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Departamento de Fisiología, Facultad de Medicina, Universidad Complutense, Madrid 28040, Spain
(Requests for offprints should be addressed to A López-Calderón; Email: alc{at}med.ucm.es)
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Abstract |
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Introduction |
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The inflammatory response can be experimentally induced by lipopolysaccharide (LPS) administration. LPS is a component of Gram-negative bacteria and its administration triggers the sepsis response (Karima et al. 1999). A decrease in serum concentrations of IGF-I has been described after LPS administration in experimental animals and humans (Fan et al. 1994, Lang et al. 1997). Although LPS or inflammation also modifies pituitary growth hormone (GH) secretion (Kasting & Martin 1982, Soto et al. 1998), LPS administration is able to decrease liver IGF-I gene expression, acting independently of pituitary GH and liver GH receptors (GHR; Priego et al. 2003).
In the liver, the endotoxin response in vivo is mediated by complex cellular interplay between the different cell types. LPS activates Kupffer cells by increasing the release of nitric oxide (NO) and various cytokines (Decker 1990). Furthermore, Kupffer cell inactivation prevents the inhibitory effect of LPS injection in vivo on IGF-I gene expression in the liver (Granado et al. 2006). Taking into account that a direct inhibitory effect of cytokines on IGF-I gene expression in hepatocyte cultures has been reported (Wolf et al. 1996, Thissen & Verniers 1997), the inhibitory effect of LPS on liver IGF-I can be mediated by the release of mediators from Kupffer cells.
Another possibility is that LPS directly decreases IGF-I gene expression in hepatocytes. LPS receptors have been reported in hepatocytes and they respond to LPS through the toll-like receptor pathway (Liu et al. 2002). Recent experimental observations from our laboratory (Priego et al. 2004) suggest that overproduction of NO by inducible NO synthase (iNOS) is one of the mechanisms responsible for LPS-induced decrease in circulating IGF-I and its gene expression in the liver. One of the major features in endotoxic shock is the induction of iNOS in the liver. iNOS is induced by LPS or inflammatory stimuli in macrophages and hepatocytes, and it is responsible for the production of most NO release during endotoxemia. Inhibition of NO from iNOS protects from liver injuries induced by both mycobacterial infection and endotoxin administration (Guler et al. 2004). iNOS is expressed by hepatocytes, and LPS is able to induce iNOS gene expression and NO release in hepatocyte cultures (Geller et al. 1993). In addition, microarray studies revealed that iNOS in hepatocytes suppresses proliferation and protein synthesis (Zamora et al. 2002), effects which are IGF-I dependent.
The aim of this study was to analyze whether LPS is able to decrease IGF-I gene expression directly in rat hepatocytes or if it is mediated by the activation of Kupffer cells, and also to study the role of NO in this effect.
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Materials and Methods |
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Hepatocyte cultures
Hepatocytes were isolated by modification of the in situ collagenase perfusion technique as previously described (Seglen 1976). Rats were anesthetized with pentobarbital (Sigma, Chemical Co, St Louis, MO, USA). The portal vein was cannulated after opening the abdomen, perfused with calcium-free buffer 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 cells were obtained by gentle ranking with a comb and filtered through a 100 µm mesh. Hepatocytes were separated from non-parenchymal cells by differential centrifugation at 400 r.p.m. (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 (5 x 106 in 5 ml medium) were plated on 100 mm gelatin-coated Petri dishes. Medium consisted of Williams’ medium E (GIBCO, Cedex, France) with L-glutamine (2 mM), insulin (1 µM), HEPES (15 mM), penicillin+streptomycin (100 units/ml+ 100 g/ml), and 10% low endotoxin calf serum.
Hepatocyte and Kupffer cell cocultures
Hepatocytes were isolated as described earlier. Kupffer cells were isolated from supernatants of the differential centrifugations and centrifuged at 2200 r.p.m. for 6 min at 4 °C. The pellet was suspended in 5 ml cold Gey’s balanced salt solutions (GBSS; Invitrogen) buffer with NaCl 8%, and 7 ml GBSS buffer with 30% 5-(N-2,3-dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihydroxypropyl) isophthalamide (HISTODENZ, Sigma). Six milliliters of this mixture were transferred to glass tubes, 1 ml GBSS buffer with 8% NaCl was added, and tubes were centrifuged at 2800 r.p.m. at 4 °C for 20 min. After centrifugation, Kupffer cells were carefully collected from the interphase, suspended in 50 ml culture medium and centrifuged at 2200 r.p.m. for 7 min. Kupffer cells were finally suspended and plated with hepatocytes in a proportion of 3:2 (3.106 hepatocytes/2.106 Kupffer cells in 5 ml medium).
Experimental design
After 24-h incubation (37 °C in 95% air/5%CO2), the culture medium was removed and different stimuli were added in serum-free medium. Cells were incubated with the stimuli for another 24 h. On the following day, the medium was removed and stored at –80 °C for nitrite+nitrate determination. Total RNA from cells was isolated to measure IGF-I mRNA by northern blot hybridization.
The LPS response was analyzed after adding LPS (serotype 055:B5, Sigma) at different concentrations (0, 0.1, 1, 10, and 50 µg/ml) to the culture medium. In order to elucidate the effect of NO on hepatocytes, different concentrations (10, 100, and 1000 µg/ml) of a NO donor, S-nitroso-n-acetyl-D,L-penicillamide (SNAP; Alexis Corporation, Lausanne, Switzerland), were used. Finally, two selective iNOS inhibitors, L-N6-(1-iminoethyl)lysine (L-NIL), 0.1 mM and aminoguanidine hemisulfate, 0.1 mM (Alexis Corporation), were used in order to test their ability to prevent the effect of 50 µg/ml LPS on hepatic cells IGF-I mRNA. Experiments were performed at least twice.
Nitrite+nitrate determination
Nitrite+nitrate concentration in the culture medium was measured by a modified method of the Griess assay, described by Miranda et al.(2001). One hundred microliters were mixed with 100 µl vanadium chloride, rapidly followed by the addition of the Griess reagents. 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 micromolar.
RNA extraction
Total RNA was extracted using a commercial kit (Real Total RNA, C.E. Durviz S.L., Valencia, Spain) according to the protocol supplied by the manufacturer. Total RNA was dissolved in 0.1% SDS diethylpyrocarbonate-treated water and quantified at 260 nm. The integrity and the concentration of the RNA were confirmed using agarose gel electrophoresis.
IGF-I northern blot
For northern blotting, 10 µg denatured RNA were separated by formaldehyde–agarose gel electrophoresis, transferred to nylon membranes (Hybond-N+, Amersham) by overnight capillary blotting and fixed by UV crosslinking (Fotodyne, Hartland, WI, USA). The rat IGF-I cDNA (Roberts et al. 1987) was generously supplied by Dr LeRoith. To generate radiolabeled cRNA, the plasmid vector (pGEM-3, Promega) was linearized with HindIII. 32P-labeled RNA antisense probes were generated from linearized plasmid with [
-32P]CTP (Nuclear Ibérica, Madrid, Spain) and T7 RNA polymerase (Roche Molecular Biochemicals). Prehy-bridization was performed for 30 min at 68 °C in ULTRA-hyb buffer (Ambion, Austin, TX, USA) followed by hybridization for 16 h at the same temperature with 1 x 106 c.p.m./ml IGF-I labeled riboprobe. The membranes were washed twice with 2 x SSC, 0.1% SDS at 68 °C for 10 min, and twice with 0.1 x SSC, 0.1% SDS at 68 °C also for 10 min. To verify loading, control hybridization was performed with a 28 S DNA probe labeled with 32P-dCTP by random primer amplification. The membranes were exposed at –80 °C for 1–4 days to X-ray film (Kodak X-Omat AR, Eastman Kodak) and with two intensifying screens. Autoradiographs were analyzed by densitometric scanning using a Gengenius (Syngene, Cambridge, UK). The intensities of autoradiograph signal levels were measured and expressed as the percentage of the mean intensity of the control cultures.
Real-time PCR
For reverse transcriptase (RT)-PCR analysis, 2 µg mRNA were reverse-transcribed using the instructions in the commercial kit Quantitec Reverse Transcription Kit (Qiagen). Primers for PCR were obtained from previously published sequences (Peinnequin et al. 2004), suppressor of cytokine signaling-3 (SOCS-3) forward: CCTCCAG-CATCTTTGTCGGAAGAC; SOCS-3 reverse: TACTGG-TCCAGGAACTCCCGAATG; hypoxanthine–guanine phosphorybosyl transferase (Hprt) forward: CTCATG-GACTGATTATGGACAGGAC; Hprt reverse: GCAGGT-CAGCAAAGAACTTATAGCC. They were designed from spanning a single sequence derived from two exons (i.e., separated by an intron in genomic DNA and primary RNA transcripts to minimize amplification). All primers were obtained from Qiagen. Each real-time PCR consisted of 10 ng total RNA equivalents, 1 x Takara SYBR Green Premix Ex Taq (Takara Bio, Inc., Shiga, Japan), and 300 nM forward and reverse primers in a reaction volume of 25 µl. Reactions were carried out on a SmartCycler (Cepheid, Sunnyvale, CA, USA).
Parameters included 40 cycles of denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. Specific amplification was confirmed by the presence of one single peak in the melting curve plots. In addition, the PCR products were analyzed by agarose gel electrophoresis. Results were calculated as percent of control cultures, using the cycle threshold method (Livak et al. 2001) with the Hprt as reference gene.
Statistical analysis
All data are presented as the mean ± S.E.M. Simple linear regression was used to determine correlations betweenvariables. Differences among experimental groups were analyzed by one-or two-way ANOVA. Where there were differences among groups, the individual means were compared by Student’s t-test. Significance was assumed when P<0.05.
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shows the IGF-I mRNA and nitrite+nitrate levels in hepatocytes incubated with different LPS concentrations (from 0.1 to 50 µg/ml LPS). The nitrite+nitrate concentrations in the culture medium increased in parallel with the increase in the LPS concentration. The IGF-I mRNA in the cells also showed a dose-related response to LPS, being significantly decreased by all LPS concentrations employed. In the groups of cells incubated with LPS, there was a negative correlation between the nitrite+nitrate concentrations in the culture medium and the IGF-I mRNA (r=–0.61, P<0.01), but not in the cells cultured with medium alone (Fig. 1).
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). Incubation of cells with LPS decreased the IGF-I mRNA levels in both types of cultures, but the inhibitory effect of LPS was higher in cocultures than in hepatocytes alone (P<0.01). When the hepatocytes were cocultured with Kupffer cells, both LPS concentrations induced a significant decrease in IGF-I gene expression (P<0.01). However, in hepatocyte cultures only the higher LPS concentration tested (1 µg/ml) induced a significant decrease in IGF-I gene expression (Fig. 2).
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shows that SNAP treatment resulted in an increase in nitrite+nitrate levels in the culture medium, which was only significant at the concentration of 1000 µM (P<0.01). In addition, exogenous NO donated by SNAP also decreased IGF-I gene expression (P<0.01) at the SNAP concentration of 1000 µM, whereas it was not modified by the other SNAP concentrations (Fig. 3).
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). The IGF-I mRNA was decreased by LPS (P<0.01), but it was not modified by either incubation with L-NIL or aminoguanidine. The addition of L-NIL to the incubation medium prevented LPS-induced decrease in IGF-I mRNA (P<0.01). Aminoguanidine attenuated the decrease in IGF-I mRNA induced by LPS; the cells incubated with LPS+aminoguanidine had higher IGF-I mRNA levels (P<0.01) than the cells incubated with LPS alone.
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. In the cultures incubated with LPS, there was a notable increase in SOC-3 mRNA (P<0.01, Fig. 5). Neither L-NIL nor aminoguanidine modified the SOCS-3 gene expression basally or after LPS stimulation.
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, LPS increased the nitrite+nitrate levels (P<0.01), and L-NIL blocked this stimulatory effect. LPS induced a significant decrease in IGF-I mRNA levels (P<0.01) in hepatocytes cultured with Kupffer cells (Fig. 6). L-NIL alone did not modify the IGF-I gene expression, but it attenuated the inhibitory effect of LPS on IGF-I.
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Discussion |
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The fact that the inhibitory effect of LPS on IGF-I is higher in cocultures than in hepatocytes alone is not unexpected, since stimulated Kupffer cells release many inflammatory mediators that can potentially inhibit IGF-I gene expression. The release of these mediators, tumour necrosis factor (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6), after LPS stimulation is higher in cocultures than in Kupffer or hepatocyte cultures (Hoebe et al. 2001, Yao et al. 2004). In addition, suppression of Kupffer cell function in vivo by gadolinium chloride injection blocks Kupffer cell phagocytosis and the subsequent release of its mediators, such as cytokines and NO after LPS administration, as well as the decrease in IGF-I gene expression in the liver (Hardonk et al. 1992, Granado et al. 2006). The increased inhibitory effect of LPS in vitro when incubated with Kupffer cells does not seem to be due to NO release, since nitrite+nitrate levels after LPS were similar in the medium of hepatocyte cultures and in cocultures. In addition, the blockade of LPS-induced NO release by L-NIL only partially prevents LPS-induced decrease in IGF-I mRNA when Kupffer cells were present in the cultures. All these data indicate that Kupffer cells increase the inhibitory effect of LPS on IGF-I mRNA by a pathway that is different to NO release. One of these pathways can be the release of cytokines, since an inhibitory effect of cytokines on in vitro IGF-I gene expression has been reported. IL-6 inhibits IGF-I gene expression both in hepatocyte and in hepatocyte with Kupffer cell cocultures (Lelbach et al. 2001). IL-1 is also able to decrease basal IGF-I mRNA levels and IGF-I release to the culture medium in isolated hepatocytes (Wolf et al. 1996). However, this effect is weak, and other authors were not able to find a decrease in IGF-I mRNA levels after IL-1 addition, basally in the absence of GH (Thissen & Verniers 1997).
As it has been described (Saad et al. 1995), LPS alone is able to increase NO production in cultured hepatocytes. The fact that there was an inverse correlation between nitrites and IGF-I mRNA in the hepatocytes incubated with LPS, but not in controls, suggests an inhibitory effect of NO on IGF-I gene expression. Moreover, when a NO donor, SNAP, was added to hepatocytes, a decrease in IGF-I mRNA was also observed, once again emphasizing the involvement of NO in the inhibition of IGF-I gene expression. The effect of NO donors on hepatocyte function is not well known. It has been reported that SNAP pretreatment protects hepatocytes from TNF-induced apoptosis by inducing heat shock protein (Kim et al. 1997). However, in another study (Liang et al. 1997) NO from NO donors induced cell death by inhibiting mitochondrial respiration, but at a higher concentration than those used in the present study. An inhibitory effect of SNAP on IGF-I gene expression has previously been described in myoblasts (Frost et al. 2003). In those cells, TNF- induces iNOS expression and decreases IGF-I mRNA, but the NOS inhibitors (L-NMMA or 1400W) are not able to prevent the inhibitory effect of TNF- on IGF-I mRNA (Frost et al. 2003). Differences can be due to the fact that IGF-I gene regulation is different in myoblasts than in hepatocytes, or to the inhibitors employed; in our data, both inhibitors totally prevent the effect of LPS on nitrite release to the culture medium. Another possibility is that in addition to inducing iNOS gene expression, TNF- might inhibit IGF-I gene expression through a pathway different to NO.
The fact that the inhibitory effect of LPS can be prevented by two specific iNOS inhibitors suggests that the effect of LPS on IGF-I is related to the generation of NO through induction of iNOS. All these data suggest that during sepsis, the increase in NO release by iNOS induction inhibits basal IGF-I gene expression in the liver and that the inhibitory effect of NO on IGF-I gene expression is exerted directly at a hepatocyte level. The overproduction of NO during septic shock has been demonstrated to contribute to circulatory failure, myocardial dysfunction, organ injury and multiple organ failure. Taking into account the effect of IGF-I in tissue regeneration, iNOS inhibition might prevent liver injury in septic human patients as it has been reported in experimental animals (Guler et al. 2004).
Specific information on the effects of iNOS-derived NO on hepatocyte IGF-I gene expression is limited. We have previously reported that iNOS inactivation by aminoguani-dine administration prevents the LPS-induced decrease in serum concentrations of IGF-I as well as in its mRNA in the liver (Priego et al. 2004). Aminoguanidine also has additional pharmacological properties unrelated to the NO pathway (Nilsson 1999), and therefore, its beneficial effects could arise from a mechanism different to iNOS inhibition. This does not seem to be the case, since in the present data L-NIL was even more potent than aminoguanidine in preventing LPS-induced decrease in IGF-I gene expression.
SOCS-3 is an acute-phase protein gene that is upregulated in the liver by LPS and inflammatory cytokines. It has been suggested that upregulation of SOCS-3 is involved in LPS-induced inhibition of STAT5b and IGF-I in vivo (Denson et al. 2003). This hypothesis is supported by previous studies in rodents showing that impairment of GH signaling in the liver induced by LPS, IL-6, chronic renal failure, and sepsis is accompanied by elevated expression of SOCS-2 and SOCS-3 (Mao et al. 1999, Denson et al. 2003). Our data do not support this hypothesis, since inhibition of NO release prevents LPS-induced decrease in IGF-I gene expression, but not the increase in SOCS-3. Similarly, it has been reported that the inhibitory effect of IL-1 on IGF-I mRNA, following GH induction, is not caused by JAK/STAT signaling or increased SOCS-3 expression (Shumate et al. 2005). Additional studies will be required to determine the mechanism by which NO inhibits IGF-I gene expression in the hepatocyte.
In conclusion, our data suggest that LPS decreases IGF-I gene expression in hepatocytes through the induction of iNOS and through the release of other inhibitory mediators in Kupffer cells.
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Acknowledgements |
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Received 28 April 2006
Received in final form 5 June 2006
Accepted 20 June 2006
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P<0.05, 
P<0.01 versus aminoguanidine incubated with medium. Two-way ANOVA and Student’s t-test.
