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Department of Physiology, Faculty of Medicine, University Complutense of Madrid, 28040 Madrid, Spain
(Correspondence should be addressed to A López-Calderón; Email: alc{at}med.ucm.es)
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Abstract |
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gene expression both in vivo and in vitro. All these data suggest that LPS-induced Ptgs2 activation decreases Igf1 gene expression in liver cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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Sepsis can be experimentally induced in rats by endotoxin (lipolysaccharide, LPS) administration, since it reproduces most of the physiological responses triggered by sepsis. One of the mechanisms by which endotoxin inhibits serum concentrations of Igf1 is by inducing GH resistance and decreasing its gene expression in the liver (Defalque et al. 1999, Priego et al. 2003a). The inhibitory effect of LPS can be exerted directly on the hepatocyte, since LPS is able to decrease Igf1 gene expression in hepatocyte cultures (Priego et al. 2006).
We have previously reported that LPS-induced decrease in serum and liver Igf1 seems to be due to inducible nitric oxide synthase (Nos2) activation, since Nos2 inhibitors are able to prevent the inhibitory effect of LPS on liver Igf1 gene expression both in vivo and in vitro (Priego et al. 2004, 2006). However, the Nos2 inhibition was not able to prevent the LPS-induced decrease in Igf1 mRNA when the hepatocytes were cultured with Kupffer cells (Priego et al. 2006). These data indicate that nitric oxide release is not the only factor responsible for the inhibition of Igf1 gene expression after endotoxin exposure.
The role of Kupffer cells on LPS-induced Igf1 inhibition was also evidenced by the fact that Kupffer cell depletion by gadolinium administration prevents the inhibitory effect of LPS on GH receptor (Ghr), Igf1 and Igf binding protein-3 (Igfbp3) gene expression (Granado et al. 2006). Kupffer cells (resident liver macrophages) have a pivotal role in liver inflammatory response; they release cytokines and prostanoids after LPS exposure, which might decrease Igf1 gene expression. Tumour necrosis factor- (Tnf) has been reported to inhibit Igf1 mRNA induction after GH stimulation both in vivo (Yumet et al. 2002) and in vitro (Ahmed et al. 2006). However, it has been demonstrated that suppression of Tnf by pentoxiphylline fails to restore both circulating Igf1 levels and GH sensitivity in rats injected with LPS (Colson et al. 2003).
When activated by LPS, Kupffer cells release the cyclooxygenase-2 (Ptgs2) products, prostaglandin E2 (Ptges2) and thromboxane A2 (Tbxa2r) (Keller et al. 2005, Bezugla et al. 2006). We have shown that Ptgs2 inhibition is able to prevent chronic arthritis-induced decrease in serum concentration of Igf1 and its gene expression in the liver (Granado et al. 2007). Taking into account that Ptgs2 is expressed in Kupffer cells, and not in hepatocytes, incubated with LPS (Martín-Sanz et al. 1998), it is possible that Ptgs2 activation can contribute to the LPS-induced decrease in Igf1 gene expression in the liver.
The present study was designed in order to elucidate the possible inhibitory effect of Ptgs2 activation on LPS-induced inhibition of Igf1 and Igfbp3 gene expression in the liver. For this purpose, the effect of meloxicam on the LPS-induced decrease in Ghr, Igf1 and Igfbp3 has been studied. In addition, studies have been performed using primary cultures of hepatocytes with non-parenchymal cells in order to analyse the possible interaction between Nos2 and Ptgs2 induction by LPS stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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In vivo experiment
Rats were randomly assigned to the treatment groups. Twenty rats were i.p. injected with 250 µg/kg LPS (serotype 055:B5, Sigma Chemical Co.) in 250 µl sterile saline. Another 20 rats were injected with saline. Half of the rats in each group were simultaneously i.p. injected with the selective Ptgs2 inhibitor, meloxicam (1 mg/kg, Sigma), while the other half were injected with 250 µl sterile saline. Meloxicam at this dosage decreased serum concentrations of Ptges2 and prevented arthritis-induced decrease in liver Igf1 gene expression (Granado et al. 2007).
Rats received the treatments at 1700 h and at 0800 h the following day. This LPS administration protocol was shown to decrease levels of serum Igf1 and its mRNA in the liver (Priego et al. 2003a). Meloxicam administration was not able to modify the LPS-induced decrease in food and water intake. All animals were killed by decapitation at 1200 h, 19 h after the first, and 4 h after the second LPS and/or meloxicam injection. Liver was removed, frozen immediately in liquid nitrogen, and stored at –80 °C for isolation of liver RNA. Blood was allowed to clot, and the serum was stored at –20 °C for Igf1, Igfbp3 and nitrite/nitrate assays.
In vitro experiments
The role of Ptgs2 and Nos2 induction on the inhibitory effect of LPS on Ghr and Igf1 gene expression was studied in hepatocytes and non-parenchymal cell cultures, as previously reported (Granado et al. 2008). Both hepatocytes and non-parenchymal cells from one culture were isolated from the same rat liver. Rats were anaesthetized with pentobarbital (Sigma, Chemical Co). The liver was perfused in situ through the portal vein at a flow rate of 40 ml/min with calcium-free buffer at 37 °C for 15 min, and then the liver was digested with a 0.04% collagenase (Roche) for another 5–10 min at 37 °C. The liver was transferred to a Petri dish and the cells were obtained by gentle raking with a comb and filtered through a 100 µm mesh to remove cell aggregates and tissue debris. Hepatocytes were separated from non-parenchymal cells by differential centrifugation at 25 g (three times, 5 min each). Hepatocyte purity was assessed by microscopy and was >90%; viability was measured by trypan blue exclusion and was >80%. Hepatocytes were kept on ice while the non-parenchymal cells were isolated.
Non-parenchymal cells were isolated from supernatant of the differential centrifugations and were then centrifuged at 650 g for 6 min at 4 °C. The pellet was suspended in a 50 ml culture medium and centrifuged at 650 g for 7 min. Medium consisted of Williams' medium E (Gibco) with L-glutamine (2 mM), insulin (1 µM), HEPES (15 mM), penicillin+streptomycin (100 units/ml+100 µg/ml) and 10% low-endotoxin calf serum. Non-parenchymal cells were finally suspended and plated with hepatocytes in a proportion of 3:2 (3.106 hepatocytes/2.106 non-parenchymal cells in 5 ml medium).
The culture medium was replaced with fresh medium 24 h before starting all experiments. A concentration of 100 ng/ml LPS was used; in earlier experiments we used a higher dose to stimulate hepatocytes, but when hepatocytes were cultured with non-parenchymal cells the effect of LPS on Igf1 can be observed at a lower LPS dose (Priego et al. 2006), since the effect of LPS was enhanced.
Real-time PCR
RNA was extracted by the guanidine thiocyanate method using a commercial kit (Ultraspec RNA, Biotecx Laboratories Inc., Houston, TX, USA). The integrity and the concentration of the RNA were confirmed using agarose gel electrophoresis. For RT-PCR analysis, 1 µg liver or cocultures cell total RNA was reverse-transcribed with Quantiscript Reverse Transcription kit (Qiagen Combh Hilden). Primers for PCR (Table 1) were obtained from Roche by using the Exiqon Universal Probe Library (Igfbp3 and Ghr) or from previously published sequences of Igf1, Tnf (Dehoux et al. 2004), Ptgs2 (Bianchi et al. 2005) and hypoxanthine-guanine phosphorybosyl transferase (Hprt1) (Peinnequin et al. 2004).
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IGF1, PTGES2 and nitrite+nitrate determinations
Serum IGF1 concentrations were measured by a double-antibody RIA. The IGF1 antiserum (UB2-495) was a gift from Dr Underwood and Dr Van Wik, and it is distributed by the Hormone Distribution Program of NIDDK through the National Hormone and Pituitary Program. Levels of Igf1 were expressed in terms of IGF1 from Gropep Ltd (Adelaide, Australia). The intra-assay coefficient of variation was 8%. All samples were run in the same assay.
Serum concentrations of PTGES2 were measured by an enzymeimmunoassay system using a commercial kit (Amersham Biosciences) following the manufacturer's instructions.
Nitrite+nitrate concentration in the serum and culture medium was measured by a modified method of Griess assay, described by Miranda et al. (2001). Serum was deproteinized to reduce turbidity by centrifugation through a 30 kDa molecular mass filter using a Centrifree micropartition device with a YM-30 ultrafiltration membrane (Amicon Division, Millipore Corporation, Bedford, TX, USA) at 300 g for 1 h at 37 °C for 300 µl samples. To 100 µl vanadium chloride, 25 µl serum or 100 µl medium were mixed; the Griess reagents were added very soon after. The determination was performed at 37 °C for 30 min. The absorbance was measured at 540 nm. Nitrite+nitrate concentration was calculated using a NaNO2 standard curve and expressed as µM.
Western ligand blot
Serum concentrations of IGFBP3 were measured by western blot. Two microliters of serum were diluted in sample buffer and boiled for 2 min at 90 °C, loaded onto 1% SDS-12.5% polyacrylamide gels, and electrophoressed under non-reducing conditions. Proteins were transferred onto nitrocellulose sheets (Hybond-C extra, Amersham). The membranes were dried and blocked for 1 h with 5% non-fat dry milk, 0.1% Tween (Sigma), in Tris-buffered saline. Membranes were probed overnight at 4 °C with 125I-labelled Igf1 (1.5x106 c.p.m./ml). The nitrocellulose sheets were then washed, dried and exposed at –80 °C to X-ray film (Kodak X-Omat AR, Eastman Kodak) and to two intensifying screens for 1–4 days according to the signal obtained. The signals of the film were quantified by densitometry using a PC-Image VGA24 program for Windows. The density of the IGFBP3 band in each lane was expressed as the percentage of the mean density of sera from control rats injected with saline.
Statistical analysis
Statistics were computed using the statistics program STATGRAPHICS plus for Windows (Statpoint Inc., Madrid, Spain). Data are presented as means±S.E.M. and differences among experimental groups were analysed by two-way ANOVA. Post hoc comparisons were made by using a subsequent LSD multiple range test. Serum concentrations of nitrites+nitrates and liver Tnf mRNA data in the in vivo experiment were subjected to log transformation, since variances showed a log-normal distribution. Statistical significance was set at P<0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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The serum concentrations of IGF1 and its gene expression in the liver, in rats injected with saline, LPS, meloxicam or LPS and meloxicam are shown in Fig. 1. LPS injection induced a significant (P<0.01) decrease in serum concentrations of IGF1 and mRNA in the liver (P<0.01). The Ptgs2 inhibitor meloxicam prevented the inhibitory effect of LPS on IGF1, since the rats injected with LPS and meloxicam had higher serum IGF1 (P<0.01) and Igf1 mRNA in the liver (P<0.05) than the control rats injected with LPS. In the control rats, meloxicam decreased serum concentrations of Igf1 (P<0.01), whereas the Igf1 mRNA in the liver was similar in the rats injected with saline and those injected with meloxicam. In the control rats, LPS injection decreased Ghr mRNA in the liver (P<0.01, Fig. 1C). LPS also decreased Ghr mRNA in the rats injected with meloxicam (P<0.01). However, this decrease was lower in the rats injected with meloxicam than in the control rats (36.6±7% of the control rats injected with meloxicam versus 58±3.4% of the control rats injected with saline, P<0.05).
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mRNA was similar to the effect on serum nitrites+nitrates (Fig. 3B). Both LPS and meloxicam increased the Tnf mRNA in the liver, but the increase was only significant in the control rats injected with LPS (P<0.05). In the rats injected with meloxicam, LPS administration did not modify Tnf mRNA.
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LPS at the concentration of 100 ng/ml induced a significant increase in Ptgs2 mRNA in liver cells (463±57 vs 100±10 in the cells incubated with medium alone, P<0.01) and in the PTGES2 in the culture medium (P<0.01, Fig. 4). The Ptgs2 inhibitor, meloxicam, prevented the stimulatory effect of LPS on Ptges2 release (Fig. 4).
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The nitrite+nitrate concentration in the culture medium was higher in the cells cultured with meloxicam than in the cells cultured with medium alone (P<0.01, Fig. 6). LPS induced a similar increase in the concentrations of nitrite+nitrate in control cells and in the cells incubated with meloxicam (Fig. 6A). Tnf mRNA in the liver cells was also increased by LPS (P<0.01). Meloxicam addition to the culture medium increased Tnf gene expression (P<0.05), and LPS was not able to further increase Tnf mRNA in the liver cells cultured with meloxicam (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDeclaration of InterestReferences |
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The lack of a significant effect of LPS on Igfbp3 gene expression in the cultured liver cells suggests that the inhibitory effect of LPS on Igfbp3 in vivo seems to be mediated by a non-hepatic factor. The data indicate that LPS or cytokines do not directly inhibit Igfbp3 gene expression. It has been reported that IL6, IL1b and TNF increase the biosynthesis of IGFBP3 in primary cocultures of hepatocytes and Kupffer cells, whereas the biosynthesis of IGF1 is inhibited (Lelbach et al. 2001). The stimulatory in vitro effect of cytokines on Igfbp3 has been described in other cell types such as salivary gland tumour (Katz et al. 1999) and human monocytic cell line GLI1 (Agnese et al. 2002). Similar to our data, no effect of LPS on Igfbp3 has been observed in primary rata microglia cultures, whereas LPS decreased Igfbp5 and -6 (Chesik et al. 2004). All these data indicate that the inhibitory effect in vivo of LPS on Igfbp3 is not exerted directly by endotoxin or cytokines on a hepatic level.
The fact that LPS decreased Igf1 gene expression directly in the liver cell cultures indicates that LPS has an inhibitory effect on Igf1 gene expression per se. However, we cannot exclude that part of the inhibitory effect of LPS administration in vivo can be secondary to a decrease in food intake. Nevertheless, we have previously reported that LPS administration is able to decrease both serum IGF1 and Igf1 mRNA in fasted rats (Granado et al. 2008).
The Ptgs2 activation seems to directly suppress liver Igf1 gene expression, since meloxicam attenuated the inhibitory effect of LPS on liver cells in culture. However, meloxicam in vivo prevented the inhibitory effect of LPS on Igf1 gene expression in the liver, whereas in vitro it only attenuated the LPS-induced decrease in Igf1 mRNA. This difference cannot be explained by the fact that meloxicam was not able to block Ptgs2 activity, since LPS-induced increase in Ptges2 release to the culture medium was prevented by meloxicam. One possible explanation could be that part of the in vivo meloxicam effect is an indirect effect, exerted outside the liver. In this sense, we have reported that meloxicam prevents the inhibitory effect of chronic arthritis on both pituitary GH and liver Igf1 gene expression (Granado et al. 2007). In the present data we did not evaluate pituitary GH, since LPS at the dose of 0.25 mg does not significantly decrease GH mRNA (Priego et al. 2003a).
In contrast to the Igf1 response, meloxicam did not modify the LPS-induced decrease in Ghr mRNA in the liver cells in vitro, although the decrease was attenuated in vivo. Taking into account that one of the factors that controls Ghr gene expression is GH (Flores-Morales et al. 2006), and that LPS is able to inhibit pituitary GH secretion (Kasting & Martin 1982), by increasing hypothalamic somatostatin (Spangelo et al. 1990, Priego et al. 2005a), it is logical to think that pituitary GH can mediate the LPS-induced decrease in Ghr mRNA. In fact, LPS at a low dose (10 µg/kg) increases pituitary GH release, does not modify liver Ghr mRNA and decreases Igf1 mRNA (Priego et al. 2003a). However, at higher doses, LPS decreases pituitary GH, liver Ghr and Igf1 (Priego et al. 2003a). These data support the hypothesis that some of the changes in liver Ghr and Igf1 genes during endotoxin administration are secondary to modifications in pituitary GH, whereas others are exerted directly on liver cells. In the liver LPS very rapidly induces a GH resistance state, since 4 h after LPS injection there is a decrease in GH post-receptor signalling (Jak2–Stat5a phosphorylation), in spite of the fact that the GHR protein levels are still unchanged (Chen et al. 2007). It has been recently reported that LPS can also directly suppress Ghr expression in vitro by inhibiting Ghr promoter activity (Dejkhamron et al. 2007). However, these authors find that LPS in vivo inhibits Ghr gene expression by a cytokine-dependent mechanism (Denson et al. 2001). It is also possible that the effect of meloxicam in the LPS-induced decrease in Ghr and Igf1 in vivo are partly mediated by changes at the pituitary GH.
TNF and nitric oxide were measured as possible mediators of the meloxicam effect, since they inhibit Igf1 gene expression in hepatocytes (Thissen & Verniers 1997, Ahmed et al. 2006, Priego et al. 2006). Although meloxicam prevented the inhibitory effect of LPS on Igf1 gene expression in the liver, it was not able to prevent the stimulatory effect of LPS on the release of nitrites+nitrates in vivo and in vitro. On the contrary, meloxicam increased nitrites+nitrates in basal conditions both in vivo and in vitro. These paradoxical results are in accordance with those recently reported (Razzak et al. 2008), in which Ptgs2 inhibition increases NO production in macrophages. Ptgs2 inhibitors have also been reported to increase nitric oxide production in oestrogenized rat uterus (Cella et al. 2006). These data suggest that PTGS2 products decrease nitric oxide synthesis.
Prostaglandins also have an inhibitory effect on Tnf gene expression and secretion in macrophages (Kunkel et al. 1988, Treffkorn et al. 2004) and Kupffer cells (Karck et al. 1988), whereas prostaglandin inhibition by indomethacin increases the Tnf response to LPS stimulation in the rat glial cells (Shemi et al. 2001).
The fact that meloxicam did not prevent Tnf and nitric oxide response to LPS suggests a direct effect of Ptgs2 products on Igf1 gene expression, rather than being mediated by nitric oxide or Tnf. In hepatocyte cultures it has been reported that Tnf was not able to alter basal Igf1 mRNA, but TNF- inhibits the induction of Igf1 mRNA by GH (Ahmed et al. 2006). Inhibition of Tnf release by pentoxifylline administration prevents LPS-induced IL-1, whereas it is not able to prevent the increased release of PTGES2 (Shemi et al. 2001), or the decrease in serum IGF1 and liver Igf1 mRNA (Colson et al. 2003). These data support the hypothesis that Tnf does not seem to be implicated in the LPS-induced decrease in basal Igf1 gene expression in the liver.
We have previously reported that Nos2 activation plays an important role in LPS-induced inhibition of GH–Igf1. Aminoguanidine, an Nos2 inhibitor, prevents the LPS-induced activation of hypothalamic somatostatin (Priego et al. 2005a), and the decrease in Igf1 in the liver (Priego et al. 2004). It has been reported that nitric oxide or iNOS activation positively regulates Ptgs2 gene expression (Salvemini et al. 1993, Yang et al. 2006). In addition, the Nos2 inhibitor aminoguanidine decreases LPS-induced Ptgs2 activation in Kupffer cells (Ahmad et al. 2002), and suppresses prostaglandin production in the LPS-treated animals (Salvemini et al. 1995). Accordingly, it is possible that part of the inhibitory effect of nitric oxide on the GH–Igf1 system can be due to the activation of Ptgs2 gene expression.
In summary, our data suggest that Ptgs2 activation plays an important role in the inhibitory effect of LPS on the Igf1 system by directly decreasing Igf1 gene expression in liver cells. On the contrary, Ptgs2 activation does not seem to be involved in the LPS-induced decrease in Igfbp3 gene expression in the liver.
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Declaration of Interest |
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Funding |
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Acknowledgements |
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References |
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Received in final form 16 May 2008
Accepted 20 May 2008
Made available online as an Accepted Preprint 20 May 2008
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