (1, 11, 13). From our previous works, it is clear that both basal and insulin-stimulated CaM gene transcriptions are mediated by Sp1 (14, 15, 18). Thus, it would almost be predictable that in the absence of insulin, i.e. diabetic ketoacidosis, Sp1 protein levels would be low, and multiple genes dependent on Sp1 protein for stimulation of transcription, including the CaM gene, inactivated. This is precisely what the data presented in our STZ-DM animal studies demonstrate by both analytical technique (Western blot) and specific immunohistochemical staining. In a similar vein, the cellular experiments with TNF{alpha}, which clearly antagonizes insulin’s actions on hepatocytes, mimic the results seen in diabetic animals, i.e. decrease in Sp1 and other Sp family proteins by both Western blot and immunohistochemical staining. In many ways the in vitro cellular experiments with TNF{alpha} could be considered to reflect the type II or noninsulin-dependent diabetic model (32).

Studies of the signal transduction mechanisms involved are presented in Fig. 8Go. Here, in general, when cAMP is stimulated, insulin’s stimulatory effect on Sp1 is attenuated. This is true for cAMP, (Bu)2AMP, and IBMX, but not forskolin. Forskolin appears to stimulate, not inhibit, Sp1 in the presence of insulin. In light of the data for cAMP and (Bu)2AMP, this would probably appear to be through effects other than those mediated by the cyclic nucleotides. However, because high dose cAMP appears to have less of an inhibitory effect than low dose cAMP on insulin activation of Sp1, it is possible that this explains the forskolin effect, which probably generates higher, rather than lower, doses of cAMP. Wortmannin inhibits what is thought to be a major pathway of insulin signaling, i.e. inositol trisphosphate 3-kinase, and therefore, abrogation of insulin’s effect on Sp1 by wortmannin is predictable.

Only limited data exist on the identities of transcription factors regulated by insulin (26). In addition to the regulation of Sp1 by insulin shown here, sterol response element-binding protein-1c (SREBP-1c) is an important downstream target of insulin (33). Regulation of SREBP-1c proteolysis and enhanced transcription in response to insulin have been documented in both cultured hepatocytes as well as in livers of STZ-DM rats (33, 34). However, the relationship between Sp1 and SREBP-1c has not been elucidated.

Questions of tissue specificity, i.e. only insulin-sensitive tissue for this effect, levels of Sp1 that are needed to turn on transcription of the CaM gene, timing, etc. need further definition to fully understand the exact mechanisms involved. Similarly, the roles of other Sp protein family members in this process, i.e. Sp3 and Sp4, need to be assessed. However, for now at least it appears clear that both acute insulin deprivation and some aspects of the pathophysiology of diabetic ketoacidosis are related functionally to insulin’s ability to stimulate Sp1 transcription factor and subsequent regulatory effects of this factor on gene transcription.


   Acknowledgments
 
The authors thank Drs. Suleiman Bahouth and Edwards Park for insightful comments and advice concerning the work, and Ms. Ann M. Oswalt for secretarial support. Thanks also to Drs. Stan Laulederkind and Anthony Cheung for assistance with the gel data, and Mr. Paul Markowitz and Ms. Laura Finley for insulin assays and animal work.


   Footnotes
 
1 This work was supported by Merit Review Grants from the Department of Veterans Affairs (to S.S.S. and R.R.) and the NIH (to S.S.S. and R.R.). Back

2 Supported by a Medical Student’s Short-Term Research Training Grant (T35-DK-07405–15, NIH). Back

3 Senior Research Career Scientist of the Department of Veterans Affairs. Back

Received August 28, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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