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Molecular Endocrinology, doi:10.1210/me.2005-0407
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Molecular Endocrinology 20 (5): 1138-1152
Copyright © 2006 by The Endocrine Society

The Genomic Analysis of the Impact of Steroid Receptor Coactivators Ablation on Hepatic Metabolism

Jae-Wook Jeong, Inseok Kwak, Kevin Y. Lee, Lisa D. White, Xiao-Ping Wang, F. C. Brunicardi, Bert W. O’Malley and Francesco J. DeMayo

Molecular and Cellular Biology (J.-W.J., I.K., K.Y.L., B.W.O’M., F.J.D.), Microarray Core Facility Department of Molecular and Human Genetics (L.D.W.), and Michael E. DeBarkey Department of Surgery (X.-P.W., F.C.B.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to Francesco J. DeMayo, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030. E-mail: fdemayo{at}bcm.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the steroid receptor coactivator (SRC) family, which include SRC-1 (NcoA-1/p160), SRC-2(TIF2/GRIP1/NcoA-2) and SRC-3(pCIP/RAC3/ACTR/pCIP/ AIB1/TRAM1), are critical mediators of steroid receptor action. Gene ablation studies previously identified SRC-1 and SRC-2 as being involved in the control of energy homeostasis. A more precise identification of the molecular pathways regulated by these coactivators is crucial for understanding the role of steroid receptor coactivators in the control of energy homeostasis and obesity. A genomic approach using microarray analysis was employed to identify the subsets of genes that are altered in the livers of SRC-1–/–, SRC-2–/–, and SRC-3–/– mice. Microarray analysis demonstrates that gene expression changes are specific and nonoverlapping for each SRC member in the liver. The overall pattern of altered gene expressions in the SRC-1–/– mice was up-regulation, whereas SRC-2–/– mice showed an overall down-regulation. Several key regulatory enzymes of energy metabolism were significantly altered in the liver of SRC-2–/– mice, which are consistent with the prior observation that SRC-2–/– mice have increased energy expenditure. This study demonstrates that the molecular targets of SRC-2 regulation in the murine liver stimulate fatty acid degradation and glycolytic pathway, whereas fatty acid, cholesterol, and steroid biosynthetic pathways are down-regulated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
STEROID RECEPTOR coactivator (SRC) proteins function as signaling intermediates between the nuclear receptor and the basal transcriptional machinery to enhance transcription activation. The family is composed of three distinct but functionally and structurally related members: SRC-1 (NcoA-1/p160) (1, 2, 3, 4, 5), SRC-2 (TIF2/GRIP1/NcoA-2) (6, 7, 8, 9), and SRC-3 (RAC3/ACTR/ pCIP/AIB1/TRAM1) (10, 11, 12, 13, 14, 15). The SRC family members enhance the transcriptional activity of a variety of nuclear receptors, including estrogen receptor (ER) {alpha} and ß and progesterone receptor (PR) (1, 9, 12, 13, 16, 17) and are expressed in a variety of hormone-responsive tissues, such as uterus, brain, prostate, and breast (2, 18, 19, 20, 21, 22). However, genetic ablation of genes encoding the members of the SRC family in mice has demonstrated that they also play a critical role in general metabolism (23, 24, 25).

A mouse model with disruption of the SRC-1 gene exhibited a partial resistance to steroid and thyroid hormones, as well as a delayed development of cerebellar Purkinje cells (2, 26, 27). In this mouse model, the function of peroxisome proliferator-activated receptor {gamma} in brown fat is partially impaired causing lower energy expenditure and higher sensitivity to diet-induced obesity (23, 24). SRC-2–/– mice are hypofertile because of restricted growth of the placenta in females and partial impairment of spermatogenesis in males (28) and, unlike SRC-1–/– mice, SRC-2–/– mice exhibit higher energy expenditure and are resistant to diet-induced obesity (23). SRC-3–/– mice are growth retarded, have lower levels of IGF 1 and estradiol, and display a delayed puberty and reduced female reproductive function (10, 29). The vasoprotective functions of the ER after vascular injury also are partially impaired in SRC-3–/– mice (30). Altogether, these genetic studies indicate that SRC family members function to regulate general metabolism and that the individual members possess relative functional specificity.

To investigate the molecular mechanisms regulating metabolism that are altered in the ablation of the SRC genes, microarray analysis was conducted on the livers of these mice. The liver was chosen as the focus for the microarray analysis because of its pivotal role in the regulation of energy metabolism. The liver is the principal organ responsible for the conversion of excess dietary carbohydrates into triglycerides. Glucose can be metabolized in the liver to provide substrates, such as acetyl coenzyme A (CoA), for fatty acid synthesis. Fatty acids are incorporated into triglycerides that function as a long-term energy reservoir. In addition, excess carbohydrate also results in the activation of several genes encoding glycolytic and lipogenic enzymes involved in carbohydrate and lipid metabolism, including glucokinase (GCK) (31) and liver pyruvate kinase (32) for glycolysis, and ATP citrate lyase, acetyl CoA carboxylase and fatty acid synthase for lipogenesis (33), thereby promoting long-term storage of carbohydrate and triglycerides (34). Aside from metabolizing glucose, hepatic glucose production is essential for maintaining normal circulating glucose levels. During low glucose states, the liver serves as the primary site for glucose production through glycogenesis and gluconeogenesis. However, when glucose levels rise, as in the postprandial state, hepatic glucose production is rapidly reduced to low levels in response to elevated circulating insulin (35, 36, 37, 38, 39, 40).

Microarray analysis of hepatic RNA in mice with ablation of specific SRC genes demonstrated that these coactivators play a critical role in regulating the expression of hepatic genes essential for energy homeostasis. The overall pattern of altered hepatic gene expressions in the SRC-1–/– mice was one of up-regulation as compared with wild-type mice. Analysis of differential hepatic gene expression in SRC-2–/– mice showed an overall down-regulation. The impact of SRC-3 ablation on liver gene expression was minimal. The groups of genes that were validated were those involved in glucose uptake, glycolysis, fatty acid catabolism and synthesis, cholesterol synthesis, and steroid synthesis. The identification of these target genes as targets of SRC regulation provides mechanistic information on the role of the SRC family in liver physiology.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hepatic Expression of SRC-1, SRC-2, and SRC-3 Genes
SRC-2–/– mice have resistance to dietary-induced obesity and display enhanced adaptive thermogenesis, whereas SRC-1–/– mice are prone to obesity due to reduced energy expenditure (23). Because the liver plays a principal role in short-term energy storage and metabolism, our hypothesis is that SRC-1 and SRC-2 play important roles in the murine liver. Before conducting microarray analysis in the liver, we confirmed the expression of the SRCs in the liver. We performed immunohistochemistry to detect the expression of SRC-1 and SRC-2 in the murine liver. SRC-1 and SRC-2 proteins were expressed in all of the types of cells in the liver (Fig. 1AGo, a and c, respectively). In the livers of control SRC-1–/– and SRC-2–/– mice, SRC-1 or SRC-2 protein was not detected (Fig. 1AGo, b and d, respectively). SRC-3 expression in the liver was assayed using the SRC-3+/– mice. In the processes of ablating the SRC-3 gene, a promoter-free lacZ (ß-galactosidase) reporter gene was inserted in frame to the N-terminal coding region (10). This lacZ "knock in" approach allowed us to assay for SRC-3 expression by using X-gal staining. ß-gal activity was detected in the liver of 8-wk-old SRC-3+/– mice (Fig. 1BaGo). These results demonstrate that all three SRC proteins are expressed in the liver.


Figure 1
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  		Fig. 1. The Expression of SRC-1, SRC-2, and SRC-3 in the Liver

A, Immunohistochemical analysis for SRC-1 (a and b) and SRC-2 (c and d) in the liver. Liver from wild-type (a and c), SRC-1–/– (b), and SRC-2–/– (d) mice were fixed with 4% paraformaldehyde (vol/vol) and embedded in paraffin. Tissue sections were stained with anti-SRC-1 and anti-SRC-2 antibodies, respectively, and counterstained with methyl green. Biotin-peroxidase immunodetection system was used with 3,3'-diaminobenzidine as a chromogen. Liver of SRC-1–/– (b) and SRC-2–/– (d) mice were used as negative control, respectively. B, Galactosidase staining of liver sections in SRC-3+/– heterozygotes (a) mice were used to detect SRC-3. Liver sections of wild-type mice (b) were used as negative control. Sections were counterstained with Nuclear Fast Red. Scale bar, 50 µm.

 
Identification of Significantly Regulated Genes in the Liver of SRC-1–/–, SRC-2–/–, and SRC-3–/– Mice
SRC-1, SRC-2, and SRC-3 regulated genes in the murine liver were identified by comparing the mRNA expression profile of liver from 8-wk-old ovariectomized female SRC-1–/–, SRC-2–/–, and SRC-3–/– and wild-type mice. We used ovariectomized female mice because our purpose was to identify SRC-regulated genes not influenced by the cyclic fluctuations in ovarian hormones, such as estrogen. It has been showed that estrogen plays an important role in the control of liver cell proliferation (41) and that ER{alpha} inhibits induction of gene expression through a coactivator based mechanism in the mouse liver (42). Total RNA was isolated from five different SRC-1–/–, SRC-2–/–, SRC-3–/–, and wild-type mice and pooled. The experiments were performed in triplicate using three independent pools of RNA. As generally accepted for oligonucleotide microarray profile analysis, differentially expressed genes in SRC-1–/–, SRC-2–/–, and SRC-3–/– were selected using a two sample comparison according to the following criteria: lower bound of 90% confidence interval of fold change greater than 1.2 and an absolute value of difference between group means greater than 80. This paradigm was applied to select increased and decreased genes. A summary of the number of differentially expressed genes in the SRC-1–/–, SRC-2–/–, and SRC-3–/– mice compared with wild-type mice is shown in Table 1Go. In the SRC-1–/– mice, 270 genes were identified as significantly differentially expressed genes compared with wild-type mice. Of the 270 genes, 245 were more highly expressed in the SRC-1–/– mice and only 25 genes demonstrated a decreased expression pattern when compared with wild-type mice. However, in the livers of SRC-2–/– mice, 492 genes were identified as differentially expressed. Of these 492 genes, 154 genes showed an increased level of expression in the SRC-2–/– mice, and 338 genes showed a decreased level of expression when compared with wild-type mice. In the livers of the SRC-3–/– mice, only 22 genes were identified as differentially expressed as compared with the livers of wild-type mice. The above analysis demonstrates that ablation of SRC-3 has a minimal impact on hepatic gene expression. The overall impact of ablation of SRC-1 was that the number of genes with increased gene expression was greater than genes with decreased expression when compare to wild type. However, the impact of SRC-2 gene ablation was an overall down-regulation of gene expression. The list of the regulated genes is shown in supplemental Tables 1–3.


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  		Table 1. Number of the Significantly Increased and Decreased Genes in the Liver of SRC-1–/–, SRC-2–/–, and SRC-3–/– Mice as Compared with Wild-Type Mice

 
SRC-1–/–, SRC-2–/–, and SRC-3–/– Mice Exhibit Specific and Nonoverlapping Functions in the Liver
To explore whether SRC-1, SRC-2, and SRC-3 share a common function in the regulation of hepatic gene expression, we analyzed which differentially regulated genes were in common in our microarray results. This result is graphically represented in Fig. 2AGo. Interestingly, our results reveal a remarkably limited number of genes that overlap among the three groups. Only one gene, leukocyte cell-derived chemotaxin 2 (Lect2), was common in the analysis of SRC-1, SRC-2, and SRC-3 in the liver. The expression of Lect2 was increased 1.44-fold in the SRC-1–/– mice, whereas Lect2 mRNA was decreased 55.8% and 71.4% in the SRC-2–/– and SRC-3–/– mice, respectively. The limited number of altered genes shared between the individual SRC knockouts could be explained by a degree of functional compensation. Changes in the expression of target genes due to the ablation of one of the SRC family members could be dampened by compensation by one of the other family members. However, the large number of genes whose expression is differentially altered between the individual knockout models clearly demonstrates that SRC-1, SRC-2, and SRC-3 exhibit a high degree of specific and nonoverlapping functional activities in the liver. The specificity in function among the SRC genes may be due in part to differential interactions of the individual SRCs with other members of the nuclear receptor family.


Figure 2
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  		Fig. 2. Relationship and Functional Categorization in the Liver of SRC-1, SRC-2, and SRC-3

A, Venn diagrams demonstrate the relationship between genes modulated in the SRC-1–/–, SRC-2–/–, and SRC-3–/– mice. The numbers, displayed within the intersections of the circles, indicate the common genes by two (or three) comparisons. The number at the bottom right corner of the panel indicates total genes analyzed. B and C, The functional categorization of increased and decreased genes in the liver of SRC-1–/– (B) and SRC-2–/– mice (C). Significantly changed genes were annotated and assigned to various functional categories using the Gene Ontology. Increased genes are shown by red bars and decreased genes are shown by blue bars.

 
Functional Categorization of Significantly Regulated Genes in the Liver of SRC-1–/–, SRC-2–/–, and SRC-3–/– Mice
After identifying the genes whose expression was significantly altered by the ablation of SRCs, we next categorized the SRCs-regulated genes into specific functional groups by gene ontology terms. The GeneSpring and MAPPFinger-based gene ontology annotation was used to examine the categorization of significantly altered genes in the SRC-1–/– and SRC-2–/– mice (43, 44). The results of the gene sorting are shown in Fig. 2Go, B and C, respectively. In the SRC-1–/– mice, signal transducer, enzyme, and structural genes were increased markedly compared with wild-type mice (Fig. 2BGo). In contrast, several enzyme, structural protein, transport, and immunity, signal transducer and transcription factor genes were decreased in the SRC-2–/– mice (Fig. 2CGo). The SRC-3 regulated genes are not shown because of their relatively low impact on hepatic gene expression observed in SRC-3–/– liver mRNA.

Energy Storage Genes and Cell-Cycle Genes Are Altered in the Liver of SRC-1–/– Mice
The expression of several genes involved in glycolysis and glycogen synthesis pathways was significantly increased in the livers of SRC-1–/– mice. The microarray results show significant increases in pyruvate kinase (Pklr), aldolase 1 (Aldoa), 2,3-bisphosphoglycerate mutase (Bpgm), carbonic anhydrase 5a (Car5a), and glycogenin 1 (Gyg) in the liver of SRC-1–/– mice (Table 2Go). These genes are involved in glycolysis and glycogen synthesis. Also, acyl-CoA synthetase long chain family member 4 (Acsl4), a fatty acid synthesis enzyme, has an increased expression level in the SRC-1–/– mice. We used real-time PCR to validate the microarray results for Pklr, Aldoa, Bpgm, and Gyg using additional preparations of RNA. The expression of Pklr, Aldoa, Bpgm, and Gyg mRNA was increased 8.13-, 5.30-, 6.38-, and 3.95-fold in the liver of SRC-1–/– mice compared with wild-type mice (Fig. 3AGo). These results suggest that the gene expression alterations in the livers of SRC-1–/– mice results in a shift in metabolism toward increased energy storage. This result is consistent with our previous report of an obesity phenotype in the SRC-1–/– fed a high-fat diet (23).


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  		Table 2. Regulation of Energy Metabolism Genes in the Liver of SRC-1–/– Mice

 

Figure 3
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  		Fig. 3. Regulation of Energy Storage and Cell-Cycle Genes in the Liver of SRC-1–/– Mice

A, Validation of microarray results for Pklr, Aldoa, Bpgm, Gyg, p21, c-myc, Egr1, and Cyp7a1 by real-time RT-PCR analysis in the liver of SRC-1–/– and wild-type mice. Ovariectomized 8-wk-old SRC-1–/– (n = 3) and wild-type (n = 3) female mice were killed after 24 h fasting and RNA was isolated from the liver. The results represent the mean ± SE of three independent RNA sets. *, P < 0.05; **, P < 0.01. B, The progression of the cell cycle in the liver of SRC-1–/– mice. Immunohistochemical analysis for phosphorylated histone H3 as mitotic cell marker in the liver of wild-type (a) and SRC-1–/– (b) mice. Liver tissue were fixed with 4% paraformaldehyde (vol/vol) and embedded in paraffin. Tissue sections were stained with an anti-phosphorylated histone H3 antibody and counterstained with methyl green. Biotin-peroxidase immunodetection system was used with 3,3'-diaminobenzidine as a chromogen. The number of mitotic cells was counted from the same area of immunohistochemistry slides (c). All of the photomicrographs are x100 (a and b) and x400 (inset) magnification. Scale bar, 200 µm (a and b) or 50 µm (inset).

 
Many cell cycle-related genes involved in the G1/S checkpoint were differentially expressed in the livers of SRC1–/– mice. For example, p21 was decreased in the SRC-1–/– mice, whereas myelocytomatosis oncogene (c-myc), FBJ osteosarcoma oncogene (Fos), Jun-B oncogene (Junb), and stromal antigen 2 (Stag2) were increased (Table 2Go). These microarray results were validated by real-time PCR in that the level of p21 mRNA had 55.0% decrease in expression and c-myc mRNA had a 4.12-fold increase in the liver of SRC-1–/– mice compared with wild-type mice (Fig. 3AGo). We also validated Egr1 and Cyp7a1 (Fig. 3AGo). All showed excellent agreement between the array expression profile and real-time PCR results.

To analyze cell cycle progression in the liver of SRC-1–/– mice, we performed immunohistochemistry on antiphosphorylated histone H3 (Phospho H3) as a mitotic marker. The number of mitotic cells in the liver of SRC-1–/– mice were 7.06-fold greater than wild-type mice (Fig. 3BGo). These results showed that the cell cycle progression is accelerated in SRC-1–/– mice compared with wild-type mice.

The Expression of Energy Expenditure Genes Is Increased, and the Expression of Energy Storage Genes Is Decreased in the Liver of SRC-2–/– Mice
Analysis of the specific genes altered in the livers of SRC-2–/– mice indicated a decrease in energy storage and an increase in energy use (Table 3Go). The pathways affected by SRC-2 gene ablation are shown graphically in Fig. 4Go. Glucose transporter 2 (Glut2) and glucokinase (Gck; hexokinase) were decreased –1.44- and –3.81-fold in the SRC-2–/– mice, respectively. Glut2 transports blood glucose to the liver, and Gck catalyzes the initial step in the use of glucose by the liver at physiological glucose concentrations. Hepatic Gck helps to facilitate the uptake and conversion of glucose by acting as an insulin-sensitive determinant of hepatic glucose usage. The decrease of these two enzymes suggests that glucose uptake is significantly decreased in the liver of SRC-2–/– mice. However, triosephosphate isomerase (Tpi), one of the steps of glycolysis, and lipoprotein lipase, a component of fatty acid degradation, are increased in the SRC-2–/– mice. Glycolysis and fatty acid degradation are important sources of energy. The results suggest that energy generation through glycolysis and fatty acid degradation is facilitated in the liver of SRC-2–/– mice and are consistent with the observed phenotype in living SRC-2–/– mice in that SRC-2–/– mice have increased energy expenditure (23).


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  		Table 3. Regulation of Energy Metabolism Genes in the Liver of SRC-2–/– Mice

 

Figure 4
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  		Fig. 4. A Chart of Energy Metabolism Genes in the Liver of SRC-2–/–

Significantly changed genes in fatty acid degradation, glycolysis, glucose uptake, fatty acid synthesis, cholesterol biosynthesis, and steroid biosynthesis pathways are represented in the chart. The number indicates fold change of gene expression in the liver of SRC-2–/– compared with wild-type mice. A light red box represents increase of mRNA expression level, and a light blue box represents decrease of gene expression. *, Validated by real-time PCR.

 
The phenotype of the SRC-2–/– mouse, which has reduced energy storage, and the microarray analysis show that genes involved in fatty acid synthesis, cholesterol biosynthesis, and steroid synthesis were decreased in the SRC-2–/– mice as compared with wild-type mice. Specifically, fatty acid synthase (Fasn), Elovl6, and Cyp2c40, which are enzymes necessary for fatty acid synthesis, were decreased in the SRC-2–/– mice. Cholesterol biosynthesis enxymes, 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), 3-hydroxy-3-methylglutaryl-CoA synthetase 1 (Hmgcs1), low-density lipoprtotein receptor (Ldlr), and sterol regulatory element binding factor 1 (Srebf1) were significantly down-regulated in the SRC-2–/– mice. We also identified that isopentenyl-diphosphate delta isomerase (Idi1) and aldo-keto reductase family 1 c6 (Akr1c6), which are important enzymes for steroid biosynthesis, were decreased in the SRC-2–/– mice. The location of these genes in the metabolic pathway and their alterations in the SRC-2–/– mice are shown graphically in Fig. 4Go. The results demonstrate that SRC-2–/– mice are defective in long-term energy storage metabolism in the liver.

Nine genes from the SRC-2 microarray analysis were validated. The metabolic enzyme genes Tpi and Lpl had increased expression (2.34- and 1.62-fold, respectively), whereas Glut2, Gck, G6pc, Fasn, Idi1, Cyp17a, and Hmgcr had decreased expression in the SRC-2–/– mice (71.6%, 32.5%, 66.0% 62.9%, 38.3%, 46.7%, and 40.0%, respectively; Fig. 5Go). The expression profiles of these genes also demonstrated an excellent correlation between the microarray expression profile and real-time PCR results.


Figure 5
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  		Fig. 5. Real-time RT-PCR Analysis of Energy Metabolism Genes in the Liver of SRC-2–/– Mice

Ovariectomized 8-wk-old SRC-2–/– and wild-type female mice were killed after 24 h fasting and RNA was isolated from liver. The results represent the mean ± SE of three independent RNA sets. *, P < 0.05; **, P < 0.01.

 
We also confirmed the protein expression profile for glucose uptake enzymes. Glut2 and Gck transcripts were significantly down-regulated in the SRC-2–/– mice. We investigated the expression level of GLUT2 and GCK proteins in the mouse liver by employing Western blot analysis and immunohistochemistry. As shown by Western blot analyses using specific GLUT2 and GCK antibodies, levels of GLUT2 and GCK in the SRC-2–/– mice were significantly lower than wild-type and SRC-1–/– mice (Fig. 6AGo). Liver sections from SRC-1–/–, SRC-2–/–, and wild-type mice were stained with antibodies specific for GLUT2 and GCK. Distinct cell membrane staining of GLUT2 was localized in the liver cells of wild-type mice. SRC-1–/– mice had the same expression pattern as the wild-type mice (Fig. 6BbGo). However, the expression of GLUT2 proteins was significantly decreased (Fig. 6BcGo). The level of GCK protein was also decreased in the SRC-2–/– mice as compared with the wild-type and SRC-1–/– mice (Fig. 6BfGo). The decreased expression of the glucose transporter and glucokinase in the liver confirm the phenotypic and microarray analysis that the SRC-2–/– mice have a defect in the ability to uptake and store glucose.


Figure 6
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  		Fig. 6. Regulation of GLUT2 and GCK Protein Level in the Liver of SRC-2–/– Mice

A, Western blot analysis for GLUT2 and GCK in the liver of wild-type, SRC-1–/–, and SRC-2–/– mice. Liver tissue from wild-type, SRC-1–/–, and SRC-2–/– mice were lysed, and equal amounts of protein were subjected to SDS-PAGE and Western blot analysis with anti-GLUT2 and anti-GCK antibodies. Quantitation of GLUT2 and GCK protein level shows changes in the SRC-2–/– mice relative to the wild-type control. The data are expressed as average ± SE. **, P < 0.01. B, Immunohistochemical analysis for GLUT2 (a–c) and GCK (d–f) in the liver of wild-type (a and d), SRC-1–/– (b and e), and SRC-2–/– (c and f) mice. Liver tissue were fixed with 4% paraformaldehyde (vol/vol) and embedded in paraffin. Tissue sections were stained with an antiphosphorylated histone H3 antibodies and counterstained with methyl green. Biotin-peroxidase immunodetection system was used with 3,3'-diaminobenzidine as a chromogen. All of the photomicrographs are x100 (a, c, and e) and x400 (inset) magnification. Scale bar, 200 µm (a, c, and e) or 50 µm (inset).

 
Altered Glycogen Storage in SRC-1–/– and SRC-2–/– Mouse Livers
Because glycogen biosynthetic enzymes were shown by microarray analysis to be decreased in the liver of SRC-2–/–, we used PAS staining to assess endogenous glycogen levels in the livers of SRC-1–/– and SRC-2–/– mice. SRC-1–/– mice had a slight increase in PAS staining when compared with the livers of wild-type mice (Fig. 7AbGo). However, SRC-2–/– mice had significantly lower levels of PAS staining compared with wild-type and SRC-1–/– mice (Fig. 7AcGo). The changes in carbohydrate content was quantified in the liver of SRC-1–/–, SRC-2–/–, and wild-type mice (n = 3) by measuring glycogen content. The level of glycogen in the liver of SRC-1–/– was significantly increased by 1.33-fold, whereas the level of glycogen in the liver of SRC-2–/– was significantly decreased compared with wild-type mice (Fig. 7BGo). These results were consistent with our microarray data indicating that SRC-2–/– mice have a glycogen storage defect in their livers.


Figure 7
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  		Fig. 7. Alteration of Glycogen Level in the Liver of SRC-1–/– and SRC-2–/– Mice

A, Analysis of glycogen level in the liver of wild-type (a), SRC-1–/– (b), and SRC-2–/– (c) mice by periodic acid Schiff (PAS) staining and counterstained with hemotoxylin. All of the photomicrographs are x100 (a, c, and e) and x400 (inset) magnification. Scale bar, 200 µm (a, c, and e) or 50 µm (inset). B, Glycogen level in the liver of SRC-1–/–, SRC-2–/–, and wild-type mice. The results represent the mean ± SE of three independent RNA sets. *, P < 0.05; **, P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we have identified SRC-regulated genes in the liver of ovarectomized SRC-1–/–, SRC-2–/–, and SRC-3–/– female mice using high-density DNA microarray analysis. In this analysis, gonadectomized mice were chosen to avoid any confounding influences of fluctuating steroid hormones on hepatic gene expression. It has been shown that estrogens play an important role in the control of liver cell proliferation (41). Several estrogen-regulated genes have been reported in the liver (45, 46). Our purpose was to identify SRC-regulated genes, not estrogen-responsive genes. To this end, we employed ovariectomized female mice to remove the effect of cycling endogenous estrogens.

Ablation of SRC-1 results in an overall activation of the genes that are regulated in the liver by SRC-1. The majority of genes activated in the SRC-1–/– mice are regulatory genes. Overall, up-regulation of gene expression may be a resulting compensation for the loss of SRC-1. Alternatively, this observation may indicate SRC-1 may serve as a repressor of gene expression. However, unlike the results found in the SRC-1–/– livers, the major impact of SRC-2 ablation was a decrease in target gene expression. Because the microarray analysis was conducted on mice with germ line ablation of these genes, the results observed may be due to either the direct actions of SRC-1 and SRC-2 regulation of target gene transcription or may be the end result of the developmental and systemic impact of SRC-1 and SRC-2 ablation on hepatic physiology. Because the expression of all three SRC genes was detected in the livers, the genomic actions of the SRCs gene ablation may be due in part to direct actions of these coactivators on target gene transcription. However, to determine whether the alterations in gene expression resulting from SRCs ablation are due to the direct action of the SRCs on gene transcription or the result of the cascade of developmental and systemic consequences of SRCs ablation, tissue-specific and temporal ablation of these genes must be accomplished. However, these microarray results do show the final impact of SRC-1 and SRC-2 on murine liver gene expression.

SRC-1, SRC-2, and SRC-3 proteins have 50~55% similarity among them (11). SRCs coactivate gene transcription with little selectivity among nuclear receptors in cell transfection experiments. Therefore, this analysis cannot identify genes regulated by each SRC in which functional compensation has occurred. To determine the overlapping functions of the SRC genes in the murine liver, combined ablation of these genes must be conducted. This too must be performed when using conditional gene ablation because combined ablation of the SRC genes results in an embryo lethal phenotype (47).

Although functional compensation may occur when one member of the SRC family is ablated, mice deficient in specific SRC coactivators exhibit distinct phenotypes. This may be impart due to the fact that each SRC member displays a specific expression pattern in vivo (10, 21, 27, 48, 49, 50). However, when these coactivators are expressed in the same tissue, distinct phenotypes resulting from their ablation are observed. In brown fat, SRC-1–/– mice show lower energy expenditure and higher sensitivity to diet-induced obesity (23, 24). SRC-2–/– mice exhibit higher energy expenditure and are resistant to obesity (23). SRC-3–/– mice are growth retarded, have lower levels of IGF-I, and display delayed puberty and reduced female reproductive tissue function (10, 29). The differences in phenotypes observed in tissues in which all the SRCs are expressed and one member is ablated may represent specificity in the interactions of the SRC with other members of the nuclear receptor transcription factor family or differential interactions with other members of the transcription complex. Our study demonstrates that specific genes are altered in the liver of mice in which the specific SRC has been ablated and the alteration in gene expression directly corresponds to the metabolic phenotype observed in these mice.

The metabolic pathways identified by our SRC-2–/– microarray analysis are those of genes involved in energy metabolism (see Fig. 8Go). Lpl degrades fatty acids and was increased in the liver of SRC-2–/–. Triacylglycerols are hydrolyzed to fatty acids and glycerol by intracellular lipases. The release of the first fatty acid from a triacylglycerol, the rate-limiting step, is catalyzed by a hormone-sensitive Lpl that is reversibly phosphorylated. The gylcolytic enzyme, Tpi, whose expression was increased, catalyzes the conversion of glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. Glyceraldehyde 3-phosphate is in the direct pathway of glycolysis, whereas dihydroxyacetone phosphate is not.


Figure 8
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  		Fig. 8. Summary of Energy Metabolism Effect in the Liver of SRC-2–/– Mice

SRC-2–/– mice represent a change of energy metabolism pathways. Fatty acid degradation and glycolysis pathways are up-regulated, whereas fatty acid, cholesterol, and steroid biosynthesis pathways are down-regulated in the liver.

 
Additionally, we identified a decrease of GLUT2 and GCK in the SRC-2–/– mice. These two enzymes catalyze the initial step in the use of glucose by ß-cells and liver at physiological glucose concentrations (51). Hepatic glucokinase facilitates the uptake and metabolic conversion of glucose by acting as an insulin-sensitive determinant of hepatic glucose usage (52, 53, 54). In glycolysis, the reactions catalyzed by glucokinase, phosphofructokinase, and pyruvate kinase are virtually irreversible; hence, these enzymes would be expected to have regulatory as well as catalytic roles.

We identified a decrease of Hmgcs1 expression. Hmgcs1 is a transmembrane glycoprotein and the rate-limiting enzyme for cholesterol biosynthesis. Squalene is synthesized from isopentenyl pyrophosphate by the reaction sequence. This stage in the synthesis of cholesterol starts with the isomerization of isopentenyl pyrophosphate to dimethylallyl pyrophosphate by Hmgcs1.

Cpy17a1, an enzyme involved in steroid biosynthesis, also was expressed at lower levels in the SRC-2–/– mice. This enzyme is involved in the conversion of pregnenolone and progesterone to their 17-{alpha}-hydroxylated products and subsequently to dehydroepiandrosterone and androstenedione which are involved in sexual development during fetal life and at puberty. Altogether, SRC-2–/– mice reveal a marked alteration in energy metabolism pathways. Fatty acid degradation and glycolysis pathways are up-regulated and fatty acid, cholesterol, and steroid biosynthesis pathways are down-regulated in the liver. These results can explain why SRC-2–/– mice are protected against obesity due to an increase in energy expenditure (23).

In summary, we have identified that 270, 492, and 22 genes were differentially expressed in the liver of SRC-1–/–, SRC-2–/–, and SRC-3–/– mice, respectively. The results demonstrate that SRC-1, SRC-2, and SRC-3 exhibit specific and nonoverlapping activities in the liver. The overall pattern of altered gene expressions in the SRC-1–/– mice was one of up-regulation, indicating that SRC-1 is largely involved in gene repression in the liver tissue. SRC-1-induced gene repression has been observed previously in the thyroid metabolic pathway (55). SRC-2–/– mice revealed an overall down-regulation of target genes, indicating a largely promotional activity on target gene expression. The molecular targets of SRC-2 regulation in the murine liver were fatty acid degradation and glycolysis pathways that were up-regulated and fatty acid, cholesterol, and steroid biosynthesis pathways that were down-regulated. This analysis of downstream genes regulated by SRC-1, SRC-2, and SRC-3 has delineated the pathways these coactivators control liver physiology and explains much of the observed phenotypes in SRC-1 and SRC-2 knockout mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals and Tissue Collection
Mice were maintained in the designated animal care facility at the Baylor College of Medicine according to the institutional guideline for the care and use of laboratory animals. SRC-1–/–, SRC-2–/–, SRC-3–/–, and wild-type mice were ovariectomized at 6 wk of age. Two weeks later, the mice were killed by cervical dislocation after placing the mice under anesthesia, Avertin (2,2-tribromoethyl alcohol; Sigma-Aldrich, St. Louis, MO). Liver tissues were flash frozen at the time of dissection and stored at –80 C.

RNA Isolation and Microarray Hybridization
Total RNA was extracted from the liver tissues using the QIAGEN (Valencia, CA) RNeasy total RNA isolation kit. The RNA was pooled from the liver of five mice per genotype. All RNA samples were analyzed with a Bioanalyser 2100 (Agilent Technologies, Palo Alto, CA) before microarray hybridization. The fragmented, labeled cRNA (15 µg) was hybridized to Affymetrix mouse genome U74Av2 and 430 2.0 arrays (Affymetrix, Santa Clara, CA). All experiments were performed in triplicate with independent pools of RNA.

Data Analysis
Microarray data analysis was performed as described previously (56). After scanning and low-level quantification using Microarray Suite (Affymetrix), DNA-Chip analyser dChip version 1.3 was used to adjust arrays to a common baseline using invariant set normalization (57). To estimate expression, the Li and colleagues’ Perfect Match-only model was used (58, 59). All n =18 Genechips were normalized to the same baseline and modeled together. Data quality was reviewed using present call rates from the MAS5 program (average = 51.2%, range = 47.4 to 53.4%), ratios of 3' to 5' glyceraldehyde-3-phosphate dehydrogenase probe sets from MAS5 (average = 1.02, range = 0.91 to 1.16), and array outlier rates (average = 0.28%, range = 0.007 to 1.121) from dChip. Based on the above parameters, all chips were considered of good quality and were included in subsequent analyses. We selected differentially expressed genes in SRC-1–/–, SRC-2–/–, and SRC-3–/– mice using a two-sample comparison according to the following criteria: lower bound of 90% confidence interval of fold change greater than 1.2 and an absolute value of difference between group means greater than 80. The software employs resampling and SE of model parameters to partially account for measurement error and unstable estimates of variability (60). Finally, the median number of detected genes in 50 permuted samples was used as an overall estimate of the false discovery rate. After excluding expressed sequence tags with no functional annotation, differentially expressed genes were classified according to Gene Ontology function using GeneSrping version 7.0 (Silicon Genetics, Redwood City, CA) and GenMAPP (43).

Quantitative Real-Time PCR
Expression levels of selected genes found to be regulated by microarray analysis were validated by real-time RT-PCR TaqMan analysis using the ABI Prism 7700 Sequence Detector System according to the manufacturer’s instructions (PE Applied Biosystems, Foster City, CA). Prevalidated probes and primers for real-time PCR were purchased from Applied Biosystems. RT-PCRs were performed using One-Step RT-PCR Universal Master Mix reagent and TaqMan Gene Expression Assays (PE Applied Biosystems) according to the manufacturer’s instructions. Standard curves were generated by serial dilution of a preparation of total RNA isolated from mouse liver. All real-time PCR was performed by using the three independent RNA sets. mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents.

Western Blot Analysis
Mouse liver tissue were washed with PBS solution and homogenized in a buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2.5 mM EDTA, and 0.125% Nonidet P-40 (vol/vol). Cellular debris was removed by centrifugation at 14,000 rpm for 15 min at 4 C. Protein concentration was determined by Bradford’s method using BSA as the standard. Samples containing 30 µg protein were applied to 10% SDS-PAGE. The separated proteins were then transferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Membranes were blocked overnight with 5% skim milk (wt/vol) in PBS with 0.1% Tween 20 (vol/vol) (Sigma-Aldrich) and probed with anti-GLUT2 or anti-GCK diluted to 1:1000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoreactivity was visualized by incubation with a horseradish peroxidase-linked second antibody and treatment with ECL reagents. To control for loading, the membrane was stripped, probed with anti-actin (Santa Cruz Biotechnology, Inc.) at 1:1000 dilution and developed again. Quantification of the scanned bands was accomplished by digital image analysis using Kodak 1D Scientific Imaging Systems (Kodak, New Haven, CT).

Immunohistochemistry
For immunohistochemistry, liver was fixed overnight in 10% formalin, followed by thorough washing in 70% ethanol and tissues were processed, embedded in paraffin, and sectioned. Liver sections from paraffin-embedded tissue were cut at 6 µm and mounted on slides, deparafinized, and rehydrated in a graded alcohol series. Sections were preincubated with 10% normal rabbit or goat serum in PBS (pH 7.5) and then incubated with primary antibodies diluted in 10% normal rabbit or goat serum in PBS (pH 7.5). Immunohistochemistry for SRC-1 was performed using a rabbit polyclonal antibody to mouse (Santa Cruz Biotechnology, Inc.). Anti-SRC-2 antibody was kindly provided by Jun Qin of Baylor College of Medicine. Anti-GLUT2 and anti-GCK antibodies were obtained from Santa Cruz Biotechnology, Inc. Immunoreacitivity was detected by using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA); the immunorectivity was visualized as brown staining.

ß-Galactosidase Activity Staining
For ß-gal detection, frozen liver sections were fixed in ice-cold 4% paraformaldehyde for 5 min. Fixed sections were subsequently rinsed in PBS. Liver sections were then immersed in lacZ staining solution [2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6.3H2O, 0.02% Nonidet P-40, 0.01% deoxycholate and 1 mg/ml 4-chloro-5-bromo-3-indolyl-ß-D-galactopyranoside in 100 mM phosphate buffer (pH 8.0)] at room temperature in the dark, for at least 2 h and up to 16 h depending on the degree of color reaction (Sigma-Aldrich).

Determination of Glycogen Content
Hepatic glycogen was purified from whole liver according to an earlier report (61). Frozen tissue samples (30–50 mg) from SRC-1–/– (n = 3), SRC-2–/– (n = 3), and wild-type mice (n = 3) were digested in a suitable volume of 30% KOH (wt/vol) saturated with Na2SO4 for 30 min at 100 C. Glycogen was precipitated by the addition of 1.2 vol absolute ethanol and incubation on ice for 30 min. The samples were centrifuged at 900 x g for 30 min and the pellet was dissolved in 3 ml distilled water. Glycogen was then hydrolyzed by digestion in 5 ml of sulfuric acid for 10 min at 100 C. The glycogen content was measured to read at 490 nm on a spectrophotometer. Glycogen standard solutions from bovine liver were used to calculate the concentration of glycogen (Sigma-Aldrich).


   ACKNOWLEDGMENTS
 
We thank Jinghua Li, Bryan Ngo, and Janet DeMayo, M.S., for technical assistance. We gratefully acknowledge Jun Qin for the antibody against SRC-2. TIF2 knockout mice (referred to herein as SRC-2–/– for clarity) were constructed by the P. Chambon laboratory and given to us as a gift.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK59820.

Jae-Wook Jeong, Inseok Kwak, Kevin Y. Lee, Lisa D. White, Xiao-Ping Wang, F. C. Brunicardi, Bert W. O’Malley, and Francesco J. DeMayo have nothing to declare.

First Published Online January 19, 2006

Abbreviations: CoA, Coenzyme A; ER, estrogen receptor; GCK, glucokinase; SRC, steroid receptor coactivator.

Received for publication October 11, 2005. Accepted for publication January 9, 2006.


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