Suppressor of Cytokine Signaling-2 Deficiency Induces Molecular and Metabolic Changes that Partially Overlap with Growth Hormone-Dependent Effects
Elizabeth Rico-Bautista,
Christopher J. Greenhalgh,
Petra Tollet-Egnell,
Douglas J. Hilton,
Warren S. Alexander,
Gunnar Norstedt and
Amilcar Flores-Morales
Department of Molecular Medicine, Karolinska Institute (E.R.-B., P.T.-E., G.N., A.F.-M.), Stockholm 17176, Sweden; and Walter and Eliza Hall Institute of Medical Research and the Cooperative Research Center for Cellular Growth Factors (C.J.G., D.J.H., W.S.A.), Melbourne, 3050 Victoria Australia
Address all correspondence and requests for reprints to: Dr. Amilcar Flores-Morales, CMM, L8:01, Karolinska Hospital, 17176 Stockholm, Sweden. E-mail: amilcar.flores{at}cmm.ki.se.
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ABSTRACT
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Suppressor of cytokine signaling-2 (SOCS2)-deficient (SOCS2/) mice grow significantly larger than their littermates, suggesting that SOCS2 is important in the negative regulation of the actions of GH and/or IGF-I. The aim of this study was to identify genes and metabolic parameters that might contribute to the SOCS2/ phenotype. We demonstrate that although SOCS2 deficiency induces significant changes in hepatic gene expression, only a fraction of these overlap with known GH-induced effects in the liver, suggesting that SOCS2 might be an important regulator of other growth factors and cytokines acting on the liver. However, an important role of GH and IGF-I in the phenotype of these animals was demonstrated by an overexpression of IGF-binding protein-3 mRNA in the liver and increased levels of circulating IGF-binding protein-3. Other GH-like effects included diminished serum triglycerides and down-regulation of lipoprotein lipase in adipose tissue. Interestingly, SOCS2/ mice did not differ from their wild-type littermates in glucose or insulin tolerance tests, which is in contrast with the known diabetogenic effects of GH. Furthermore, there was no evidence of impaired insulin signaling in primary hepatocytes isolated from SOCS2/ mice. Moreover, increased expression of peroxisome proliferator-activated receptor-
coactivator-1
mRNA was detected in skeletal muscle, which might contribute to normal glycemic control despite the apparent overactivity of the GH/IGF-I axis. Our data indicate that SOCS2 deficiency partially mimics a state of increased GH activity, but also results in changes that cannot be related to known GH effects.
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INTRODUCTION
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THE SUPPRESSORS OF cytokine signaling (SOCS) are a family of intracellular proteins involved in the negative regulation of cytokine signal transduction (1, 2). The finding that SOCS2-deficient (SOCS2/) mice (3) display a considerable (3040%) increase in mature body size compared with their wild-type littermates indicates that SOCS2 is important for the negative regulation of body growth. The enhancement in body weight was associated with increased muscle mass and skeletal size, changes linked to increased cell number rather than cell size. This phenotype is very similar to those observed in human acromegaly and in mice with elevated levels of GH (4) or IGF-I (5). It has also been shown that the molecular basis for high growth (hg) phenotype, a spontaneous mutation in mice that causes a 3050% increase in postnatal growth, is caused by a genomic deletion that spans a part of the SOCS2 gene, rendering it nonfunctional (6). Although a large number of nutritional and hormonal factors can regulate growth, only GH is able to stimulate longitudinal bone growth in a specific- and dose-dependent manner (7). Taken together, various data from the literature indicate that increased growth in SOCS2-deficient animals is a consequence of increased GH and/or IGF-I signaling, although definitive evidence is lacking.
Unraveling the mechanism of SOCS2 action in relation to GH receptor signaling has been complicated by the fact that the SOCS proteins display a high degree of promiscuity in vitro. For example, SOCS2 has been shown to be active against GH, prolactin (8, 9), and IGF-I receptors (10, 11), and several SOCS proteins (SOCS1, SOCS2, SOCS3, and cytokine-inducible SH2-domain-containing protein) have been shown to inhibit GH signaling when forcedly expressed in cell cultures (8, 12). SOCS2-deficient animals offer a way to study the role of SOCS2 in vivo. The various investigations of SOCS2/ mice have already shown a number of features that support a role for altered GH signaling in relation to their altered phenotype, such as increased local concentration of IGF-I in extrahepatic tissues. Moreover, signal transducer and activator of transcription-5b, a GH-activated transcription factor, has been shown to be required for the growth phenotype of SOCS2/ mice (13). A small but significantly increased sensitivity toward GH-induced STAT5 activation was also evident in primary hepatocytes from SOCS2/ mice compared with cells from normal animals (13). Other features, such as normal levels of circulating IGF-I (mainly liver-derived), speak against a general increase in GH signaling. More studies are therefore required before a definitive conclusion can be reached regarding SOCS2 in relation to GH signaling in different tissues.
The liver expresses high amounts of GH receptors and is considered one of the main targets for GH actions (14). Consequently, if SOCS2 is a regulator of hepatic GH action, a detailed analysis of hepatic gene expression in SOCS2/ mice should reveal potential similarities to known GH-dependent transcriptional effects in rodent liver. In addition, because GH has well documented and distinct effects on hepatic and extrahepatic metabolic pathways (15), studying the metabolic features of SOCS2/ mice might provide clues regarding the involvement of GH-dependent and independent changes that could contribute to the SOCS2/ phenotype. The aim of this study was to identify the molecular consequences of SOCS2-deficiency in terms of alterations in hepatic gene expression and metabolic parameters and to examine whether putative changes might be related to altered hepatic GH receptor signaling. We therefore analyzed the effect of deleting SOCS2 from male mice on hepatic gene expression using cDNA microarray analysis. With the array data as a point of entry, additional experiments were conducted related to the metabolic state of the animals. We show that SOCS2 deficiency induces specific molecular and metabolic changes that partially overlap with known GH-dependent effects.
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RESULTS
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Absence of SOCS2 Induces Changes in Hepatic Gene Expression with Limited Relation to Known Hepatic GH Actions
To obtain more information about the consequences of SOCS2 deficiency, microarray analysis was used to define the set of hepatic genes differentially expressed between SOCS2-deficient and wild-type mice. The liver was chosen because it is one of the main targets of GH action and is known to express high levels of SOCS2 (16). Of 6000 genes printed on the microarray, approximately 3100 were detected in the mouse liver, but only 105 were differentially expressed in a statistically significant manner. Among these, 30 unique transcripts displayed a greater than 50% induction in expression levels in mice lacking the SOCS2 gene, whereas 21 were reduced. Table 1
shows the list of genes differentially expressed in mice lacking SOCS2. Six genes were chosen for validation by realtime RT-PCR. Lipoprotein lipase (Lpl), IGF-binding protein-3 (IGFBP3), and stearyl coenzyme A desaturase (SCoAD) were increased in mice lacking SOCS2, whereas serine threonine kinase PIM-3, phosphoenolpyruvate carboxykinase (PEPCK) and 12
-hydroxylase (CYP8B1) were decreased, confirming the microarray data (Fig. 1
).

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Fig. 1. Real-Time Quantitative RT-PCR Measurement
Six genes with altered expression in SOCS2/ mice according to the microarray results were selected for real-time RT-PCR analysis. The figure represents the results obtained from duplicate measurements from individual animals (n =4). The values obtained for wild-type and SOCS2/ animals were normalized using ß-actin expression measurements. To calculate the fold differences, each measurement for each animal (both wild-type and SOCS2/) was divided by the average of the control group. Statistical analysis of the data was performed by unpaired, two-tailed t test. *, P < 0.05. AU, Arbitrary units.
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The changes in gene expression described above might be due to SOCS2-dependent alterations in GH receptor signaling, but they could also be unrelated to GH or, most likely, a mixture of both. To investigate this, the gene expression profile derived from mice lacking SOCS2 was compared with previously obtained microarray data regarding the effects of various hormone (GH and T3) and cytokine [IL-6 and interferon-
(IFN-
)] on the liver, and a newly generated profile of hepatic gender differences in mice. Average linkage hierarchical clustering using Pearson correlation coefficient, as a measurement of similarity, was used to evaluate the relationship between the experiments (17) (Fig. 2A
). In the present study we found that gene expression changes due to SOCS2 deficiency bear similarity to the transcript profile obtained by comparing female and male hepatic expressions. Other profiles with similarities to SOCS2 deficiency were found in experiments investigating the effects of 1) hepatic SOCS3 deficiency, 2) IL-6, or 3) GH treatments. This meta-analysis of expression profiles obtained from different platforms has previously been used by others in the classification of different types of tumors (18). Although this method has been successfully used in numerous studies to discover mechanistic relationships between different biological samples, there are limitations to this approach (see Materials and Methods for more information). The validity of this approach in the present investigation is strengthened, however, by the fact that we obtained results that have already been described in studies using other techniques (19, 20). The correlation between SOCS2- and GH-dependent changes was not as strong as we would have expected based on the SOCS2/ growth phenotype (Fig. 2B
). Several well known GH-regulated hepatic gene products (IGF-I, acid-labile subunit, carbonic anhydrase 3, serine protease inhibitor, or P450 enzymes) were not affected by the absence of SOCS2. However, other GH-regulated genes, such as major urinary protein (MUP), glucose-6-phosphate dehydrogenase (G6PD), SCoAD, and PEPCK (Table 1
), did have altered expression levels in SOCS2/ mice, suggesting limited similarities to GH-dependent long-term effects. A small positive correlation existed between SOCS2-dependent genes and gender-related gene expression (Fig. 2B
). Sex differences in hepatic gene expression have a well established relationship to the sexually differentiated pattern of GH secretion. GH treatment in a continuous manner (minipumps) mimics the female GH secretory pattern, whereas pulsatile GH (injections) controls a male type of hepatic gene expression (21). Of 200 genes showing gender-differentiated expression levels in the mouse, 17 were also significantly altered in the SOCS2/ mouse (Table 1
). Fourteen of these genes were shown to be expressed in a female-predominant manner. These 14 genes constitute 35% of all genes showing an altered expression in SOCS2-deficient mice. This proportion is higher than expected, considering that less than 5% of the genes measured in mouse liver showed gender-related differences in expression levels. A lack of SOCS2 results in the down-regulation of MUP and G6PD mRNA, which are known to be regulated by gender and GH (19, 22). It is also known that hepatic fatty acid (FA)-binding protein levels are normally higher in female than in male mice (23). These results may indicate that SOCS2 does influence a subset of GH actions in the liver. Additional analysis would be needed to determine to what extent the changes in gene expression detected in SOCS2-deficient animals result in a feminized hepatic function.

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Fig. 2. Hierarchical Clustering
The figure represents a hierarchical cluster of hepatic gene expression profiles using average linkage analysis and Pearson correlation coefficient as distance metric. A, Similarities between profiles are shown as a hierarchical tree. The data include gene probes where expression changes of more than 50% were detected in at least one of the experiments. The expression profiles included are described in Materials and Methods. Briefly, the gene expression profiles of GH treatments, female/male in rat and mouse, SOCS2 deficiency, SOCS3 deficiency, and the effects of IL-6 and IFN- were used. B, The figure represents the Pearson correlation coefficient calculated for each of the gene expression profiles compared with the SOCS2-deficient mice gene expression profile.
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Increased Expression of IGFBP3 in SOCS2-Deficient Mice
It is well established that regulation of body growth depends on circulating IGF-I (24, 25). In agreement with previous findings showing no difference between SOCS2/ mice and wild-type in IGF-I serum levels, hepatic IGF-I mRNA expression was not affected in SOCS2/ mice (3). Nevertheless, we found that hepatic expression of IGFBP3 mRNA was higher in these animals. This finding is of particular interest because IGFBP3 expression is known to be induced by GH, and only a few GH-regulated hepatic genes were found to be overexpressed in the present study of giant SOCS2/ mice. IGFBP3 is known to exert effects on cell proliferation independently of IGF-I and has regulatory roles on IGF-I action in target tissues (26). Furthermore, IGFBP3 transgenic mice show organomegaly (27), one of the features observed in SOCS2/ mice. Hepatic synthesis is the most important contributor to circulating levels of IGFBP3 (28), but because IGFBP3 is expressed in a variety of tissues where it can exert local actions, we decided to quantify its expression in extrahepatic tissues (Fig. 3A
). No significant differences in IGFBP3 mRNA expression levels were observed in adipose tissue or cardiac muscle. Although there was a tendency for higher (2-fold) levels in skeletal muscle in SOCS2/ animals, this was not statistically significant. Consequently, we performed Western blot analysis using serum from wild-type and SOCS2/ animals and found significantly higher levels of circulating IGFBP3 in SOCS2/ animals (Fig. 3B
), in agreement with hepatic gene expression data.

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Fig. 3. IGFBP3 Expression in Extrahepatic Tissues and Levels of Circulating Protein
A, Quantification by real-time RT-PCR was used to measure IGFBP-3 expression in adipose tissue and skeletal and cardiac muscle. The figure represents the results from replicated measurements of samples obtained from individual animals (n = 4). Values for wild-type animals were set at 1, as described in Fig. 1 . Statistical analysis of the data was performed by unpaired, two-tailed t test. *, P < 0.05. B, IGFBP-3 levels were measured in serum from individual animals, either wild-type (lanes 14) or SOCS2/ (lanes 58), by immunoblotting. The blot is representative of at least three independent experiments.
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Analysis of Metabolic Parameters in SOCS2/ Animals
Increased growth is associated with defined changes in metabolism in models of GH overexpression. Some of these changes are due to the direct effects of GH, whereas others are mediated by IGF-I. Prompted by the finding that a limited number of GH-dependent genes were found to be regulated in SOCS2/ mice, and that several of the identified SOCS2-dependent gene products are involved in metabolic control, we decided to investigate the metabolic phenotype of these animals. Serum levels of creatinine, total protein, albumin, globulin, cholesterol, triglycerides (TG), glucose, and insulin were measured in animals fed ad libitum (Table 2
). TG levels were significantly reduced in the knockout mice compared with their wild-type littermates (P < 0.05). These characteristics overlap with the phenotype of bovine GH transgenic animals, in which circulating levels of TG are also low compared with those in wild-type mice (29).
Lower circulating levels of TG can be the result of impaired absorption of dietary lipids, diminished hepatic synthesis, or increased TG clearance in the liver or extrahepatic tissues. Lpl is a key enzyme in the regulation of uptake of FA in adipose, skeletal, and cardiac muscle tissues. SOCS2-deficient livers expressed significantly elevated levels of Lpl mRNA, a feature also seen in livers of bovine GH transgenic mice (30). This prompted us to quantify relative Lpl concentrations in cardiac and skeletal muscles and in fat tissue by real-time RT-PCR (Fig. 4
). In contrast to that in liver, Lpl mRNA expression was decreased in adipose tissue and cardiac muscle, whereas no changes were observed in skeletal muscle in SOCS2-deficient animals. When the hepatic content of lipids in SOCS2/ mice was measured, TG levels were significantly lower in knockout animals (Fig. 5A
). The decreased concentration of TG in liver is contradictory to the known lipogenic effect of GH treatment (19) and indicates that the metabolic profile in SOCS2/ mice is distinct from those described in GH overexpression models. A reduced production and secretion of hepatic TG might explain the reduced levels in serum. Alternatively, increased biosynthetic requirements due to increases in skeletal muscle mass could also contribute to increased utilization of FA as an energy source in SOCS2/ mice. The peroxisome proliferator-activated receptor-
(PPAR
) coactivator-1
(PGC-1
) is a key regulator of energy homeostasis in skeletal muscle through the regulation of mitochondrial biogenesis and oxidative phosphorylation. Therefore, we measured the expression of PGC-1
in skeletal muscle of SOCS2/ mice and found it to be increased more than 2-fold compared with muscle from control mice (Fig. 5B
). In contrast, the expression of carnitine palmitoyl transferase I, a key regulator of FA ß-oxidation, did not show differences between the two models (Fig. 5C
), suggesting that the levels of skeletal muscle ß-oxidation do not differ between SOCS2/ mice and controls.

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Fig. 4. Lpl Expression in Extrahepatic Tissues
Lpl expression was quantified by real-time RT-PCR in adipose tissue and skeletal and cardiac muscle. The figure represents the results from duplicated measurements of samples obtained from individual animals (n = 4). Values for wild-type animals were set at 1, as described in Fig. 1 . Statistical analysis of the data was performed by unpaired, two tailed t test. *, P < 0.05.
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Fig. 5. Lipid Content in Liver and PGC-1 Expression
A, Total cholesterol and triglycerides were determined in hepatic lipid extracts. Data are expressed as micromoles of lipid per gram of liver and represent the mean ± SD. A t test was used for determination of statistical significance. *, P < 0.01. Real-time PCR was used to quantify the expression of PGC-1 (B) and carnitine palmitoyl transferase I (C) in skeletal muscle. The figures represent the results obtained from duplicated measurements from individual animals (n =4). Statistical analysis of the data was performed by unpaired, two-tailed t test. *, P < 0.05.
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GH transgenic mice are hyperinsulinemic and resistant to insulin, as judged from the insulin/glucose ratio measurements (30). IGF-I is known to oppose the antiinsulin-like effects of GH. The circulating IGFBP-3 complex is important for the insulin-like actions of IGF-I. Our finding of increased circulating levels of IGFBP-3 in SOCS2/ mice suggests an important role for IGF-I in the metabolic phenotype of SOCS2/ mice that may explain the absence of diabetogenic features. This hypothesis is reinforced by the finding that PGC-1
, a positive regulator of glucose utilization, had increased expression levels in muscle from SOCS2/ mice. To test this hypothesis, we performed glucose and insulin tolerance tests in SOCS2/ mice (Fig. 6
, A and B). In contrast to what is found in acromegalic patients and GH overexpressing models, no evidence of glucose intolerance or insulin resistance was found in SOCS2/ compared with wild-type littermates. Accordingly, no differences were observed in phosphorylation of the insulin receptor after insulin treatment of primary hepatocytes derived from wild-type or SOCS2/ mice (Fig. 6C
). Similar levels of phosphorylation of the insulin receptor were evident at 10 min poststimulus in both models. No differences existed in AKT (or protein kinase B) phosphorylation, strongly suggesting that SOCS2-deficient mice are not insulin resistant in liver.
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DISCUSSION
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The microarray analysis comparing hepatic gene expression in SOCS2/ and wild-type mice showed that SOCS deficiency induces changes that are only partly overlapping with GH-dependent expression changes. Altered expression levels of well known GH-regulated hepatic transcripts, such as IGFBP3 and MUP, were indeed found in the SOCS2/ mice, but most of the SOCS2-dependent gene products have not been shown to be regulated by GH. This is paradoxical given the known antagonistic effects of SOCS2 overexpression on GH receptor signaling (12) and the phenotypical similarities between SOCS2/ mice and GH overexpression models regarding somatic growth. An initial interpretation is that the excessive growth phenotype of SOCS2/ animals may be due to the activation of a small subset of hepatic GH-regulated genes. Alternatively, increased GH receptor signaling in extrahepatic target tissues, such as bone and muscle, might be more important than the hepatic effects of SOCS2 deficiency. The regulatory role of SOCS2 in terms of GH signaling in these tissues might be greater than expected.
A large body of evidence supports the idea that a discrete number of GH-regulated genes have major roles in the regulation of somatic growth. Of special importance is the role of IGF-I. Inactivation of this gene or its receptor severely impairs growth (31). The increased circulating IGFBP-3 levels in SOCS2/ mice described here could be highly relevant, because these animals show elevation of IGF-I expression in several extrahepatic tissues (cardiac muscle, lung, and spleen) (3). IGFBP-3 is the most abundant IGFBP in the circulation and modulates the actions of IGF-I (26). Previous findings indicate that GH regulates IGFBP-3 expression in liver (32, 33). IGFBP-3 acts as a reservoir and carrier for IGF-I, resulting in an increased half-life in the circulation and delivery to peripheral tissues. In cell models, IGFBP-3 can have positive and negative effects on IGF-I actions (34, 35). The idea that IGFBP-3 plays a positive role in organ growth comes from the finding that IGFBP-3 transgenic mice show organomegaly of the liver, spleen, lung, and epididymal fat pad, although the size of these animals was not different from that of their littermates (27). Moreover, an elevation of IGFBP-3 in the circulation positively correlates with growth velocity in children treated with GH (36). The increased circulating IGFBP-3 levels in SOCS2/ mice are most likely liver-derived, which, in combination with locally increased IGF-I production, could contribute to the excessive growth of SOCS2/ animals. This might also have important consequences for the metabolic features of these mice.
GH has well known diabetogenic effects. Long-term GH treatment in rats, GH transgenic mice, and acromegalic patients show hyperinsulinemia, insulin resistance, and hyperglycemia (37). In contrast, SOCS2/ mice have normal glucose tolerance and insulin sensitivity. Animals that have reduced circulating levels of IGF-I due to hepatic-specific gene inactivation grow normally, but have a 4-fold increase in circulating GH, with marked insulin resistance. The insulin insensitivity can be reversed by treatment with IGF-I or GH-releasing hormone antagonist or by antagonizing the GH receptor (38). It has also been demonstrated that in acromegalic patients, treatment with the IGF-I/IGFBP-3 complex improves insulin sensitivity (39). Therefore, the normal glycemic control in SOCS2/ mice could be a consequence of the insulin-like actions of IGF-I, supporting an important role for IGF-I/IGFBP-3 in SOCS2/ mice. The liver is the main gluconeogenic tissue, and liver-specific changes detected in SOCS2/ mice may also contribute to the phenotype independently of IGF-I. SOCS2-deficient mice have reduced levels of PEPCK expression, a rate-limiting enzyme in the gluconeogenic process, and increased expression of hexokinase, the first enzyme in the glycolytic pathway. This could result in reduced hepatic glucose output and increased intrahepatic glucose oxidation, contributing to normal glycemic control in SOCS2/ mice. Studies have revealed that acute exposure to IGF-I in parallel with insulin decreases hepatic glucose output and increases glucose uptake (40). Finally, the increased expression of PGC-1
in muscle of SOCS2/ mice could be important in the regulation of glucose homeostasis in these animals. Recent studies showed that diabetic and prediabetic individuals with impaired glucose tolerance have moderately reduced muscle expression of genes involved in oxidative phosphorylation, which is linked to diminished levels of PGC-1
(41). Increased PGC-1
expression and oxidative phosphorylation might compensate for the increased energy requirements linked to enhanced skeletal muscle growth in SOCS2/ mice. An increased catabolism of glucose, amino acids, FA, or ketone bodies would be required for increased oxidative phosphorylation, which might have positive consequences for glucose homeostasis. Preliminary results from our laboratory show that PGC-1
is rapidly induced by GH in skeletal muscle of GH-deficient mice (data not shown). This suggests that increased PGC-1
expression in SOCS2/ mice could be a consequence of increased GH receptor signaling in muscle.
Gigantism, as observed in SOCS2/ mice, is a unique feature of GH oversecretion that cannot be mimicked by IGF-I or IGFBP-3 overexpression. In the latter cases, excessive growth is limited to organomegaly of several organs, but not to longitudinal growth. In contrast, a minimal amount of circulating IGF-I is required for normal growth, and increased IGF-I and IGFBP-3 levels are commonly associated with gigantism (42). It has been proposed that a direct action on the bone growth plate accounts for the effects of GH on longitudinal growth (43). Alternatively, the unique diabetogenic actions of GH in metabolism, such as increased lipolysis in adipose tissue and lipid mobilization from liver (44), could constitute a requirement that cannot be mimicked by IGF-I alone. Here we show that SOCS2/ mice have significantly reduced levels of triglycerides compared with to their littermates in both serum and liver. This suggests that FA uptake and usage might be increased, in line with the proposed actions of GH (15). The increased expression of liver Lpl in SOCS2/ mice might contribute to the increased hepatic clearance of circulating TG and lead to increased intrahepatic supply of nonesterified FA. The up-regulation of FA-binding protein and SCoAD, two genes involved in the transport and desaturation of FA (45, 46), supports the hypothesis that hepatic FA uptake and usage might be increased. Because there was no increased concentration of hepatic TG levels, FA might be used for energy and/or biosynthetic purposes within the liver. Alternatively, FA could be exported from the liver and used in extrahepatic tissues, as either FA or ketone derivatives. As explained above, the increased expression of PGC-1
in muscle would favor increased fuel catabolism. The decreased Lpl expression in heart and adipose tissue observed in SOCS2/ mice mimics the lipolytic antiinsulin-like action of GH in adipose tissue. GH treatment has been found to diminish Lpl activity in adipose tissue of GH-deficient adults (47). The relevance of these changes is highlighted by the observation that abdominal adipose tissue is one of few tissues that does not increase in mass in SOCS2/ male animals.
The excessive growth of SOCS2/ animals is characterized by increases in tissue and organ size due mostly to an increase in the number of cells instead of the size of the cells, inferring that SOCS2 plays a central role in regulating cell proliferation. This hypothesis is supported by microarray analysis of liver expression. The profile reveals significant inhibition of negative regulators of cell proliferation, such as inhibitor of DNA binding 3 and growth arrest-specific-1 (48, 49), and the induction of positive regulators, such as hepatocellular carcinoma-enriched 33 kDa (Keg1 in mouse) (50), apoptosis inhibitor 6 (also known as CD5-like) (51), retinol-binding protein (52), and G6PD (53). Most of these genes are not known to be regulated by GH or IGF-I, suggesting that other, as yet unidentified, pathways could be targeted by SOCS2.
In summary, we have analyzed hepatic gene expression changes induced by SOCS2 deletion in mice as well as some physiological and metabolic features. Despite obvious phenotypic similarities to GH overexpression models, SOCS2-deficient mice display specific expression and metabolic changes. Similarities to known GH effects are limited to a subset of hepatic GH-regulated genes and metabolic parameters, such as serum triglyceride levels. Other features, such as the unaltered glycemic control, not found in GH overexpression models could be mediated by increased activity of IGF-I through increased expression of IGFBP-3. Furthermore, significant changes in the expression levels of a number of genes involved in regulating cell proliferation suggest that SOCS2 plays an important regulatory role in the actions of other growth factors and cytokines. The metabolic changes in relation to glucose and lipid metabolism suggest that the growth-promoting actions of GH can be dissociated from its diabetogenic effects. It seems rational to suggest that the inhibition of SOCS2 would result in beneficial effects that mimic some of the GH anabolic, lipolytic, and growth-promoting activities while avoiding the diabetogenic actions associated with direct GH treatment.
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MATERIALS AND METHODS
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Animals
Wild-type C57BL/6 and SOCS2-deficient male littermates (3) were maintained under standardized conditions of light and temperature with free access to animal chow and water. All animals were kept in the same environment until they were killed. Blood samples were taken, and serum was obtained and stored at 80 C. Liver, adipose tissue, and skeletal and cardiac muscle were removed, frozen in liquid nitrogen, and stored at 80 C for additional analysis. The animal experiments were performed in accordance with guidelines of the National Health and Medical Research Council of Australia.
cDNA Microarray Analysis
Generation of cDNA microarrays has been described previously (54). The arrays represented approximately 6200 cDNA clones selected from the TIGR Gene Index (www.tigr.org), Research Genetics (www.resgen.com/), and our own collection of rat and mouse cDNA libraries enriched with hepatic genes of known function. Total RNA was isolated by homogenization of liver tissue using a Polytron PT-2000 (Kinematica AG, Basel, Switzerland) and TRIzol reagent (Invitrogen Life Technologies, Inc., Grand Island, NY), according to the protocol supplied by the manufacturer. The quality of the RNA samples was examined on a denaturing agarose gel. Four independent hybridizations were performed, comparing individual animals from the different experiment groups (four wild-type and four SOCS2/ mice) each time. The protocol employed for probe labeling and purification was essentially as described previously (55). Total liver RNA (25 µg) was used in each of the labeling reactions. Labeled cDNA was produced by an oligo(deoxythymidine)-primed RT reaction using Superscript II (Invitrogen Life Technologies, Inc.). Fluorescent nucleotides (Cy3 or Cy5; Pharmacia Biotech, Uppsala, Sweden) were used to label RNA samples from SOCS2/ and wild-type mice. Dye swaps were included in the experimental design. The labeled and purified cDNA was added to the array and placed in a sealed hybridization chamber (Corning Glass, Corning, NY) for 1518 h at 65 C, after which the array was washed and dried.
The arrays were scanned immediately after hybridization using a GMS 418 scanner (Affimetrix, Palo Alto, CA). Image analysis was performed using the GenePix Pro software (Axon Instruments, Union City, CA). Automatic flagging was used to localize absent or very weak spots, which were excluded from analysis. Fluorescence ratios were normalized as described previously (56) using the LOWESS (Locally Weighted Scatter Plot Smoother) method in the SMA (Statistics for Microarray) package. SMA is an add-on library written in the public domain statistical language R (57). The variability of the analysis was estimated using SAM software (Significance Analysis of Microarray) (58). A q value was assigned for each of the detectable genes in the array. This value is similar to the P values, measuring the lowest false discovery rate at which the differential expression (the ratio between wild-type and SOCS2 knockout cDNA) of the gene is considered significant. In this study, genes with a false discovery rate of less than 12% were listed as differentially expressed. An additional requirement was added to this statistically based criterion to correct for differential expression based on the absolute changes in gene expression ratios. A value of 1.5 (50%) was chosen to denote differences (increased or decreased expression) in the level of hybridization between wild-type and knockout cDNA. Although lower significant levels of changes in gene expression may have important biological consequences, insufficient information exists regarding reproducibility by independent methodologies. The complete list of significantly regulated genes is available at www.cmm.ki.se/EndoGED.
Total RNA (25 µg) from six male and six female mice was pooled and hybridized in a dye swap protocol. Gender-predominant genes in mice were determined by microarray as described above, and the results were compared with those obtained from the SOCS2 knockout analysis. The comparison was performed only with genes that demonstrated average gender differences of 50% or more.
Gene Expression Analysis by Real-Time Quantitative RT-PCR
The expressions of Lpl, SCoAD, IGFBP-3, serine-threonine kinase PIM-3, PEPCK, and 12
-hydroxylase (CYP8B1) were measured using Light-Cycler quantitative real-time PCR (Roche, Basel, Switzerland) in RNA samples from individual animals. The experimental procedure was carried out as described previously with some modifications (56). Briefly, 5 µg total RNA were treated with deoxyribonuclease I (Promega Corp., Madison, WI) and then reverse transcribed using a first strand cDNA synthesis kit from Invitrogen Life Technologies, Inc., in a 20-µl reaction. Gene-specific primers corresponding to the target genes were used to generate amplicons (Table 3
).
A relative standard curve was constructed with serial dilutions (1:1, 1.10, and 1:100) using a pool of cDNA generated from all eight animals used in the study. To measure the relative concentration of gene expression, 2 µl each of the unknown samples (dilution, 1:3) were analyzed in duplicated. Real-time PCR was performed in a 20-µl volume with 2 µl of the respective cDNA, 0.4 µM primers, and 4 mM MgCl2. Nucleotides, Hot-Start Taq polymerase, reaction buffer, and SYBR Green I dye were supplied in the Light-Cycler DNA Master SYBR Green I Kit (Roche). The amplification program consisted of one cycle of 95 C for 10 min (hot start), followed by 45 cycles of 95 C for 15 sec, with annealing at 55 C for 10 sec and 72 C for 30 sec; fluorescence intensity was measured at a specific acquisition temperature for each gene. The level of individual mRNA measured in the real-time RT-PCR was normalized with the level of ß-actin in each sample.
Serum Analysis
Serum samples from 10 wild-type and 10 SOCS2/ 6-wk-old male mice were analyzed using standardized protocols (IDEXX-Central Clinical Diagnostic Laboratory, Mount Waverly, Australia). All animals were fed ad libitum. Blood was taken from the orbital sinus, and glucose concentrations were measured using an Advantage glucometer and Advantage II glucose blood test strips (Roche). Serum insulin concentrations were measured by RIA (Insulin RIA Kit, Pharmacia-Upjohn, Uppsala, Sweden) using a rat insulin standard (Novo Nordisk, Copenhagen, Denmark). The results are expressed as the mean ± SD. A t test was used for determination of statistical significance.
Glucose and Insulin Tolerance Tests
For glucose tolerance, male age-matched mice (five SOCS2/ and six wild type) fasted overnight were given an ip injection of 1.2 g D-glucose/kg body weight in a solution of 0.25 g/ml. For insulin tolerance, age-matched male mice fed ad libitum were given an ip injection of 0.75 IU (Ultratard) insulin/kg body weight in a solution of 0.25 IU/ml. Blood was collected from the orbital sinus at regular intervals, and glucose concentrations were determined as described above.
Hepatic Lipid Content
The liver content of total cholesterol and triglycerides was determined after homogenization of frozen liver (50 µg) in 1 ml methanol, followed by extraction of total lipids with 2 ml chloroform. The extracts were evaporated until dry and dissolved in 200 µl isopropanol. The lipid contents were determined using kits for total cholesterol (ABX Diagnostic) and triglycerides (Roche). Samples were analyzed from individual animals in duplicate, and the results were determined as micromoles of lipid per gram of liver tissue. The results are expressed as the mean ± SD. A t test was used for the determination of statistical significance.
Immunoblotting for IGFBP3 in Serum
Serum samples (1 µl) from four wild-type and four SOCS2-deficient animals were resolved by SDS-PAGE on 10% premade polyacrylamide gels and Western blotted with a Trans-Blot SD semidry transfer cell (Hoefer, Pharmacia Biotech) to polyvinylidene difluoride membranes. The filters were blocked for 1 h at room temperature in 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween, pH 7.4, containing 5% skim milk powder. Membranes were incubated overnight at 4 C with antimouse IGFBP-3 (1:1000; GroPep, Adelaide, Australia). After three 10-min washes in 20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween, pH 7.4,, binding of primary antibody was visualized using horseradish peroxidase-conjugated secondary antibodies, and immunolabeling was detected by the enhanced chemiluminescence method according to the manufacturers instructions (Pierce Chemical Co., Rockford, IL).
Insulin Signaling in Primary Hepatocytes
Primary hepatocytes from wild-type and SOCS2/ animals were obtained using a collagenase perfusion as described previously (13) and plated on Matrigel. Isolated hepatocytes were allowed to attach to the dishes for 3 h in RPMI 1640 medium containing 10% fetal calf serum. Cells were then washed with PBS and incubated overnight with RPMI 1640 medium containing 0.5% BSA and 50 µM ß-mercaptoethanol. The next day, the cells were stimulated with 10 nM insulin (Ultratard, Novo Nordisk) for various periods. Whole cell extracts were obtained using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 10% glycerol, 1 mM Na3VO4, 20 mM NaF, 1 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, and protease inhibitor; from Roche, Indianapolis, IN), followed by centrifugation at 13,000 rpm for 10 min at 4 C. Equal amounts of protein were immunoprecipitated for 3 h at 4 C using an antiinsulin receptor (ß-chain; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by addition of protein G-Sepharose beads. Sepharose was washed three times with RIPA buffer (without inhibitors) before being boiled in Laemmli buffer and centrifuged. For the detection of AKT phosphorylation, equal amounts of protein were loaded and separated by electrophoresis. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and blocked with 10% nonfat dried skim milk powder in PBS containing 0.1% Tween 20 for 1 h at room temperature. Western blotting was performed with monoclonal antiphosphotyrosine clone 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY; 1:8,000) overnight at 4 C or with anti-phospho-AKT (Ser473; Cell Signaling Technology, Beverly, MA; 1:1000) overnight at 4 C. After 30-min washes, the membranes were incubated during 1 h at room temperature with the secondary antibody conjugated to horseradish peroxidase. Membranes were then extensively washed and developed by the enhanced chemiluminescence method according to the manufacturers instructions (Pierce Chemical Co.). Membranes were reblotted using antiinsulin receptor antibody (1:1000) or anti-AKT (Cell Signaling Technology).
Hierarchical Cluster Analysis of Hormonally Regulated Hepatic Expression Profiles
EndoGED is an integrated system for automation of processes involved in microarray-based experimentation. The system is based on a relational database that complies with the MIAME guidelines for microarray data. A complete description of the database is available at www.cmm.ki.se/EndoGED. We used the EndoGED database to create an expression matrix where the total number of detected genes in the SOCS2 experiment (
3000) was used as the baseline. Because each experiment has several hybridizations, corresponding to biological replicates, the mean expression ratio for each experiment was used. Average linkage hierarchical clustering using the Pearson correlation coefficient as a measurement of similarity was used to evaluate the relationship between the experiments (17). Although this methodology has been successfully used in numerous studies to discover mechanistic relationships between different biological samples, there are limitations to this approach. They arise from the use of different experimental models and technical platforms as well as from limitations in the expression profiles available for analysis. To minimize those effects we selected experiments from our own database (www.cmm.ki.se/EndoGED) that bear relevance/similarity to the phenotype of SOCS2/ mice. Moreover, we included public data from investigations in which the effects of SOCS3 deletion in hepatic gene expression as well as the effects of IFN-
and IL-6 treatments (20) were studied. The latter profiles were selected because SOCS3 is also known to negatively regulate GH signaling, and the intracellular pathways activated by IFN-
and IL-6 overlap those activated by GH.
The experiments used to study the generation of the expression matrix are described in detail in the respective publication or in the web page (www.cmm.ki.se/EndoGED). Briefly, 11 different studies, including the gene expression profile for SOCS2 male knockout, gender in young mice, gender in young rats, young male rats treated with GH for 1 wk in a continuous (minipump) manner (19), old male rats treated with GH in injections (33), hypothyroid wild-type male mice treated with T3 (55), male rat primary hepatocytes treated with GH for 24 h, and mice with IL-6 induction, IFN-
induction and SOCS3 deficiency (20) were used in the analysis. The data from the last three experiments were obtained from Gene Expression Omnibus (GEO) public database (available at www.ncbi.nlm.nih.gov/geo/) and were generated using Affymetrix MGU74Av2 Gene Chip. Genes with extremely low expression levels (average difference, <100) were filtered away before expression ratios were calculated (59). To compare the different platforms, ortholog probes in both arrays were identified using the TIGR orthologue gene alignment database (TOGA) as implemented in RESOURCERER (60), a software for annotating and linking microarray resources within and across species (available at www.tigr.org/tigr-scripts/magic/r1.pl). The expression matrix generated contained information for 2100 gene probes. Cluster analysis was performed using the TIGR Multiple Experimental Array Viewer (www.tigr.org) (61).
 |
ACKNOWLEDGMENTS
|
---|
We thank Anne Thaus for excellent technical assistance. Matrigel for primary hepatocytes culture was kindly provided by Dr. Leandro Fernandez (Universidad de Las Palmas de Gran Canaria, Spain).
 |
FOOTNOTES
|
---|
This work was supported by grants from Swedish Research Council (K2003-31SX-14782-01A and K2002-72X-14281-01A), the Magnus Bergvall Foundation, the Swedish Society for Medical Research, the Journal of Cell Science (Traveling Fellowship to E.R.-B.), Australian Postdoctoral Research Fellowship (to C.J.G.), Amrad Corp. Ltd., and UDCA University (Bogota, Colombia; to A.F.-M.).
First Published Online November 24, 2004
Abbreviations: FA, Fatty acid; G6PD, glucose-6-phosphate dehydrogenase; IFN-
, interferon-
; IGFBP-3, IGF-binding protein-3; Lpl, lipoprotein lipase; MUP, major urinary protein; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1
, peroxisome proliferator-activated receptor-
coactivator-1
; PPAR
, peroxisome proliferator-activated receptor-
; SCoAD, stearyl coenzyme A desaturase; SOCS, suppressor of cytokine signaling; TG, triglycerides.
Received for publication January 30, 2004.
Accepted for publication November 16, 2004.
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