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1 Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation,
2 Department of Medicine and Clinical Science, Graduate School of Medical Sciences, University of Kyushu, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
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Abstract |
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and IL-6, has been implicated in inflammation-mediated insulin resistance in the liver and adipocytes. However, no genetic evidence has been provided for the involvement of SOCS3 on insulin resistance. Here, we generated hepatocyte-specific SOCS3-deficient (L-SOCS3 cKO) mice and examined insulin sensitivity. Being consistent with a previous idea, the loss of SOCS3 in the liver apparently improved insulin sensitivity. However, unexpectedly, L-SOCS3 cKO mice exhibited obesity and systemic insulin resistance with age. Insulin signaling was rather suppressed in muscles, suggesting that deletion of the SOCS3 gene in the liver modulates insulin sensitivity in other organs. Anti-inflammatory reagent, sodium salicylate, partial improved insulin resistance of aged L-SOCS3 cKO mice, suggesting that enhanced inflammatory status is associated with the phenotype of these mice. STAT3 was hyperactivated and acute-phase proteins were elevated in L-SOCS3 cKO mice liver, which were reduced by sodium salicylate treatment. We conclude that hepatic SOCS3 is a mediator of insulin resistance in the liver; however, lack of SOCS3 in the liver promotes systemic insulin resistance by mimicking chronic inflammation. |
Introduction |
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Type 2 diabetes mellitus is the most common metabolic disorder worldwide. Insulin resistance and relative insulin deficiency are characteristics of type 2 diabetes mellitus. Insulin resistance is caused by a decrease in the ability of insulin to stimulate glucose disposal and inhibit hepatic glucose production (HGP) (Kahn 1994). However, precise mechanism on how chronic inflammation links to type 2 diabetes remains to be investigated. Proinflammatory cytokines, such as interleukin-6 (IL-6) (Klover et al. 2003) and the tumor necrosis factor- (TNF) (Hotamisligil et al. 1996), have been shown to play a critical role in insulin resistance in case of obesity, infection, injury, and aging.
These proinflammatory cytokines up-regulate the expression of the suppressor of cytokine signaling 3 (SOCS3) through activation of the signal transducers and activators of transcription (STAT) and nuclear factor 
B (NFB)-mediated pathways. SOCS3 is a member of CIS/SOCS family proteins, a group of proteins characterized by their ability to down-regulate cytokine signal transduction. The SOCS family is composed of SOCS1 to –7 and the cytokine-inducible src homology 2 domain-containing protein (CIS), and each member contains a central src homology-2 (SH2) domain and a conserved C-terminal SOCS box (Yasukawa et al. 2000). SOCS3 binds to the gp130 through the SH2 domain, while SOCS1 binds to the Janus kinase (JAK) tyrosine kinase domain, and both SOCS1 and SOCS3 inhibit JAK tyrosine kinase activity (Nicholson et al. 2000; Lehmann et al. 2003).
In addition to the regulation of cytokine signaling, recent studies have shown that both SOCS1 and SOCS3 participate in the regulation of insulin signaling. SOCS3 expression, in particular, is shown to be high in insulin-resistant obese mice (Emanuelli et al. 2001). A two-hybrid system and molecular reconstitution in culture cells have shown that SOCS3 can bind an insulin receptor (IR) (Emanuelli et al. 2000) and inhibit phosphorylation of the insulin receptor substrate 1 (IRS1) and IRS2 (Senn et al. 2003). Over-expression of SOCS3 causes a reduction of IRS1 and IRS2 by ubiqutin/proteosome-mediated degradation (Rui et al. 2002). Over-expression of SOCS3 in the liver induced insulin resistance in vivo, while suppression of SOCS3 expression by antisense-oligonucleotide treatment in obese diabetic mice improved insulin sensitivity (Ueki et al. 2004a,b). Therefore, SOCS3 has been suggested to be a target for the improvement of insulin resistance induced by obesity or chronic inflammation. However, there is not genetic evidence to support the idea of using SOCS3-gene therapy for type 2 diabetes, and the side effects of SOCS3 reduction in the liver have not been investigated.
In the current study, we generated hepatocyte-specific SOCS3-deficient (L-SOCS3 cKO) mice to assess the biological function of SOCS3 in the liver for insulin resistance. We confirmed that hepatic SOCS3 is a mediator of insulin resistance in the liver. However, L-SOCS3 cKO mice exhibited obesity and systemic insulin resistance with age. Hyperactivation of STAT3 was evident in the SOCS3-deficeint liver, and the expression of acute phase proteins was dysregulated. Sodium salicylate treatment of L-SOCS3 cKO mice resulted in reduced STAT3 activation and partial improvement of insulin resistance. These data suggest that hepatic SOCS3 is a mediator of insulin resistance in the liver; however, lack of SOCS3 in the liver in aged mice promotes systemic insulin resistance by inflammatory factor production from the liver.
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Results |
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To investigate the connection between reduced SOCS3 expression and insulin resistance in vivo, we used a tissue-specific SOCS3 knockout (cKO) approach, since SOCS3-deficient mice die as a result of placental defects during embryonic development (Roberts et al. 2001). As the liver plays important role in glucose and lipid homeostasis, we generated hepatocyte-specific SOCS3-deficient (L-SOCS3 cKO) mice by crossing SOCS3-flox mice and Albumin-Cre (AlbCre) transgenic mice, in which Cre recombinase is specifically expressed in liver parenchymal cells under the albumin promoter/enhancer. Genomic DNA extracted from the liver and other tissues was analyzed with PCR to verify the deletion of the socs3 gene. The PCR product obtained with a and b from the Socs3WT locus was 280 bp, and that from the Socs3-flox locus was 380 bp. A 250-base pair band obtained with primers a and c corresponded to the deleted socs3 allele (Fig. 1a); such a base pair was observed in the PCR analysis of the liver DNA from L-SOCS3 cKO mice but not in that of the control SOCS3flox/flox mice (WT). We confirmed Socs3 deletion in the liver but not in other tissues, including the pancreas, fat, muscle, and hypothalamus in L-SOCS3 cKO mice (Fig. 1b). Lack of SOCS3 mRNA and protein in the liver, but not in other tissues, of L-SOCS3 cKO mice was confirmed by RT-PCR and Western blotting (Figs 1c, 3a and 5e). Interestingly, SOCS3 expression in the liver was enhanced with age (Fig. 1c). The histology of the livers of young (2 months old) L-SOCS3 cKO mice appeared normal and the alanine transaminase (ALT) in serum was not elevated (data not shown). The histology and triglyceride and total cholesterol contents of the liver also did not differ between aged (6 months old) L-SOCS3 cKO and WT mice (Table 1 and histology data not shown).
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First, we examined the basal glucose metabolism of L-SOCS3 cKO mice. The blood glucose concentrations were similar in 2-month-old WT and cKO mice (Table 1). A glucose-tolerance test (Fig. 2a) and an insulin-tolerance test (Fig. 2b) revealed no significant differences in the systemic insulin sensitivity and glucose metabolism between young WT and L-SOCS3-cKO mice. However, glucose- and insulin-tolerance tests may not be sensitive enough to assess insulin sensitivity of the liver. Therefore, we used a euglycemic-hyperinsulinemic glucose clamp to quantify HGP and glucose disposal rates (GDR) in 2-month-old male mice (Fig. 2c–f). As expected from the insulin-tolerance test, we did not observe any significant difference in the glucose infusion rate and GDR in the skeletal muscles of WT and L-SOCS3 cKO mice (Fig. 2c,d). In the liver, HGP in the clamp period was lower in L-SOCS3 cKO mice than in WT mice, while the basal HGP was not so different (Fig. 2e). Thus, insulin infusion reduced the HGP rate more efficiently in L-SOCS3 cKO mice (83.7%) than in WT mice (61.7%) (P < 0.05) (Fig. 2f). These data suggest that the SOCS3-deficient liver was more sensitive to insulin than the WT liver.
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IRS1 degradation was dependent on SOCS3 in hepatocytes
SOCS3 has been implicated in the proteosomal degradation of IRS1 and IRS2. To demonstrate this, primary cultured hepatocytes were prepared from mice and then pretreated with or without IL-6 or insulin for 16 h and stimulated with or without insulin (Fig. 3b). Insulin-mediated IRS1 and Akt phosphorylation was always stronger in SOCS3-deficient hepatocytes than in WT hepatocytes, which is consistent with in vivo data (Fig. 3a). Pretreatment with IL-6 or insulin for 16 h enhanced the SOCS3 protein levels but reduced the IRS1 protein levels and suppressed insulin-induced IRS1 and Akt phosphorylation in primary cultured hepatocytes of WT mice. Importantly, such a reduction of the IRS1 protein levels and IRS1 and Akt phosphorylation levels by IL-6 or insulin pretreatment was attenuated in the primary cultured hepatocytes of L-SOCS3 cKO mice. These data are consistent with a previously proposed hypothesis that SOCS3 functions as E3 ubiqutin-ligase for IRS1.
Systemic insulin resistance in aged L-SOCS3 cKO mice
Because of improved insulin sensitivity, SOCS3-deficient mice are expected to be resistant to diabetes. However, unexpectedly, 4 months after birth, the body weight of L-SOCS3 cKO mice was greater than that of WT mice under both normal diet (ND) and high fat diet (HFD) conditions (Fig. 4a,b). Macroscopic (Fig. 4c,d) and computed tomographic analysis (Fig. 4e) of 9-month-old mice demonstrated that both visceral and subcutaneous fat deposits were significantly larger in L-SOCS3 cKO mice than in WT mice. The blood glucose concentration was slightly elevated in cKO mice (WT, 72.7 ± 3.2; L-SOCS3 cKO, 83.6 ± 4.2 mg/dL), and the plasma insulin concentration was significantly higher in 6-month-old L-SOCS3 cKO mice than in WT mice (WT, 0.20 ± 0.05; L-SOCS3 cKO, 0.50 ± 0.11 ng/mL, P < 0.05) (Table 1). When mice were fed a HFD, both the plasma insulin and blood glucose concentrations of L-SOCS3 KO mice were higher than those of WT mice (insulin: WT, 1.86 ± 0.32; L-SOCS3 cKO, 3.81 ± 0.83 ng/mL; glucose: WT, 184.6 ± 5.7; L-SOCS3 cKO, 215.5 ± 12.1 mg/dL, P < 0.05). Moreover, serum triglycerides and total cholesterol were higher in L-SOCS3 cKO mice (Table 1).
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To define the mechanism of insulin resistance in L-SOCS3 cKO mice, we examined insulin-induced Akt phosphorylation in the muscle, which is a major source of glucose uptake in response to insulin. The SOCS3 protein levels in the quadriceps muscles of L-SOCS3 cKO mice were similar to those in WT mice (Fig. 5e). Akt activation in the muscle of young (2 months old) L-SOCS3 cKO mice was similar to that in WT mice, suggesting normal insulin signaling in the muscle of young mice (Fig. 5e). However, the insulin-induced Akt phosphorylation level was lower even in 4-month-old L-SOCS3 cKO mice (which did not show strong obesity) than in age-matched WT control mice (Fig. 5e). However, insulin-induced Akt phosphorylation in the liver of aged L-SOCS3 cKO mice was still stronger, although this effect in aged mice liver was not as strong as in young mice liver (data not shown).
SOCS3 deficiency in the liver modulated gene expression related to obesity, diabetes and inflammation
Since L-SOCS3 cKO mice showed obesity and secondary insulin resistance, we suspected that a loss of SOCS3 gene expression resulted in the modulation of gene expression related to obesity and diabetes in the liver. Thus, we compared the gene expression profiles in the liver of 6-month-old L-SOCS3 cKO mice and WT mice. A DNA microarray analysis demonstrated that the enzymes involved in glucose and fatty acid metabolism were variably changed (Supplementary Table S1). The expression of gluoconeogenic enzymes were relatively up-regulated, probably reflecting insulin resistant status of aged L-SOCS3 cKO mice. In secreted proteins, we found a strong up-regulation of acute-phase proteins, such as serum amyloid A 1(SAA1), SAA2, and CRP in L-SOCS3 cKO mice. CRP was recently found to bind to leptin and inactivate it (Chen et al. 2006). High levels of CRP have been shown to be associated with diabetes and obesity (Visser et al. 1999). SAA has been implicated in the cholesterol metabolism (Manley et al. 2006). We confirmed the up-regulation of SAA and CRP in L-SOCS3 cKO mouse livers by RT-PCR (P < 0.05) (Fig. 6a,b). It has been shown that acute phase proteins are regulated by STAT3. We confirmed that STAT3 were hyper activated in the liver of aged L-SOCS3 cKO mice (Fig. 6a,b). Thus, deletion of SOCS3 in the liver mimicked chronic inflammatory status.
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B activation and reduces inflammatory cytokine production. STAT3 inhibitors are not suitable, because STAT3 inhibitors have been shown to induce hyperglycemia (Netea et al. 2006). Insulin resistance in aged L-SOCS3 cKO mice was improved when the mice were treated with sodium salicylate (Fig. 6c). A sodium salicylate treatment blocked hyper STAT3 phosphorylation and reduced SAA and CRP production in L-SOCS3 cKO mouse livers to the same levels observed in WT mouse livers (Fig. 6d,e). These data suggest that inflammatory status in L-SOCS3 cKO mice liver is responsible for systemic insulin resistance of these mice. We also noticed that an angiopoietin-like growth factor (AGF) was reduced in L-SOCS3 cKO mice in our microarray search. We confirmed AGF reduction in L-SOCS3 cKO mouse livers by RT-PCR (P < 0.05) (Fig. 6a, b). Reduced expression of AGF by gene targeting resulted in obesity and insulin resistance (Oike et al. 2005). However, sodium salicylate treatment did not affect AGF mRNA levels (Fig. 6d,e). This suggests that AGF expression could be down-regulated by a STAT3-independent mechanism in the SOCS3-defcient livers, and may not play an essential role in insulin resistance in aged L-SOCS3 cKO mice.
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Discussion |
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leads to inhibitory phosphorylation of IRS1 at Ser307 via activation of c-jun amino-terminal kinase (JNK1). Chronic TNF exposure has also been demonstrated to lead to IRS1 degradation (Hirosumi et al. 2002). SOCS3 has been implicated as the mediator whereby inflammatory cytokines negatively regulate the insulin signaling cascade. In 3T3-L1 cells, TNF, IL-6 as well as resistin induce SOCS-3 mRNA (Shi et al. 2004; Steppan et al. 2005). Shi et al. (2004) found that SOCS3 deficiency increases insulin-stimulated IRS1 and IRS2 tyrosine phosphorylation, enhances downstream PI 3-kinase activity, and leads to increased insulin-stimulated glucose uptake. Moreover, SOCS3 deficiency blocks the TNF-induced inhibition of insulin signaling, and this is largely attributed to the suppression of TNF-induced IRS1 and IRS2 protein degradation. Using our L-SOCS3 cKO mice, we clearly demonstrated that the reduction of IRS1 resulting from a pretreatment with IL-6 or insulin was abolished without SOCS3. Thus, our results confirmed an earlier hypothesis that SOCS3 is an E3 ligase for IRS1. Phosphorylation and protein levels of IRS2 in response to insulin were not very different between WT and L-SOCS3-cKO mice (Fig. 3a). SOCS3 may have difference effect on IRS1 and IRS2, which was not apparently observed in over-expression studies. In addition to SOCS3, SOCS1 and SOCS7 have been implicated as a therapeutic target for insulin resistance in obesity. Inhibition of SOCS1 as well as SOCS3 in obese diabetic mice improves insulin sensitivity (Ueki et al. 2004a). In addition, studies of SOCS1-deficient mice demonstrate that SOCS1 also regulates IRS1 phosphorylation following insulin treatment (Kawazoe et al. 2001; Jamieson et al. 2005). Recently, Banks et al. (2005) showed that SOCS7-deficint mice were hypersensitive to insulin and SOCS7-deficeint fibroblasts showed dramatically enhanced adipocyte differentiation. SOCS1 and SOCS7 are shown to interact with IRSs. Thus, SOCS family members other than SOCS3 may play also important roles in insulin-sensitivity and diabetes.
As predicted by previous studies, our study confirmed that a short-term decrease of SOCS3 in the liver actually improves insulin sensitivity. However, long-term suppression of SOCS3 may not be appropriate because it induces metabolic syndromes, such as hyperglycemia and obesity. A similar dual effect of SOCS3 was observed in adipocytes. SOCS3 deficiency in adipocytes enhanced insulin sensitivity in vitro (Shi et al. 2004); however, over-expression of SOCS3 in adipose tissue in vivo caused local insulin resistance and improved systemic insulin sensitivity.
Our data suggest that systemic insulin resistance in aged L-SOCS3 cKO mice is due to hyperactivation of STAT3, resulting in hyperproduction of acute-phase proteins. Hyper STAT3 phosphorylation was evident in aged L-SOCS3 cKO mice compared with young mice (Figs 3a and 6a). We think that this situation resembles human metabolic syndrome. It has been shown that high CRP levels without apparent infection are associated with metabolic syndrome, which includes obesity, type 2 diabetes, insulin resistance, and dyslipidemia with age. Additionally, it was observed that individuals with a more severe disease/syndrome would show higher CRP levels. Although the mechanism of chronic inflammation in metabolic syndrome has not been clarified, proinflammatory cytokines from adipose tissues may be primarily involved in up-regulation of acute phase proteins in the liver. Acute phase proteins may directly induce the dysregulation of lipid and glucose metabolism, which further promotes obesity and pro-inflammatory cytokine production from adipose tissues. Thus, anti-inflammatory drugs might be able to break this circuit and could be useful for disease therapy. Indeed, we showed here that sodium salicylate treatment blocked hyper STAT3 phosphorylation and reduced SAA and CRP production in L-SOCS3 cKO mouse livers and corrected insulin-resistance (Fig. 6c,d).
The insulin resistant and obese phenotype of L-SOCS3 cKO mice is in contrast to previous studies using liver-specific STAT3-deficent mice (Inoue et al. 2004). Liver-specific STAT3 cKO mice showed insulin resistance associated with increased hepatic expression of gluconeogenic genes. Furthermore, liver-specific expression of a constitutively active form of STAT3, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. This group concluded that hepatic STAT3 signaling provides new therapeutic targets for diabetes mellitus. This is consistent with the observation that suppression of SOCS3 expression by antisense-oligonucleotide in the liver of obese mice ameliorated diabetes (Ueki et al. 2004b). However, those effects may represent a short term effect of STAT3, which regulates glucogenic genes. Long term effect of hyperactivated STAT3 should be re-examined in terms of metabolic syndrome associated with inflammation.
The IL-6/STAT3 signal seems to have dual functions in the regulation of glucose homeostasis. Continuous IL-6 administration caused hyperactivation of STAT3, and resulted in insulin resistance (Klover et al. 2003; Kim et al. 2004), which may be similar to the results observed in our aged L-SOCS3 cKO mice. This may reflect the pathophysiological role of IL-6/STAT3 in inflammation. On the other hand, physiological levels of IL-6, which is induced by brain insulin, leads to systemic insulin hypersensitivity (Inoue et al. 2006), and a lack of IL-6 causes obesity (Wallenius et al. 2002). Thus, it is possible that hyperactivation of STAT3 and lack of STAT3 may provide unrelated phenotypes regarding metabolism and insulin actions.
We found reduced insulin signaling in muscles in aged L-SOCS3 cKO mice, even though insulin sensitivity was enhanced in the liver. This suggests that a circulating factor caused systemic insulin resistance in these mice. Elevated TNF (Hotamisligil et al. 1996) and IL-6 (Senn et al. 2002) from the liver could be a possible mechanism for insulin resistance. However, the serum TNF and IL-6 concentrations were not elevated in L-SOCS3 cKO mice (data not shown). Resistin and growth hormone were not affected either. To identify the factors that are involved in insulin resistant and obesity in L-SOCS3 cKO mice, we performed a DNA microarray analysis. We found that acute-phase proteins were elevated in the liver of L-SOCS3 cKO mice. Acute-phase proteins, such as CRP and SAA, are also related to obesity and insulin resistance (Visser et al. 1999). CRP levels increase dramatically in most mammals, including human; however, the major acute phase protein in the mouse is the homologous pentraxin, serum amyloid P-component (SAP), whereas CRP is a minor acute phase reactant. STAT3 plays an essential role in the transcriptional activation of CRP and SAA (Zhang et al. 1996; Hagihara et al. 2005). High-dose sodium salicylates are known to reverse insulin resistance in obese mice by inhibiting IKKß (Yuan et al. 2001). Sodium salicylates inhibit the phosphorylation of STAT3 in cardiac fibroblasts and macrophage (Wang et al. 2002; Liu et al. 2003). We also observed that, in L-SOCS3 cKO mice, a sodium salicylate treatment inhibited the hyperactivation of STAT3 and reduced CRP and SAA to the same level of those observed in WT mice. Activation of STAT3 in L-SOCS3 cKO mice is probably caused by autocrine or paracrine secretion of IL-6, whose expression is induced through NF-B. Thus, sodium salicylates could be an effective way to prevent inflammation-related diabetes.
In summary, hepatocyte-specific deletion of SOCS3 in mice leads to insulin hypersensitivity in the liver, as evident from the stronger suppression of HGP and enhanced insulin signaling. However, insulin resistance occurred secondarily in the whole body with age. We conclude that hepatic SOCS3 is a mediator of insulin resistance in the liver; however, lack of SOCS3 in the liver promotes systemic insulin resistance, probably because hyperactivation of STAT3 in the liver mimics a chronic inflammatory status of the body. We propose that SOCS3 in the liver protects metabolic syndrome associated with aging, and our L-SOCS3 cKO mice will be a useful mode for human metabolic syndrome.
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Experimental procedures |
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AlbCre mice on a C57BL/6 background expressing Cre recombinase under the control of the mouse albumin gene regulatory region were purchased from Jackson Laboratory (Wallenius et al. 2002). SOCS3flox/flox mice were generated as previously described (Yasukawa et al. 2003). AlbCre mice were crossed with SOCS3fl/fl mice, and the resulting AlbCre-SOCS3fl/fl (L-SOCS3 cKO) mice intercrossed with SOCS3fl/fl mice and their littermates were compared. Genotyping was conducted with primers as described (Yasukawa et al. 2003). All experiments on these mice were approved by and conducted in accordance with the Guidelines of the Animal Ethics Committee of Kyushu University, Fukuoka, Japan. Mice were housed in a 12 h-light/dark cycle and temperature-controlled environment. Male and female L-SOCS3 cKO mice and WT mice were fed a ND (6% calories from fat). Eight-week-old mice were then placed on a HFD consisting of 23% calories from fat (Oriental Kobo). The body weight and metabolic parameters were measured from 08:00 to 09:00. All mice we used in these experiments were male.
Metabolic measurements
Blood glucose was measured with an Antisense-II glucometer (Sankyo). The plasma insulin and C-peptide concentration was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Morinaga and Shibayagi). Serum and liver cholesterol (TC) were determined using a cholesterol C-II kit (WAKO). Serum and liver triglycerides (TG) were determined using a triglyceride G kit (WAKO). Weight of gonadal fat pads, retroperitoneal fat pads, mesenteric fat pads and inguinal fat pads were measured.
Glucose- and insulin-tolerance tests
We performed a glucose-tolerance test on 16 h-fasted mice followed by an intraperitoneal glucose injection (2 g/kg body weight). An insulin-tolerance test was performed on randomly fed mice injected intraperitoneally with human insulin (0.5 U/Kg body weight) (Novolin R, Novo Nordisk). Sodium salicylate (120 mg/kg body weight) was administered by a daily intraperitoneal injection for 2 weeks.
Euglycemic-hyperinsulinemic glucose clamp
Euglycemic-hyperinsulinemic glucose clamp were performed on 2-month-old mice. After a bolus injection (5 µCi), [3-3H] glucose was infused at a rate of 0.05 µCi/min throughout the clamp study. For the measurement of the basal glucose turnover rate, blood glucose was collected 90 min after the initiation of [3-3H] glucose infusion. Insulin was infused at a rate of 2.5 mU/kg for 90 min, and 25% glucose was infused by variable infusion pump. Plasma glucose was clamped at 100–120 mg/dL. Plasma [3-3H] glucose radioactivity was measured after deproteinization with barium hydroxide and zinc sulphate, evaporation to removed tritiated water, and counted using a liquid scintillation counter (Beckman Instruments Inc.).
CT scan analysis
The adiposity of mice was examined radiographically using LaTheta LCT-100 (ALOKA) according to the manufacturer's protocol. We performed CT scanning at 2 mm intervals from the diaphragm to the bottom of the abdominal cavity.
Histological and immunohistochemical analysis
Six 9-month-old male mice were used for each genotype. The isolated pancreases were fixed in 10% formalin. Tissues were embedded in paraffin and consecutive 5 µm sections were mounted on slides. Sections were stained with hematoxylin/eosin or anti-guinea pig insulin antibody (DAKO) and stained with a histofine kit (Nichirei) according to the manufacturer's instructions. Analyses of islet size were performed using the ImageJ software.
Primary cell culture
The liver was perfused via the portal vein with Hanks’ balanced salt solution with 0.5 mM EGTA and then with a 0.05% collagenase typeIV solution. Isolated hepatocytes were washed and purified in Hanks’ balanced salt using low-speed centrifugation at 40 g. Hepatocytes were placed on type I collagen-coated dishes. Hepatocytes were pretreated with IL-6 (50 ng/mL) or insulin (200 nM) for 16 h and then stimulated with 100 nM insulin for 15 min.
RNA extraction, RT-PCR and microarray
Total RNAs from mice liver and cultured cells were purified, and reverse transcription and PCR protocols were performed as previously described (Chinen et al. 2006). The oligonucleotides used for PCR were as follows: CRP, 5'-ggg tgg tgc tga agt acg at-3' and 5'-cca aag act gct ttg cat ca-3' SAA1, 5'-gta att ggg gtc ttt gcc-3' and 5'-ttc tgc tcc ctg ctc ctg-3' and AGF, 5’-tcg tgt agt agc cgt gtg gtg gtg t-3' and 5'-cac ctg ctg cac agg ttc ca-3'. SOCS3 and 36B4 was measured as described (Inoue et al. 2004). We performed a DNA microarray analysis using Affymetrix mouse oligonucleotide arrays and GeneSpring.
Immunoblot analysis
Mice were fasted for 16 h, anesthetized with an intraperitoneal injection of ketamine and xylazine, and injected intraperitoneally with human insulin (4 U/kg). Fifteen minutes after the insulin injection, liver and muscle were removed and frozen in liquid nitrogen. Anti-SOCS3 antibody (C204) was purchased from IBL (Tokyo, Japan). Antibodies to phosphorylated Akt (Ser 473), Akt, and phosphorylated STAT3 (Thy 705) were obtained from Cell Signaling Technology. Antibodies to IRS1, STAT3, and ERK2 were from Santa Cruz. The antibody to IRS2 was from Upstate Biotechnology Inc. The antibody to phosphorylated IRS1 (Thy 612) was from Oncogene. PY-IRS2 was immunoblotted by the anti-phosphotyrosine antibody after immunoprecipitated with the antibody to IRS2.
Statistical analysis
Data are expressed as the mean ± SEM. Statistical significance was tested with an unpaired two-tailed Student's t-test.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yakihiko{at}bioreg.kyushu-u.ac.jp
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Received: 21 September 2006
Accepted: 29 October 2006
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