NOS2 deficiency increases intestinal metabolism both in nonstimulated and endotoxemic mice

Yvonne L. J. Vissers,1 Marcella M. Hallemeesch,1 Peter B. Soeters,1 Wouter H. Lamers,2 and Nicolaas E. P. Deutz1

Departments of 1Surgery and 2Anatomy and Embryology, Maastricht University, NL-6200 MD Maastricht, The Netherlands

Submitted 2 September 2003 ; accepted in final form 1 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal studies have suggested that nitric oxide (NO) synthases (NOS) play a role in the regulation of protein metabolism in endotoxemia. We therefore investigated the role of inducible NOS (NOS2) on intestinal protein and neuronal NOS (NOS1) and endothelial NOS (NOS3) on amino acid metabolism. Three groups of mice were studied: 1) wild-type (WT), 2) NOS2 knockout (NOS2-KO), and 3) NOS2-KO + N{omega}-nitro-L-arginine methyl ester (NOS2-KO + L-NAME), both in nonstimulated and LPS-treated conditions. By infusion of the stable isotopes L-[phenyl-2H5]Phe, L-[phenyl-2H2]Tyr, L-[guanidino-15N2]Arg, and L-[ureido-13C; 2H2]citrulline (Cit), intestinal protein, amino acid, and Arg/NO metabolism were studied on the whole body level and across intestine. In nonstimulated situations, NOS2 deficiency increased whole body protein turnover and intestinal Gln uptake and Cit production. In NOS2-KO + L-NAME, the above-mentioned changes were reversed. After LPS in WT, whole body NO and Cit production increased. In contrast to this, LPS decreased net intestinal Gln uptake, whole body NO, and Cit production in NOS2-KO mice. Treatment of NOS2-KO + L-NAME with LPS was lethal in eight of eleven mice (73%). The surviving mice in this group showed a major drop in intestinal protein breakdown and synthesis to almost zero. Thus both in baseline conditions and during endotoxemia, the absence of NOS2 upregulated NOS1 and/or NOS3, which increased intestinal metabolism. The drop in intestinal protein metabolism in the endotoxemic NOS2-KO + L-NAME group might play a role in mortality in that group.

protein metabolism; stable isotope; knockout; sepsis


DURING SEPSIS, both a reduction (2) and an increase (12) in intestinal protein synthesis have been reported, whereas protein breakdown in the gut is usually not changed. In addition, we observed an increase in intestinal Arg production (3). Others (19) have reported reduced intestinal uptake of Gln during sepsis. Because Gln is the principal fuel for intestinal cells, this can be regarded as an indication of decreased intestinal metabolism. Until now, it has been unclear by what mechanism these changes in intestinal protein and amino acid metabolism are regulated.

In baseline conditions, constitutively expressed isoforms of nitric oxide (NO) synthases (NOS) produce low levels of NO. These isoforms are neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3). After administration of LPS, NOS2 is upregulated in many tissues. Therefore, NO produced by NOS2 is thought to induce the metabolic changes accompanying sepsis and endotoxin treatment. It has thus been suggested that NOS plays a role in the regulation of protein and amino acid metabolism in endotoxemia. We (9) previously demonstrated that nonspecific NOS inhibition with N{omega}-nitro-L-arginine methyl ester (L-NAME) reduced systemic protein and Arg turnover, whereas selective NOS2 inhibition with S-methylisothiourea did not. In conjunction with this, nonspecific NOS inhibition increased renal protein breakdown, whereas selective NOS2 inhibition increased renal Arg production (9). However, the role of different NOS isoforms on protein and amino acid metabolism in the gut is unknown.

Therefore, the purpose of our study was to investigate the role of NOS2 on protein and NOS1 and NOS3 on amino acid metabolism on the whole body level and in the gut, both in nonstimulated conditions and during sepsis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male C57BL6/J NOS2+/+ [wild-type (WT)] and C57BL6/J NOS2-/- [NOS2 knockout (KO)] mice (22–26 g, 2–3 mo old) were originally obtained from Jackson Laboratories and bred at the Department of Anatomy and Embryology (AMC, Amsterdam, The Netherlands). The mice were fed standard lab chow (Hope Farms, Woerden, The Netherlands) and were subjected to standard 12:12-h light-dark cycle periods (7:30 AM to 7:30 PM). Room temperature was maintained at 25°C. Water was provided ad libitum throughout the experiment. Experiments were approved by the Ethical Committee of Animal Research of Maastricht University.

Experimental protocol. All experiments started between 08:00 and 10:00 AM. Figure 1 depicts the six experimental groups included in the study. However, in the group receiving both L-NAME and LPS treatment (grey block) 8 of 11 mice died during the experiment. Therefore, this group was not included in the statistical analyses, tables, and figures, but results of the surviving mice are discussed.



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Fig. 1. Experimental groups. Six experimental groups were studied. However, in the group receiving both N{omega}-nitro-L-arginine methyl ester (L-NAME) and LPS treatment (grey block), 8 of 11 mice died during the experiment. This group was therefore not included in statistical analyses, tables, and figures. WT, wild-type; NOS2-KO, inducible nitric oxice (NO) synthase (NOS)-knockout.

 

To create the condition of blocked NOS1, NOS2, and NOS3, we chose to inhibit NOS1 and NOS3 in NOS2-KO mice by intraperitoneal injection of the nonselective inhibitor L-NAME (Sigma, St. Louis, MO; 100 µg·100 µl saline-1·10 g-1). Intraperitoneal injection of endotoxin (Escherichia coli O55:B5; 100 µg·200 µl saline-1·10 g-1; Sigma) was used as a model for sepsis. Control animals received corresponding volumes of saline. After injection with LPS or saline, food was withheld, but drinking water was provided ad libitum.

Five hours after LPS and L-NAME treatment of the mice, anesthesia and fluid maintenance was performed as described by Hallemeesch et al. (11). A primed-constant infusion of the stable isotopes L-Arg [guanidine-15N2, 99 AP], L-citrulline (Cit) [ureido-13C, 99 AP; 5,5-2H2, 98+ AP 2H1], L-Phe [phenyl-2H5, 99 AP 2H1] and L-Tyr [phenyl-3,5-2H2, 98 AP 2H1] (Mass Trace, Woburn, MA) was given in the jugular vein (Table 1). Plasma flow across the portal-drained viscera was measured by using an indicator-dilution technique with [glycyl-1-14C]-p-aminohippuric acid (11). Blood was collected from the carotid artery and portal vein as previously described (11). Amino acid concentrations and tracer/tracee ratios (TTR) were determined in plasma as described by Van Eijk et al. (20, 21). Plasma urea and ammonia were determined as previously described (6).


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Table 1. Tracer prime and infusion rates

 

Calculations. Portal-drained viscera amino acid fluxes were calculated by multiplying the portal venous-arterial concentration differences with the mean portal-drained viscera plasma flow and are referred to as "intestinal net fluxes." A positive flux indicates net release, and a negative flux reflects net uptake.

Plasma Arg, Cit, Phe, and Tyr whole body rates of appearance (Ra) were calculated from the arterial isotope TTR values of L-Arg [guanidino-15N2], L-Cit [ureido-13C; 5,5-2H2], L-Phe [phenyl-2H5] and L-Tyr [phenyl-3,5-2H2] as recently described (10). NO production was calculated as plasma Arg-to-Cit flux, and de novo Arg production was calculated as plasma Cit-to-Arg flux (10). Whole body protein breakdown, net protein breakdown, and protein synthesis were calculated as described (10).

Tracer disposal and production rates of portal-drained viscera were calculated by multiplying TTR with substrate fluxes as previously described (4) and are referred to as "intestinal kinetics." Protein metabolism of portal-drained viscera was estimated from the [2H5]Phe and [2H2]Tyr tracers (4). NO production and de novo Arg production in portal-drained viscera were calculated by using the [15N2]Arg and [13C; 2H2]Cit tracers (4). The portal-drained viscera will be referred to as "gut" or "intestine" in RESULTS and DISCUSSION.

Statistical analysis. Results are presented as means ± SE. Twoway ANOVA was used to compare differences among treatment groups, by using "group" with three levels (WT, NOS2-KO, and NOS2-KO + L-NAME) and as between-factor "treatment" with two levels (control and LPS). When significant differences were observed, post hoc analysis was done with Fisher's least significant difference test. Significance was defined as P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Baseline NOS2-KO effects. Absence of NOS2 did not have an effect on whole body NO production (Table 2, Fig. 2).


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Table 2. Whole body fluxes

 


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Fig. 2. Whole body NO production. NO production was calculated as plasma [15N2]Arg to [15N]citulline (Cit) conversion. Absence of NOS2 did not affect NO production. LPS increased NO production in WT but decreased NO production in NOS2-KO mice. Statistics were determined with 2-way ANOVA. b, LPS effect in WT, c, interactive effect vs. WT.

 

In baseline conditions, whole body protein breakdown, net protein breakdown, and protein synthesis in NOS2-KO mice were all increased compared with WT (Table 2, Fig. 3), as was the total arterial amino acid concentration (Table 3). De novo Arg production was slightly but not significantly decreased, whereas whole body Ra of Arg was unchanged (Table 2). This resulted in a decreased contribution of de novo Arg production to whole body Ra of Arg (11.6 ± 0.6 vs. 8.7 ± 1.0% in WT vs. in NOS2-KO, P < 0.05).



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Fig. 3. Baseline whole body protein metabolism: nonstimulated situation. Whole body protein breakdown, protein synthesis, and net protein breakdown were calculated from the plasma tracer/tracee ratios (TTRs) of [2H5]Phe, [2H4]Tyr, and [2H2]Tyr. NOS2-KO had higher protein breakdown, protein synthesis, and net protein breakdown. This effect was blunted when NOS2-KO mice were treated with L-NAME. Statistics were determined with 2-way ANOVA. a, Group effect vs. WT, d, group effect vs. NOS2-KO.

 

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Table 3. Arterial concentrations

 

Intestinal plasma flow was not affected by NOS2 deficiency (0.38 ± 0.13 vs. 0.51 ± 0.09 ml·10 g-1·min-1 in WT vs. in NOS2-KO). Also, intestinal protein breakdown, net protein breakdown, and protein synthesis were not changed compared with WT (Table 4). Intestinal production and disposal of Arg were unchanged. Disposal of Cit in the gut increased (Table 4), together with an almost doubled amino acid net uptake by the gut, including Gln and Arg (Table 5). Intestinal Cit production tended to increase (P = 0.07).


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Table 4. Intestinal kinetics

 

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Table 5. Intestinal net fluxes

 

Baseline NOS2-KO + L-NAME effects. When NOS2-KO mice were treated with L-NAME to inhibit NOS1 and NOS3, the increases in whole body protein turnover and intestinal metabolism were reversed (Tables 2, 4, and 5 and Fig. 3).

In the gut, L-NAME treatment did not change plasma flow (0.51 ± 0.09 NOS2-KO vs. 0.32 ± 0.09 ml·10 g-1·min-1 NOS2-KO + L-NAME). It did increase the net production of total amino acids by the gut, both compared with NOS2-KO and WT (Table 5).

LPS effects in WT. In WT, LPS increased whole body net protein breakdown, but whole body protein breakdown and protein synthesis were unchanged (Table 2). Whole body Arg production and de novo Arg did not change either (Table 2). LPS increased whole body NO production in WT (Table 2, Fig. 2).

LPS did not affect intestinal plasma flow (0.38 ± 0.13 vs. 0.25 ± 0.08 ml·10 g-1·min-1 for WT/CON vs. WT/LPS), but it decreased intestinal protein breakdown, net protein breakdown, and protein synthesis in WT (Table 4).

LPS effects in NOS2-KO. In NOS2-KO mice, the responses to LPS on whole body protein turnover were the same as in WT (Table 2). However, NO production in endotoxemic NOS2-KO did not increase as in endotoxemic WT. On the contrary, LPS treatment in NOS2-KO reduced NO production (Table 2, Fig. 2). Whereas LPS did not affect whole body Cit production in WT, it decreased whole body Cit production in NOS2-KO mice (Table 2).

In endotoxemic NOS2-KO mice, intestinal plasma flow decreased to the same extent as in endotoxemic WT (WT group: from 0.38 ± 0.13 in control to 0.25 ± 0.08 ml·10 g-1·min-1 in LPS vs. NOS2-KO group: from 0.51 ± 0.09 in control to 0.28 ± 0.05 ml·10 g-1·min-1 in LPS) LPS decreased intestinal protein breakdown, net protein breakdown, and protein synthesis in NOS2-KO to the same extent as in WT (Table 4). In contrast to WT, NOS2-KO decreased intestinal amino acid uptake, including Gln, on LPS treatment (Table 5).

LPS effects in NOS2-KO + L-NAME. Eight of eleven mice (73%) died before blood sampling could take place. In the remaining three NOS2-KO + L-NAME mice, LPS increased whole body protein breakdown and synthesis to 35.2 ± 3.5 and 26.9 ± 2.7 nmol·10 g-1·min-1, respectively, without changing net protein breakdown. Whole body NO production decreased on LPS treatment (0.2 ± 0.1 nmol·10 g-1·min-1), which was comparable with WT and NOS2-KO. Intestinal plasma flow also decreased to the same extent as in endotoxemic WT and NOS2-KO. However, intestinal protein breakdown and synthesis dropped to almost zero (2.2 ± 1.0 and 1.9 ± 0.9 nmol·10 g-1·min-1, respectively). At the same time, total intestinal amino acid production decreased to zero.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present in vivo study showed that NOS2 deficiency in baseline conditions profoundly increased whole body protein turnover and intestinal Gln uptake and Cit production, without affecting whole body NO production. During endotoxemia, NOS2 deficiency blunted the increase in whole body NO production and Cit production that was observed in endotoxemic WT. Inhibiting all NOS isoforms during endotoxemia was lethal. A drop in intestinal protein breakdown and synthesis probably preceded death in endotoxemic NOS2-KO + L-NAME.

Baseline. Deleting NOS2 in nonstimulated conditions did not affect whole body NO production. This indicates either 1) that NOS2 was not active under baseline conditions or 2) that deleting NOS2 induced an upregulation of NOS1 and/or NOS3, compensating the reduced NO production. This first possibility is not supported by our data, because deleting NOS2 clearly affected protein metabolism under baseline conditions. It also demonstrates that in nonstimulated conditions, NOS2 activity was already present in vivo, confirming previous ex vivo data (13, 15) on baseline NOS2 expression. In contrast, the second possibility is supported by the effect of L-NAME treatment on protein metabolism in the NOS2-KO mice. Remarkably, the increase in whole body protein turnover that was observed in NOS2-KO mice was blunted when both NOS1 and NOS3 were inhibited by L-NAME treatment. Apparently, these changes in protein metabolism were not directly caused by deleting NOS2 but rather were mediated by upregulation of NOS1 and/or NOS3 in the absence of NOS2, although this did not result in altered NO production. The reason for this increase in NOS1 and/or NOS3 activity is unclear, but there appears to be a mechanism between the different isoforms of NOS to regulate total NO production. This has been suggested before in intestinal mucosa of rats treated with endotoxin; increased NOS2 mRNA was accompanied by a downregulation of NOS3 mRNA (5). In a previous in vivo study (unpublished results) in endotoxemic mice, we observed a similar downregulation of NOS3-mediated NO production in conjunction with an increased NO production mediated by NOS2. In rat intestine, it has been shown that NOS1 suppresses gene expression of NOS2 and that NOS1 suppression leads to NOS2 expression (18). Altogether, this suggests interaction between NOS1 and/or NOS3 on one hand and NOS2 on the other, each regulating the expression of the other. In healthy situations, NOS1 and/or NOS3 activity might be relatively high to keep NOS2 activity low, whereas in endotoxemia, NOS1 and/or NOS3 activity might be downregulated to enable higher activity of NOS2.

Besides, it is clear that NOS and protein metabolism affect each other or may have a collective regulatory mechanism. Indications of a relationship between NO and protein synthesis have been previously given (7, 8). In our in vivo study, deleting NOS2 under baseline conditions increased whole body protein turnover and Gln uptake by the gut. The latter suggests an increased intestinal metabolism in NOS2-KO mice, because Gln is the main fuel for enterocytes and Gln uptake has been related to intestinal function. Because the gut converts Gln to Cit, the tendency (P = 0.07) toward increased Cit production by the gut points toward increased intestinal metabolism as well. Changes in arterial concentrations of Gln and Cit corresponded with these changes in Gln uptake and Cit production. Taking into consideration that the absence of NOS2 in these mice upregulated NOS1 and/or NOS3, these effects might also be caused by this upregulation of NOS1 and/or NOS3.

LPS. In WT, LPS increased NO production, concomitantly with increased net protein breakdown and total amino acid concentrations. The latter might serve as a means of increasing substrate for the NO pathway and thereby keeping up NO production in endotoxemia. In contrast, in endotoxemic NOS2-KO, a decrease in NO production was observed. This confirms numerous observations that NOS2 is involved in NO production after LPS. Strikingly, NO production in endotoxemic NOS2-KO mice was even lower than in nonstimulated NOS2-KO mice. Because NO in NOS2-KO mice can only be produced by NOS1 and/or NOS3, it also indicates that the absence of NOS2 upregulated NOS1 and/or NOS3. This occurred together with a decreased intestinal amino acid uptake, including Gln, pointing toward a decreased intestinal metabolism in endotoxemic NOS2-KO mice. However, intestinal protein metabolism after LPS was not affected by deleting NOS2.

As described above, in baseline NOS2-KO mice, net consumption of Gln in the gut was increased and NO production was maintained compared with WT. In contrast, after LPS treatment in NOS2-KO mice, NO production on the whole body level decreased, in parallel with a decreased consumption of Gln in the gut and a decreased whole body Ra of Cit. We could therefore hypothesize that in endotoxemia, Gln disposal in the gut determined the maintenance of NO synthesis on the whole body level because of the availability of Cit. It is known that Gln in the gut can be converted to Cit. In the kidney, Cit can thereafter be converted to Arg, which in turn can be used for NO synthesis. Indeed, Cit-Arg cycling was upregulated in endotoxemic rats (16), supporting regulation of NO production by Gln. On the other hand, it has also been suggested that Gln supplementation inhibits NO production (1, 14). In our study, Gln net consumption in the gut appeared to have a stimulating effect on NO production. There was no clear relationship with arterial plasma concentrations of Gln.

Because of the high mortality in the NOS2-KO + L-NAME + LPS mice, this group was not included in the statistical analysis and was also excluded from figures and tables. However, the few surviving mice might give us a clue as to which mechanisms are involved in the lethal effect of combining NOS inhibition with LPS. That a majority of animals died when L-NAME was administered during endotoxemia has been reported before by our group and others. Studies in endotoxemic rats demonstrated an increased mortality when overall NO synthesis was inhibited with L-NAME (9, 17). This may reflect the indispensability of NO during sepsis. Both Pernet et al. (17) and Hallemeesch et al. (9) have suggested that this might be related to the stimulating effects of NO on protein metabolism. Our data subscribe to this hypothesis, because the three surviving endotoxemic animals in which all NOS isoforms were inhibited showed a decrease in intestinal protein breakdown and synthesis to almost zero. One could speculate that this drop in intestinal protein metabolism plays a role in the mortality in the NOS2-KO + L-NAME group that was treated with LPS.

In conclusion, this is, to the best of our knowledge, the first in vivo study that clearly points out that different NOS isoforms interact to affect whole body protein metabolism and intestinal metabolism in nonstimulated conditions as well as in endotoxemia. It demonstrates that the absence of NOS2 upregulated NOS1 and/or NOS3 and suggests that intestinal Gln disposal is determining for whole body NO production in endotoxemia because of the availability of Cit. Besides, it confirms that NO is essential in endotoxemia. This might be related to its stimulatory effect on intestinal protein breakdown and synthesis in endotoxemia.


    ACKNOWLEDGMENTS
 
The authors thank J. L. J. M. Scheyen and H. M. H. van Eijk, for expert liquid chromatography-mass spectrometry measurements and G. A. M. ten Have for assistance with the animal experiments.

GRANTS

This study was supported by Grants 902-23-098 and 902-23-239 from the Netherlands Organization for Scientific Research.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. E. P. Deutz, Dept. of Surgery, Maastricht Univ., PO Box 616, NL-6200 MD Maastricht, The Netherlands (E-mail: nep.deutz{at}ah.unimaas.nl).

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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