NO plays a role in LPS-induced decreases in circulating IGF-I and IGFBP-3 and their gene expression in the liver
Teresa Priego,1
Inmaculada Ibáñez de Cáceres,1
Ana Isabel Martín,2
M. Ángeles Villanúa,1 and
Asunción López-Calderón1
1Departamento de Fisiología, Facultad de Medicina, Universidad Complutense 28040 Madrid; and 2Departamento de Ciencias Morfológicas y Fisiología, Universidad Europea, 28670 Madrid, Spain
Submitted 4 April 2003
; accepted in final form 12 September 2003
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ABSTRACT
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In this study, we administered aminoguanidine, a relatively selective inducible nitric oxide synthase (iNOS) inhibitor, to study the role of nitric oxide (NO) in LPS-induced decrease in IGF-I and IGFBP-3. Adult male Wistar rats were injected intraperitoneally with LPS (100 µg/kg), aminoguanidine (100 mg/kg), LPS plus aminoguanidine, or saline. Rats were injected at 1730 and 0830 the next day and killed 4 h after the last injection. LPS administration induced an increase in serum concentrations of nitrite/nitrate (P < 0.01) and a decrease in serum concentrations of growth hormone (GH; P < 0.05) and IGF-I (P < 0.01) as well as in liver IGF-I mRNA levels (P < 0.05). The LPS-induced decrease in serum concentrations of IGF-I and liver IGF-I gene expression seems to be secondary to iNOS activation, since aminoguanidine administration prevented the effect of LPS on circulating IGF-I and its gene expression in the liver. In contrast, LPS-induced decrease in serum GH was not prevented by aminoguanidine administration. LPS injection decreased IGFBP-3 circulating levels (P < 0.05) and its hepatic gene expression (P < 0.01), but endotoxin did not modify the serum IGFBP-3 proteolysis rate. Aminoguanidine administration blocked the inhibitory effect of LPS on both IGFBP-3 serum levels and its hepatic mRNA levels. When aminoguanidine was administered alone, IGFBP-3 serum levels were increased (P < 0.05), whereas its hepatic mRNA levels were decreased. This contrast can be explained by the decrease (P < 0.05) in serum proteolysis of this binding protein caused by aminoguanidine. These data suggest that iNOS plays an important role in LPS-induced decrease in circulating IGF-I and IGFBP-3 by reducing IGF-I and IGFBP-3 gene expression in the liver.
nitric oxide; inducible nitric oxide synthase; lipopolysaccharide; growth hormone; insulin-like growth factor I; insulin-like growth factor-binding protein-3; proteolysis; aminoguanidine
SEPSIS INDUCES PROTEOLYSIS and negative nitrogen balance, which leads to lean body mass loss and cachexia. Sepsis is also associated with a wide range of hormonal changes that contribute to the catabolic state; among these there is an alteration of the somatotropic axis (47). A decrease in serum concentrations of IGF-I in septic patients and in different inflammatory diseases has also been observed (12, 28).
Lipopolysaccharide (LPS), or endotoxin, triggers the initiation of host responses to sepsis by gram-negative bacterial infection. Endotoxin administration in rats increases plasma concentrations of corticosterone, whereas it decreases circulating growth hormone (GH) and IGF-I (20, 27). The decrease in circulating IGF-I is secondary to a decrease in IGF-I gene expression in the liver and other peripheral tissues such as muscle (30). The inhibitory effect of LPS on the IGF-I axis is also exerted at the insulin-like growth factor-binding protein-3 (IGFBP-3) levels, since in the LPS-treated rats there was a decrease in serum concentration of this protein and in its synthesis in the liver (37). The functional significance of this impact on IGFBPs is equivocal. The decrease in this binding protein may increase IGF-I turnover and contribute to the catabolic state in sepsis, or, on the contrary, it may increase the IGF-I bioavailability and counteract the catabolic response.
The mechanism by which endotoxin inhibits liver IGF-I and IGFBP-3 is not totally known. We have recently reported that LPS-induced decrease in both circulating IGF-I and IGFBP-3, as well as their gene expression in the liver, is independent of glucocorticoids (35, 36). It has been proposed that the increased release of TNF can be the cause of LPS-induced IGF decrease (19). However, inhibition of TNF production did not prevent the decrease in serum and liver IGF-I in LPS-treated rats (11). It has been postulated that the decrease in circulating IGF-I after endotoxin challenge is related to nitrogen free radical production and that reaction of peroxynitrite with liver proteins affects the regulation of IGF-I. This hypothesis is supported by the observation that plasma IGF-I was not affected by LPS in calves treated with vitamin E (18).
Nitric oxide (NO) is a key intermediary signal in several different responses during endotoxemia. NO is generated by constitutive nitric oxide synthase (cNOS) and inducible NOS (iNOS). iNOS is induced by inflammatory stimuli such as LPS in macrophages or hepatocytes, and it is responsible for the production of most NO during endotoxemia. Endotoxin or cytokine administration increases iNOS activity in hepatic parenchymal cells, Kupffer cells, and hepatic endothelial cells (21). The reaction of NO with superoxide anion results in the formation of cytotoxic oxygen radicals, which are responsible for several cytotoxic actions of NO. Overproduction of NO can be responsible, in part, for the toxic effect of sepsis and hepatic injury.
Therefore, the aim of this work was to study the role of LPS-induced NO release on the IGF-I system in rats. Administration of nonselective NOS inhibitors, such as NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester, had deleterious effects in experimental endotoxic shock and amplified the hepatic injury induced by LPS (10). However, aminoguanidine, a selective iNOS inhibitor, improved survival of LPS-challenged animals (15, 25, 41), and iNOS deficiency increased the resistance to LPS-induced endotoxic shock (32). For this purpose, the effect of aminoguanidine on the IGF-I and IGFBP-3 response to LPS was examined.
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MATERIALS AND METHODS
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Experimental procedures. Adult male Wistar rats (200-250 g) were purchased from Harlam (Barcelona, Spain). They were housed three to four per cage under controlled conditions of temperature (22°C) and light (lights on from 0730 to 1930). Food and water were available ad libitum. The procedures followed the guidelines recommended by the European Union for the care and use of laboratory animals. Twenty rats were injected intraperitoneally with 100 µg/kg LPS (serotype 055:B5; Sigma Chemical, St. Louis, MO) in 250 µl of sterile saline. Another 20 rats were injected with saline. One-half of the rats in each group were simultaneously injected intraperitoneally with the selective iNOS inhibitor aminoguanidine hemisulfate (100 mg/kg; Alexis, Lausanne, Switzerland) and the other half with 250 µl of sterile saline. Rats received the treatments at 1730 and at 0830 the following day, as such an LPS administration protocol was shown to decrease levels of serum IGF-I and its mRNA in the liver (unpublished data). All animals were killed by decapitation at 1230, 19 h after the first and 4 h after the second LPS and/or aminoguanidine injection. Liver tissues were 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 GH, IGF-I, IGFBP-3, insulin, and nitrite/nitrate assays.
Hormone determination. Concentrations of GH were measured by radioimmunoassay (RIA) using reagents provided by Dr. A. F. Parlow of the National Hormone and Pituitary Program (National Institutes of Health, Bethesda, MD). Levels of GH were expressed in terms of National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) rat-RP-2 standard. The GH detection level was 10 pg/tube, and the intra-assay coefficient of variation was 3%. All necessary comparisons between test and control animals were made within the same assay.
Serum IGF-I concentrations were measured by a double-antibody RIA. The IGF-I antiserum (UB-2495) was a gift from Drs. L. Underwood and J. Van Wyk, and it is distributed by the Hormone Distribution Program of NIDDK through the National Hormone and Pituitary Program. Levels of IGF-I were expressed in terms of IGF-I from Gropep (Adelaide, Australia). The intra-assay coefficient of variation was 8%. All samples were run in the same assay.
Western ligand blot of IGF-binding proteins. Western blots were prepared as previously described (39). Blood serum proteins were separated by 12.5% SDS-PAGE under nonreducing conditions and blotted onto nitrocellulose membranes (Hybond-C extra, Amersham, Little Chalfont, UK) by means of a semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA). The blots were dried and blocked for 1 h with 5% nonfat dry milk and 0.1% Tween (Sigma) in Tris buffered saline and incubated overnight at 4°C with 125I-labeled IGF-I (5 x 105 cpm/ml). The nitrocellulose sheets were washed and dried, and blots were exposed at -80°C to X-ray film (Kodak X-Omat AR; Eastman Kodak, Rochester, NY) and two intensifying screens for 1-3 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 IGFBP-3 band in each lane was expressed as the percentage of the mean density of sera from control rats injected with saline.
Proteolysis assay. To measure the IGFBP-3 proteolysis in serum, we used the Lamson et al. (29) method. Serum samples (5 µl) were mixed with 15,000 cpm 125I-rhIGFBP-3 (glycosylated recombinant human, GroPep, iodinated using the chloramine T method) in a total volume of 30 µl in 0.05 M phosphate buffer, pH 7.4. The mixture was incubated for 18 h at 37°C, and the reaction was stopped by adding 10 µl of nonreducing sample buffer and boiling. Ten microliters of the mixture were subjected to 12.5% SDS-PAGE and run at 160 mA for 2 h. Gels were fixed and dried (Bio-Rad Gel Drying System-543) and exposed to X-ray film at -80°C for 2-3 days. The intact 125I-rhIGFBP-3 (42- to 45-kDa band) from each sample was measured by densitometry. Proteolysis was expressed as percentage of the total optical density from heat inactivated serum (56°C for 30 min).
Nitrite/nitrate determination. Serum was deproteinized to reduce turbidity by centrifugation through a 30-kDa molecular mass filter by use of a Centrifree Micropartition Device with a YM-30 ultrafiltration membrane (Amicon Division, Millipore, Bedford, MA), at 1,500 rpm for 1 h at 37°C for 300-µl samples. Serum nitrite/nitrate concentration was measured by a modified method of the Griess assay, described by Miranda et al. (33). One hundred microliters of filtrated serum were mixed with 100 µl of VCl3, 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 micromoles per liter.
RNA extraction and Northern blot analysis. Total liver RNA was extracted by the guanidine thiocyanate method with a commercial kit (Ultraspec RNA; Biotecx Laboratories, Houston, TX) according to the protocol supplied by the manufacturer. The extracted total RNA was dissolved in 0.1% SDS-diethyl pyrocarbonate-treated water and quantified at 260 nm. The integrity and the concentration of the RNA were confirmed using agarose gel electrophoresis.
For Northern blotting, 30 µg of denatured RNA from each liver were separated by formaldehyde-agarose gel electrophoresis, transferred to nylon membranes (Hybond-N+, Amersham), and fixed by UV crosslinking (Fotodyne, Hartland, WI).
IGF-I and GH receptor (GHR) mRNA hepatic levels were measured by Northern blot hybridization using riboprobes (31). The rat IGF-I cDNA (38) was generously supplied by Dr. D. LeRoith, and the rat GHR cDNA probe was kindly provided by Dr. W. R. Baumbach (4). To generate radiolabeled complementary RNAs (cRNA), the plasmid vector (pGEM-3; Promega, Madison, WI) was linearized with HindIII. The 32P-labeled RNA antisense probes were generated by transcription with T7 RNA polymerase (Roche Molecular Biochemicals, Barcelona, Spain) using [
-32P]cytidine triphosphate (Nuclear Ibérica, Madrid, Spain). Prehybridization was performed for 30 min at 68°C in ULTRAhyb buffer (Ambion, Austin, TX) followed by hybridization for 16 h at the same temperature with 1 x 106 cpm/ml IGF-I-labeled riboprobe or 2 x 106 cpm/ml GHR-labeled riboprobe. The membranes were washed twice with 2x SSC and 0.1% SDS at 68°C for 10 min, and twice with 0.1x SSC and 0.1% SDS at 68°C also for 10 min.
The rat IGFBP-3 cDNA probe encodes the IGFBP-3 protein mRNA (1). The probe was obtained by cutting the PEGEM 4Z plasmid vector using EcoRI and HindIII and labeling it with [32P]dCTP (Nuclear Ibérica) by a random-priming DNA labeling kit (DECAprimeTM II; Ambion). Prehybridization was performed for 30 min at 42°C with ULTRAhyb buffer followed by hybridization for 16 h at the same temperature with 3 x 106 cpm/ml IGFBP-3-labeled probe. The membranes were washed twice with 2x SSC and 0.1% SDS at 42°C for 10 min and twice with 0.1x SSC and 0.1% SDS also for 10 min at 42°C. The membranes were exposed at -80°C for 2-4 days.
To verify loading, control hybridization was performed with a 28 S DNA probe labeled with [32P]dCTP by random primer (Roche). The intensities of autoradiogram signal levels were analyzed by densitometric scanning.
Statistical analysis. Statistics were computed using the statistics program STATGRAPHICS Plus for Windows. Statistical significance was calculated by multifactorial ANOVA with LPS and aminoguanidine as main effects. When the ANOVA indicated a significant interaction between effects, individual means were compared with Duncan's multiple range test. Correlation between different variables was calculated by linear regression. Serum concentrations of GH and IGFBP-3 data were subjected to log transformation, since variances showed a log-normal distribution. A P value of <0.05 was considered significant.
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RESULTS
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Figure 1 shows the serum concentrations of nitrite/nitrate in all groups of rats. LPS administration induced a significant increase (P < 0.01) in serum concentrations of nitrite/nitrate, whereas aminoguanidine administration prevented this effect.

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Fig. 1. Serum growth hormone (GH; A), IGF-I (B), and nitrite/nitrate levels (C) in rats treated with lipopolysaccharide (LPS, 0.1 mg/kg) and LPS + aminoguanidine (AG, 100 mg/kg). LPS administration decreased serum concentrations of GH (F1,39 = 16, P < 0.01). There was an interaction (F1,33 = 10, P < 0.01) between the effects of LPS and AG on serum concentrations of IGF-I, as LPS decreases IGF-I (P < 0.01) in control but not in AG-treated rats. There was also an interaction (F1,33 = 4.4, P < 0.05) between the effects of LPS and AG on serum concentrations of nitrite/nitrate, as LPS increased serum nitrite/nitrate levels (P < 0.01) in control but not in AG-treated rats. Results are expressed as means ± SE; n = 8-10 rats/group; **P < 0.01; *P < 0.05 vs. respective group injected with saline; ++P < 0.01 vs. control group injected with LPS (Duncan's multiple comparison test).
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LPS administration decreased serum concentrations of IGF-I in control rats (P < 0.01), and aminoguanidine treatment prevented the inhibitory effect of LPS on serum IGF-I levels (Fig. 1). Serum concentrations of GH were decreased (P < 0.05) in the rats injected with LPS. Contrary to the IGF-I results, aminoguanidine administration was not able to prevent the inhibitory effect of LPS on serum concentrations of GH (Fig. 1).
GHR mRNA in the liver was not significantly modified by LPS, although LPS-injected rats had a tendency to lower GHR mRNA levels than the rats injected with saline (89 ± 3 vs. 100 ± 3%), whereas the rats injected with aminoguanidine (either with or whithout LPS) had the same values of GHR mRNA (Fig. 2).

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Fig. 2. Effect of LPS and AG administration on hepatic GH receptor (GHR) mRNA. A: hepatic GHR mRNA levels. Data from 9-10 individual rats were quantified by densitometry and expressed as a percentage of the mean value in control rats treated with saline. B: representative Northern blot analysis showing the 4.5- and 1.2-kb GHR transcripts; 28S ribosomal RNA (28 S) in each sample is shown below.
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iNOS inhibition had a similar effect on liver gene expression of IGF-I and on serum concentrations of IGF-I, since LPS administration decreased the IGF-I mRNA levels in the liver (P < 0.05) in saline-treated rats but not in rats treated with aminoguanidine (Fig. 3).

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Fig. 3. Effect of LPS and AG administration on hepatic IGF-I mRNA. A: liver IGF-I mRNA levels. Data from 7-10 individual rats were quantified by densitometry and expressed as a percentage of the mean value in control rats treated with saline. *P < 0.05 vs. control group injected with saline (Duncan's multiple range test). B: representative Northern blot analysis showing the 7.5-, 1.7-, and 0.9-kb IGF-I transcripts; 28S ribosomal RNA in each sample is shown below.
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LPS administration decreased the IGFBP-3 mRNA in the liver of control rats (P < 0.01) but not in aminoguanidine-treated rats (Fig. 4). There was a decrease in hepatic IGFBP-3 mRNA in aminoguanidine plus saline-injected rats (P < 0.05). The effect of aminoguanidine and LPS administration on serum concentrations of IGFBP-3 is shown in Fig. 5. The serum IGFBP-3 response to aminoguanidine was very different from the liver IGFBP-3 mRNA response. Aminoguanidine administration increased serum concentrations of IGFBP-3 both in saline-treated (P < 0.05) and in LPS-treated (P < 0.01) rats. LPS decreased the serum IGFBP-3 levels in control rats (absolute values 68.6 ± 9 vs. 100 ± 10.7%, P < 0.05) but not in rats treated with aminoguanidine.

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Fig. 4. A: effect of LPS and AG administration on hepatic insulin-like growth factor-binding protein-3 (IGFBP-3) mRNA. B: representative Northern blot of IGFBP-3 mRNA hybridization. Size of the hybridization band (in kb) is indicated at left; each band corresponds to an individual animal from the indicated group. Quantitative analyses are expressed as percentages of control rats injected with saline. There was an interaction (F1,36 = 8.4, P < 0.01) between the effects of LPS and AG, as LPS decreased IGFBP-3 in control but not in AG-treated rats. Bars represent means ± SE; n = 10 rats. **P < 0.01; *P < 0.05 vs. control group injected with saline; +P < 0.05 vs. control group injected with LPS (ANOVA plus Duncan's multiple comparison test).
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Fig. 5. A: serum IGFBP-3 levels in LPS- and AG-treated rats. B: representative Western ligand blot of IGFBP-3 in 2.5-µl serum from the different groups; approximate molecular mass of the band is indicated at left. Data from 6-10 individual rats were quantified by densitometry and expressed as percentage of the mean value in control rats treated with saline. *P < 0.05 vs. control group injected with saline; ++P < 0.01 vs. control group injected with LPS (ANOVA plus Duncan's multiple comparison test).
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LPS administration tended to decrease IGFBP-3 proteolysis in serum, but this effect was not significant (Fig. 6). Aminoguanidine administration decreased (P < 0.05) the serum IGFBP-3 proteolysis in the rats injected with saline but not in LPS-treated rats (Fig. 5). There was a correlation between the serum nitrite/nitrate levels and the serum IGFBP-3 proteolysis in the rats injected with saline plus aminoguanidine (r = 0.897, P < 0.01) but not in the other three groups of rats.

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Fig. 6. A: effect of AG administration on IGFBP-3 proteolytic activity in serum of rats treated with saline or LPS. 125I-labeled recombinant human (rh)IGFBP-3 was incubated for 18 h at 37°C with sera and submitted to SDS-PAGE as described in MATERIALS AND METHODS. B: representative auto-radiograph of IGFBP-3 protease assay of rat serum samples from the different groups and heat-inactivated serum. 125I-rhIGFBP-3 abundance was determined by densitometry and proteolysis expressed as a percentage of the value of the heat-inactivated serum (B). Data represent means ± SE; n = 8-11 rats/group. *P < 0.05 vs. control rats injected with saline (Duncan's multiple comparison test).
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DISCUSSION
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As expected, LPS administration increased nitrite/nitrate serum levels, whereas it decreased serum concentrations of GH, IGF-I, and IGFBP-3. Our results show that the iNOS inhibitor aminoguanidine is able to prevent the inhibitory effect of LPS administration on serum concentrations of IGF-I and IGFBP-3 as well as their gene expression in the liver. These data suggest that overproduction of NO by iNOS is one of the mechanisms responsible for endotoxin-induced decrease in circulating IGF-I and IGFBP-3 and their mRNA in the liver.
The inhibitory effect of iNOS activation on IGF-I and IGFBP-3 does not seem to be secondary to an alteration in pituitary GH secretion, because aminoguanidine administration prevents the decrease in IGF-I and IGFBP-3 without modifying LPS-induced decrease in serum concentrations of GH. Both neuronal NOS and iNOS have been described in the pituitary (9, 44) and in the hypothalamus (3, 5), but the role of NO in the regulation of GH secretion is controversial. It has been postulated that pituitary iNOS activation decreases GH secretion, since its inhibition by L-N(6)-(l-iminoethyl)lysine administration prevents the inhibitory effect of IFN-
on GH release in pituitary cell cultures (45). However, other data in the literature suggest that NO has a stimulatory effect on GH release in vitro (3, 34), and in vivo (43). These discrepancies have been explained by a biphasic action of NO on GH secretion: a positive effect at low concentrations and a negative effect at high concentrations (7).
As previously reported, the decrease in serum concentrations of IGF-I in LPS-treated rats is concomitant with a decrease in its gene expression in the liver (30, 35). Taking into account that most of the circulating IGF-I is synthesized in the liver, the decrease in serum concentrations of IGF-I seems to be secondary to the inhibitory effect of LPS on IGF-I gene expression in the liver cells. The decrease in liver IGF-I gene expression in LPS-treated rats is associated with GH resistance (13). The lack of a significant decrease in GHR mRNA in LPS-injected rats in the present study could be due to the LPS dose, because when using a higher LPS dose (1 mg/kg) we found a significant reduction in GHR mRNA (31). The higher sensitivity of liver IGF-I mRNA over liver GHR mRNA to the inhibitory effect of LPS has recently been explained by the fact that LPS administration inhibits GH activation of signal transducer and activator of transcription 5 and by induction of postreceptor signaling inhibitors such as cytokine-inducible SH2-containing protein 1 and suppressors of cytokine signaling 3 (14).
There are few data about the role of NO on IGF-I and IGFBP-3 gene expression and serum levels. Aminoguanidine administration in the drinking water for 18 days increased serum concentrations of IGF-I but reduced bone formation in remodeling bone in normal rats (42). Treatment with aminoguanidine is able to prevent a diabetes-induced decrease in renal IGF-I mRNA, suggesting an inhibitory effect of NO in renal IGF-I synthesis (2). It is now generally accepted that NO derived from endothelial NOS is critical for maintaining the integrity of hepatic microvasculature, whereas the role of iNOS activation in LPS-induced liver injury is not well known. A protective role of iNOS in the ischemia-reperfusion-induced liver injury has been recently proposed (24). In contrast, a role of NO, derived from activated macrophage, in hepatocyte injury during inflammation has also been reported (48). In addition, inhibition of LPS-induced iNOS prevents liver injury (23). These data suggest that NO and its oxidative metabolite peroxynitrite play key roles in hepatocyte injury during inflammation and cause of subsequent DNA damage in surviving hepatocytes (48).
Kupffer cells, when exposed to LPS, can directly damage hepatocytes, but only if L-arginine, a NO precursor, is present (6). It is not only the Kupffer cells that are LPS responsive; hepatocytes can also be induced by LPS to express the molecules necessary for responses to bacteria (46). In addition, aminoguanidine is a potent selective inhibitor of iNOS in cultured rat hepatocytes (50). It is also possible, then, that the inhibitory effect of LPS on hepatic IGF-I and IGFBP-3 gene expression can be due to a direct effect of NO in the liver cells. Microarray studies revealed that iNOS in hepatocytes acts to suppress proliferation and protein synthesis (49), effects that are IGF-I dependent.
The effect of iNOS inhibition on the IGFBP-3 response to LPS injection is similar to the IGF-I response. Aminoguanidine administration prevents the inhibitory effect of LPS on circulating IGFBP-3 as well as its gene expression in the liver. However, aminoguanidine administration to control rats has a very different effect on circulating IGFBP-3 than on its gene expression in the liver. Although aminoguanidine treatment increased serum concentrations of IGFBP-3, it decreased the IGFBP-3 mRNA in the liver.
Differences between liver IGFBP-3 mRNA and IGFBP-3 serum levels have been previously reported. Normal liver IGFBP-3 mRNA with low concentrations of IGFBP-3 in serum have been observed in cirrhotic patients (16) or after administration of a potent IGF-I analog that binds poorly to IGFBP (17). The fact that in control rats aminoguanidine treatment decreases IGFBP-3 synthesis in the liver, but significantly increases the IGFBP-3 serum levels, can be explained by two causes: first for the decrease in its proteolysis and second for an increased IGFBP-3 synthesis in organs other than the liver. In our data, there was an increase in the IGFBP-3 half-life in serum, since its proteolysis was decreased in control rats treated with aminoguanidine. Proteolysis of the IGFBP-3 in the circulation can be due to several proteases, such as plasmin, thrombin, kallikrein, etc. Thus the stimulatory effect of NO on IGFBP-3 proteolysis can be exerted at the level of many different proteases. Although the increased half-life of IGFBP-3 in serum can explain its increase in serum, we cannot exclude the possibility that aminoguanidine treatment increases IGFBP-3 synthesis in organs other than the liver.
Aminoguanidine has previously been reported to inhibit proteases such as in calpain-mediated proteolysis (26). Selective thrombin- and serine protease-inhibitory properties have also been described for sulfonyl-aminoguanidine (8). In addition, NO has been reported to be involved in the activation of proteolytic enzymes (40). However, if NO had a stimulatory effect on IGFBP-3 proteolysis, proteolysis should be increased in the rats injected with LPS, and this was not the case. On the contrary, LPS administration tended to decrease IGFBP-3 proteolysis. The fact that there was a correlation between serum concentrations of nitrite/nitrate and proteolysis in saline- or aminoguanidine-treated rats but not in the rats injected with LPS suggests that the effect of aminoguanidine on serum proteases can be due to aminoguanidine pharmacological effects other than iNOS inhibition.
In conclusion, this study shows that aminoguanidine administration attenuated LPS-induced decrease in IGF-I and IGFBP-3, suggesting that induction of iNOS during sepsis is involved in the inhibition of IGF-I-IGFBP-3. Because the IGF-I system has been involved in tissue regeneration, these findings suggest one of the possible mechanisms by which iNOS inhibition prevents liver injury and metabolic derangement in septic rats.
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GRANTS
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This work was supported by a grant from Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS 00/0949) and a fellowship to T. Priego (Formacion Profesorado Universitorio, AP2001-0053).
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ACKNOWLEDGMENTS
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We are indebted to A. Carmona for technical assistance and to C. Bickart for the English correction of the manuscript. We are grateful to the US National Institute of Diabetes, Digestive and Kidney Diseases, National Hormone and Pituitary Program, for the GH reagent and IGF-I antibody.
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FOOTNOTES
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Address for reprint requests and other correspondence: A. López-Calderón, Dpt Fisiología, Fac Medicina, Univ. Complutense, 28040 Madrid, Spain (E-mail: ALC{at}med.ucm.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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