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Endocrinology, doi:10.1210/en.2007-1558
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Endocrinology Vol. 149, No. 6 3148-3157
Copyright © 2008 by The Endocrine Society

Glycemic Control Modulates Arginine and Asymmetrical-Dimethylarginine Levels during Critical Illness by Preserving Dimethylarginine-Dimethylaminohydrolase Activity

Björn Ellger1, Milan C. Richir1, Paul A. M. van Leeuwen, Yves Debaveye, Lies Langouche, Ilse Vanhorebeek, Tom Teerlink and Greet Van den Berghe

Department of Intensive Care Medicine (B.E., Y.D., L.L., I.V., G.V.d.B.), Catholic University of Leuven, 3000 Leuven, Belgium; Department of Anesthesiology and Intensive Care Medicine (B.E.), University Hospital, 48149 Münster, Germany; and Departments of Surgery (M.C.R., P.A.M.v.L.) and Clinical Chemistry (T.T.), Vrije Universiteit Medical Center, 1007 MB Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. Björn Ellger, Department of Anesthesiology and Intensive Care Medicine, University of Münster, 48149 Münster, Germany. E-mail: ellger{at}anit.uni-muenster.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the context of the hypercatabolic response to stress, critically ill patients reveal hyperglycemia and elevated levels of asymmetrical-dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthases. Both hyperglycemia and elevated ADMA levels predict increased morbidity and mortality. Tight glycemic control by intensive insulin therapy lowers circulating ADMA levels, and improves morbidity and mortality. Methylarginines are released from proteins during catabolism. ADMA is predominantly cleared by the enzyme dimethylarginine-dimethylaminohydrolase (DDAH) in different tissues, whereas its symmetrical isoform (SDMA) is cleared via the kidneys. Therefore, glycemic control or glycemia-independent actions of insulin on protein breakdown and/or on DDAH activity resulting in augmented ADMA levels may explain part of the clinical benefit of intensive insulin therapy. Therefore, we investigated in our animal model of prolonged critical illness the relative impact of maintaining normoglycemia and of glycemia-independent action of insulin over 7 d in a four-arm design on plasma and tissue levels of ADMA and SDMA, on proteolysis as revealed by surrogate parameters as changes of body weight, plasma urea to creatinine ratio, and plasma levels of SDMA, and on tissue DDAH activity. We found that ADMA levels remained normal in the two normoglycemic groups and increased in hyperglycemic groups. SDMA levels in the investigated tissues remained largely unaffected. The urea to creatinine ratio indicated reduced proteolysis in all but normoglycemic/normal insulin animals. DDAH activity deteriorated in hyperglycemic compared with normoglycemic groups. Insulin did not affect this finding independent of glycemic control action. Conclusively, maintenance of normoglycemia and not glycemia-independent actions of insulin maintained physiological ADMA plasma and tissue levels by preserving physiological DDAH activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AS AN INTEGRAL part of the body‘s response to stress, critically ill patients uniformly reveal complex endocrine and metabolic changes (1). Hypercatabolism occurs, with protein breakdown from skeletal muscle and visceral structures. Concomitantly hyperglycemia is brought about by insulin resistance, increased gluconeogenesis, and relative insulin deficiency, the so-called "diabetes of injury" (2). The notion of hyperglycemia being a beneficial adaptation to stress, to provide substrates for vital organ function from endogenous stores, was recently challenged because it is associated with a number of life-threatening complications and death (3). Moreover, strictly maintaining normoglycemia by intensive insulin therapy (IIT) improved morbidity and mortality of critically ill patients (4, 5, 6). Among other mechanisms (7), the modulation of nitric oxide (NO) metabolism emerges as a potentially important mechanism contributing to the clinical benefits (8, 9, 10).

NO is released by NO synthases (NOSs), a family of enzymes converting the amino acid arginine as the sole substrate into NO and citrulline (11). NO modulates inflammation, coagulation, and vasomotor tone, and, thus, microcirculatory blood flow and trafficking of nutrients (12). During critical illness, it appears crucial to preserve physiological NO metabolism because both excessive and impaired NO releases are associated with impaired organ function and increased mortality. Indeed, NOS activity can be up-regulated in critical illness by a number of factors such as endotoxin or cytokines (13, 14) with ensuing cardiovascular collapse (14, 15). Of special interest, hyperglycemia also can cause this overwhelming NO production (16, 17). However, reducing NO production via pharmacologically inhibiting NOS was deleterious because it impaired microcirculation, aggravated organ dysfunction, and led to increased mortality (18). In addition, elevated levels of endogenous NOS inhibitors, in particular asymmetrical-dimethylarginine (ADMA), are found in critically ill patients, and emerge as risk factors for organ failure and death (19, 20). ADMA impairs endothelial function and NO-dependent vasorelaxation by both diminishing NOS activity (21, 22) and by competing with arginine for cellular transport, leading to substrate depletion (23). It is synthesized when arginine residues in proteins are methylated by the action of protein arginine methyltransferases. During proteolysis, ADMA and the symmetrical isoform (SDMA) are released from the protein pool. Therefore, because methylarginines (MAs) are virtually not incorporated into de novo synthesized proteins, the levels in tissue might reflect the overall metabolic activity and the protein turnover rate of cells (24, 25). ADMA is eliminated in the cytosol via degradation by the enzyme dimethylarginine-dimethylaminohydrolase (DDAH). When intracellular ADMA levels surmount the degradation capacity, it is externalized via bidirectional working cationic amino acid transporters (26). Circulating ADMA is predominantly cleared by DDAH in the liver and kidney (26) or, to a minor extent, excreted in the urine (27), whereas SDMA is predominantly cleared by renal excretion (28). Apart from the impact on hemodilution, or concentration, elevated MA levels in plasma and tissues may thus result from both excessive protein breakdown and/or reduced clearance by diminished tissue DDAH activity (ADMA) (29, 30) and impaired renal function (SDMA) (28). However, impaired DDAH activity might occur in the context of kidney or liver failure (20).

Preventing hyperglycemia with IIT concomitantly lowers the plasma concentrations of ADMA in critically ill patients (9) and affects regional NO metabolism (8, 10, 31). This effect on regional NO metabolism emerges as a potential mechanism explaining part of the clinical benefits of IIT (8). Until now, it remains unclear whether an effect on regional NO metabolism is brought about by modulating the substrate availability for NOS, therefore, plasma and tissue levels of arginine and MA, and whether this effect occurs as a result of modulating protein kinetics or MA clearance. Moreover, because insulin has various actions besides its effect on blood glucose, it is of special interest whether the observed effects are directly brought about by insulin or result from the glycemic control that is obtained concomitantly.

Therefore, in our animal model of prolonged critical illness, we assessed the relative impact of glycemic control and glycemia-independent actions of insulin on levels of arginine and MA in plasma and myocardium, skeletal muscle, kidney, and liver biopsies. In addition, we evaluated surrogate markers of protein catabolism, measured DDAH activity in the biopsies, and assessed parameters of liver and kidney function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The protocol was approved by the University of Leuven ethical review board for animal research (protocol Nr. P 04058). Animals were treated according to the "Principals of Laboratory Animal Care" formulated by the U.S. National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the U.S. National Institute of Health.

In our animal model (32), metabolic and endocrine changes during a 7-d study period revealed the typical biphasic course of acute and prolonged critical illness of the human counterpart (1). Moreover, the model provided the opportunity to manipulate plasma insulin levels and blood glucose independently as described in detail previously (10). Briefly (Fig. 1Go), in male, adult New Zealand white rabbits, endogenous insulin deficiency was induced by alloxan monohydrate (150 mg/kg, Alloxan; Sigma-Aldrich, Bornem, Belgium), and critical illness was brought about by a third-degree burn injury of 20% body surface area. Via an indwelling central venous line, continuous parenteral nutrition (Clinomel N7 containing 3.22 g/liter arginine, Baxter; Clinitec, Maurepas Cedex, France) was administered to ensure basal glucose, amino acid, and fat intake. Caloric intake as well as amino acid and fat intake (each 1–1.5 g/kg·d, arginine intake 0.1 g/kg·d) met the physiological requirements of adult rabbits (33). In addition, we administered insulin (Actrapid; Novo Nordisk, Begsvaerd, Denmark) by a continuous fixed dose iv infusion to receive low and high physiological plasma insulin levels, respectively. On each insulin level, we manipulated blood glucose by adjusting the speed of a supplementary iv glucose infusion (glucose 50%; Baxter, Lessines, Belgium) to receive either normoglycemia or hyperglycemia, respectively. Thus, four study groups resulted (Fig. 1Go): group 1, normal insulin levels and normoglycemia (NI/NG) (n = 8); group 2, high insulin levels and normoglycemia (HI/NG) (n = 8); group 3, normal insulin levels and hyperglycemia (NI/HG) (n = 9); and group 4, high insulin levels and hyperglycemia (HI/HG) (n = 8). Samples from animals without alloxan injection and burn injury served as "healthy controls" (n = 8).


Figure 1
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  		FIG. 1. Flow chart of the experiment. Insulin was continuously administered at a fixed dose from d 0–7. The infusion rate of glucose 50% was adjusted to regulate glycemia from d 0–7.

 
Blood glucose (glucose-oxidase method), electrolytes, and hemoglobin were quantified in arterial whole blood sampled from an indwelling line using a blood gas analyzer (ABL analyzer; Radiometer, Copenhagen, Denmark) three to seven times daily to allow tight adjustment of glycemia. Supplementary, before the intervention (baseline), and on d 3, 5, and 7 after burn injury, respectively, 4 ml blood was collected, and plasma was stored at –80 C until further analysis.

On d 7, animals were killed, and tissue samples of myocardium, skeletal muscle (quadriceps femoris), kidney, and liver were taken, immediately snap frozen in liquid nitrogen, and stored at – 80 C until analysis.

Assays
All analyses were conducted blinded to randomization within a single assay run. All chemicals were derived from Sigma-Aldrich unless indicated otherwise.

Plasma insulin was measured by RIA using a guinea pig-derived antibody as described elsewhere, within assay variation less than 10% (34) (kindly provided by R. Bouillon, Katholieke Universiteit of Leuven, Leuven, Belgium).

Protein content in the four investigated tissues was measured using Coomassie Protein Assay Reagent (Pierce Biotechnology, Erembodegem, Belgium) according to the users manual and a standard curve of BSA as described previously (35).

Plasma levels of arginine, ADMA, and SDMA
The concentrations of arginine, ADMA, and SDMA were determined simultaneously by HPLC as described previously (36, 37). In brief, solid-phase extraction on polymeric cation-exchange columns was performed after addition of monomethylarginine as the internal standard. After derivatization with ortho-phthaldialdehyde reagent containing 3-mercaptopropionic acid, analytes were separated by isocratic reversed-phase HPLC with fluorescence detection. Intraassay and interassay coefficients of variation were 1.2 and 3.0%, respectively. The arginine over ADMA ratio was calculated.

Tissue levels of arginine, ADMA, and SDMA
For the determination of arginine, ADMA, and SDMA in myocardium, skeletal muscle, kidney, and liver, tissue was homogenized with an OMNI-2000 Homogenizer (OMNI Intl., Waterbury, CT) in 4 vol sodium phosphate buffer [100 mM (pH 6.5)]. Two hundred fifty microliters of the tissue homogenate were mixed with 250 µl 1.2 M perchloric acid. After centrifugation (10 min at 2000x g at 4 C), 200 µl supernatant was mixed with 400 µl 0.5 M disodium hydrogen phosphate, 100 µl internal standard solution, and 400 µl water. This mixture was subjected to solid-phase extraction, derivatization, and chromatography, as described previously (36, 37).

Tissue DDAH activity
Tissue DDAH activity was determined by measurement of citrulline formation during incubation of tissue homogenates with excess of ADMA. Tissue samples were homogenized with four volumes of sodium phosphate buffer [100 mM (pH 6.5)] at 4 C with an OMNI-2000 Homogenizer and subjected to a dual centrifugation procedure (10 min at 2000x g, followed by 30 min at 10,000x g, both at 4 C). One hundred sixty microliters of the supernatant were mixed with 240 µl of a 4 mM solution of ADMA in sodium phosphate buffer [100 M (pH 6.5)] containing protease inhibitors (Complete protease inhibitor cocktail; Roche Applied Science, Mannheim, Germany). Before and after incubation for 2 h at 37 C, the reaction was stopped, and proteins were precipitated by transferring 200 µl of the incubation mixture to vials containing 8 mg sulfosalicylic acid. After vortex mixing, the vials were centrifuged (10 min at 3000x g at 4 C), and citrulline in the clear supernatant was measured by HPLC as described previously (38). The increase of citrulline concentration during incubation was used to calculate DDAH activity (expressed as nmol/min·g wet weight of tissue). Control incubations were performed in the absence of either tissue homogenate or ADMA, and in the presence of an excess citrulline, as previously described (39).

Organ function parameters
By commercial kits, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in plasma according to the guidelines of the International Federation of Clinical Chemistry. Creatinine in plasma was measured by the Jaffé method, and urea in plasma was measured by a kinetic UV assay with equipment-specific reagents. Modular Roche and specific reagents by Roche (Roche/Hitachi, Bern, Switzerland; A.M. L bvba, Antwerp, Belgium) were used according to the users manual. Because protein intake was equal in all groups, the ratio of plasma urea over plasma creatinine (U/C ratio) was calculated, and the changes from baseline ({delta} U/C ratio) were determined as a surrogate marker of catabolism (40). An increase of this ratio is suggestive for increased and a decline for reduced protein breakdown.

Statistical analysis
Results are expressed as mean ± SEM. Data are presented as box plots; the central line indicates the median, the box the interquartile range, and the whiskers the 10th and 90th percentiles. Data were analyzed by multifactorial ANOVA and Fisher’s protected least significant difference post hoc testing. For nonnormally distributed data, Kruskal-Wallis and Mann-Whitney U tests were used. Correlation analysis was performed by linear or logarithmic regression (Pearson coefficient). A two-tailed P value less than 0.05 was considered significant. The statistical analyses were done with StatView 5.0.1 (SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We described previously that in our model blood glucose and insulin levels were manipulated independently from each other (10). Glucose intake inevitably differed among groups to achieve this (Table 1Go); protein and lipid intake did not differ significantly. Protein content per gram of tissue did not differ among groups and from healthy controls in any of the investigated tissues. Plasma levels of electrolytes, hemoglobin, and surrogate parameters of cardiac preload (as given in Ref. 10) did not differ significantly among groups, largely excluding intravascular fluid volume contraction or expansion.


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  		TABLE 1. Glucose intake, plasma insulin levels, blood glucose, changes of body weight (BW), and relative protein content in the investigated tissues

 
In plasma, the arginine levels were lower in the hyperglycemic groups compared with normoglycemic groups and healthy controls. Grouping both hyperglycemic and normoglycemic groups, ADMA plasma levels were elevated in hyperglycemic animals compared with normoglycemic animals (Fig. 2Go). Consequently, the arginine over ADMA ratio in plasma was significantly lower in hyperglycemic groups compared with normoglycemic animals. The pattern was largely comparable on d 3 and 7. Insulin evoked no glycemia-independent actions.


Figure 2
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  		FIG. 2. Levels of arginine (upper panel), ADMA (second panel), arginine over ADMA ratio (third panel), and SDMA (lower panel) in plasma on d 3 (left column) and d 7 (right column), gray area reflects range in healthy controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001. {dagger}, P < 0.05; {dagger}{dagger}, P < 0.001 vs. healthy controls. NS, Not significant.

 
The pattern of the tissue levels of ADMA was similar in all investigated tissues, with higher ADMA levels in both hyperglycemic groups as compared with normoglycemic groups (Fig. 3Go). ADMA in the liver was elevated in sick animals compared with healthy controls (P = 0.007), most pronounced in HI/HG animals. The arginine over ADMA ratio was reduced in both hyperglycemic groups as compared with normoglycemic groups and healthy controls in all investigated tissues. Insulin evoked no glycemia-independent actions. SDMA remained unaffected by the intervention in the investigated tissues.


Figure 3
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  		FIG. 3. Tissue levels of the arginine over ADMA ratio (left column), arginine (left-middle column), ADMA (right-middle column), and SDMA (right column) in myocardium (upper row), skeletal muscle (second row), kidney (third row), and liver (lower row); gray area reflects range in healthy controls. *, P < 0.05; ***, P < 0.001; {dagger}, P < 0.01 vs. healthy controls. {ddagger}, P < 0.05 vs. all other groups and healthy controls. NS, Not significant.

 
The arginine over ADMA ratio in all tissues correlated with the ratio in plasma (P < 0.0003; all R2 > 0.34). Plasma SDMA correlated with SDMA in myocardium, kidney, and liver (P < 0.0001; all R2 > 0.4), but not with SDMA in skeletal muscle (P = 0.228; R2 = 0.018) (data not shown).

The U/C ratio increased from baseline to d 7 (P = 0.02) in NI/NG animals, whereas in HI/NG, NI/HG, and HI/HG animals, this ratio decreased (P < 0.001) (Fig. 4Go).


Figure 4
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  		FIG. 4. Changes of the urea over creatinine ratio over time (left panel) and urea over creatinine ratio among groups on d 7 (right panel). Gray area reflects range in healthy controls. Filled circle indicates NI/NG. Open circle represents HI/NG. Filled triangle reflects NI/HG. Open triangle indicates HI/HG. *, P < 0.05; **, P < 0.01; ***, P < 0.001; {dagger}, P < 0.01 vs. healthy controls. {dagger}{dagger}, P < 0.001 vs. healthy controls.

 
The DDAH activity in myocardium and skeletal muscle did not differ among groups and was similar to healthy controls (Fig. 5Go). In the kidney and liver, both hyperglycemic groups revealed lower DDAH activity than both normoglycemic groups. In the liver both normoglycemic groups revealed higher DDAH activity than healthy controls (P < 0.05). Insulin evoked no glycemia-independent actions.


Figure 5
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  		FIG. 5. DDAH activity in myocardium (left-upper panel), skeletal muscle (right-upper panel), kidney (left-lower panel), and liver (right-lower panel). Gray area reflects range in healthy controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001; {dagger}, P < 0.05 vs. healthy controls. NS, Not significant.

 
The plasma levels of ADMA negatively correlated with DDAH activity in the kidney and liver (Fig. 6Go). Likewise, the arginine over ADMA ratio in plasma correlated with the DDAH activity in the kidney and liver. In myocardium and muscle, no correlation between DDAH activity and plasma levels of arginine and ADMA was found (data not shown).


Figure 6
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  		FIG. 6. Correlation of the DDAH activity in the kidney (left column) and liver (right column) with plasma levels of ADMA (upper row) and arginine over ADMA ratio (lower row).

 
We previously reported altered liver and kidney function in the hyperglycemic groups in our model (10). DDAH activity in the liver correlated with the plasma markers of liver function, and DDAH activity in the kidney and SDMA in plasma correlated with plasma creatinine (Fig. 7Go).


Figure 7
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  		FIG. 7. Correlation of the DDAH activity with parameters of organ function. DDAH activity in the liver vs. plasma AST (upper panel) and ALT (upper-middle panel), and DDAH activity in the kidney vs. plasma creatinine (lower-middle panel). Correlation of plasma SDMA vs. plasma creatinine (lower panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this model of prolonged critical illness, preventing hyperglycemia and not glycemia-independent actions of insulin maintained physiological arginine and ADMA levels in plasma and tissue biopsies of myocardium, skeletal muscle, kidney, and liver. This was most likely explained by preserved breakdown of ADMA via maintaining physiological DDAH activity rather than by an effect on MA release via protein catabolism.

In our experiment, plasma ADMA levels in both hyperglycemic groups were elevated compared with both normoglycemic groups, regardless of the insulin levels. Concomitantly to increased ADMA, arginine levels in plasma were decreased in both hyperglycemic groups compared with both normoglycemic groups. This resulted in a pronounced decrease in the arginine over ADMA ratio in hyperglycemic groups. The pattern of the arginine and ADMA levels in plasma largely reflected the pattern in the investigated tissue biopsies. Also in the tissues, consistently, although not always reaching statistical significance, ADMA levels were elevated in hyperglycemic compared with normoglycemic groups, and the arginine over ADMA ratio was diminished in both hyperglycemic groups. Because ADMA is not reincorporated into proteins (24, 25), our findings are compatible with the notion that plasma levels of ADMA mainly reflect cellular spillover of the amount of ADMA that surmounts intracellular clearance capacity (25, 26).

In human diabetes mellitus, elevated ADMA levels occur in the context of insulin resistance (41) and hyperglycemia (42), rendering patients at risk for cardiovascular complications (43). Equivalently, in human critical illness, increased ADMA levels occurred in plasma already on d 2 after the onset of the disease (9), and emerged as independent predictors of organ dysfunction and mortality (19). Strictly maintaining normoglycemia by IIT was shown to prevent elevated ADMA levels. In view of our current results and the findings in a rat model of type 2 diabetes mellitus (44), this is most likely brought about by strict glycemic control and not by glycemia-independent actions of insulin. However, not only preventing increased ADMA levels but also the preservation of a physiological balance between arginine and ADMA by maintaining normoglycemia appears important because the arginine over ADMA ratio is the major determinant of NOS activity in tissues (31, 45). Notably, neither maintaining normoglycemia nor insulin increased this ratio above normal values. Elevated arginine over ADMA ratio could be considered potentially deleterious because it might lead to overwhelming NO production. Therefore, maintaining normoglycemia during critical illness not only reduces ADMA levels but also largely preserves the physiological regulation of substrate availability for NOS (31). The latter property appears crucial for the survival of critically ill patients because both overwhelming NO production with ensuing cardiovascular collapse, and local NO deficiency with altered endothelial function and disturbed microcirculation, are detrimental (18).

Elevated ADMA levels during critical illness may either result from increased generation or impaired breakdown. In fact, MAs like ADMA and SDMA are extensively released from the intracellular protein pool during high protein turnover, so that high levels of ADMA (25) and SDMA (24) in plasma and tissues might be expected during hypercatabolism. Assuming that critical illness evokes hypercatabolism, protein breakdown from organs and tissues with a known high protein turnover rate, such as the liver and muscle, is expected (46). In the liver, but not in the other tissues, we found that the ADMA levels in all sick groups were moderately increased as compared with healthy controls, with hyperglycemic animals consistently revealing the highest ADMA levels. Meanwhile, SDMA levels did not increase in any group in any of the tissues, making it unlikely that elevated ADMA and differences among groups result from an effect on ADMA release from protein breakdown (24). Moreover, the U/C ratio, roughly reflecting protein breakdown from muscle (40), was reduced with the interventions in all but the NI/NG group, indicating reduced catabolism. In the NI/NG animals, the U/C ratio increased with time, suggesting increased catabolism. However, these animals revealed the lowest circulating and muscle ADMA levels. Although insulin is considered an anabolic hormone reducing protein breakdown and, thereby, hypothetically, causing less ADMA release (20), we could not detect any glycemia-independent actions of insulin on protein breakdown in muscle and on ADMA levels. In addition, protein content in tissues and body weight did not change in any of the groups during the experiment, largely excluding profound protein depletion and starvation (33). The remarkable variance in body weight changes can be interpreted as a result from variable bladder and gastric distension, an inconsistent amount of edema in the burn wound, and a variable degree of ascites. However, it remains unclear why the U/C ratio indicates catabolism in HI/HG animals only, although the other parameters did not reveal any evidence for increased catabolism in this group. Furthermore, in the other groups, the U/C ratio decreased, indicating anabolism, although the other surrogate parameters did not reveal any evidence for this notion. A possible explanation might be seen in the bias of the measures by fluid load or an impact of organ dysfunction. However, also in the clinical study of IIT, there was no clear evidence that glycemic control and/or insulin, in the doses that were used to maintain normoglycemia, affected catabolism (47). Conclusively, because our experimental setting was not designed accordingly, it does not allow to define circumstantially and precisely minor effects of the interventions on catabolism or anabolism. Although, our methodology allows the statement that the impact of glycemic control on arginine and ADMA levels is not likely explained by effects on MA generation via protein breakdown.

In contrast, it appears that blood glucose control affected ADMA levels via reduced ADMA clearance. ADMA is directly cleared by the enzyme DDAH in those tissues in which it is generated. The ADMA quantity that surmounts tissue clearance capacity is externalized from the cells and appears in the circulation. From the circulation, ADMA is cleared by DDAH in the kidney and liver, and to a minor extent by direct renal excretion (25). In a physiological control loop, DDAH activity is up-regulated when ADMA levels increase (26). We found in our experiment that in myocardium and muscle tissue of critically ill animals, DDAH activity was largely preserved at the level of healthy controls. In the presence of elevated ADMA levels, elevated DDAH activity would be required in the hyperglycemic groups; hence, the physiological control loop may be impaired by hyperglycemia in these tissues. More clearly this is the case in the kidney and liver because DDAH activity in these tissues was frankly low in both hyperglycemic groups as compared with healthy controls and normoglycemic groups, in the presence of elevated ADMA. However, in both normoglycemic groups, the control loop of ADMA and DDAH activity appeared intact because in the liver biopsies of both normoglycemic groups, moderately elevated ADMA levels and a concomitantly increased DDAH activity were present. DDAH activity in the kidney and liver appears to be the major determinant of ADMA clearance from the plasma and for the preservation of a physiological arginine over ADMA balance (25, 26, 29), as indicated by the correlation of DDAH activity in these tissues with plasma arginine and ADMA.

Hyperglycemia appeared to down-regulate DDAH activity in our model of critical illness. This is in line with what has been observed in cultured endothelial cells (48) and models of type 2 diabetes mellitus (44). Also in these studies, impaired DDAH activity and not protein turnover appeared to increase ADMA levels. A potential reason for impaired DDAH activity with hyperglycemia could be glycation of the DDAH protein (49) or increased oxidative stress (7, 44) because DDAH is an oxidant-sensitive enzyme (44). Insulin might theoretically ameliorate ADMA clearance by affecting amino acid transporter systems whereby increasing ADMA uptake into organs that eliminate ADMA (50). However, in our model we could not detect any glycemia-independent effect of insulin on DDAH activity or ADMA levels in tissue or plasma.

Reduced DDAH activity has been reported in a number of medical conditions, especially when kidney and liver function are impaired (20). Also in our experiment, DDAH activity in the kidney and liver correlated negatively with the organ function parameters. Therefore, reduced DDAH activity and hereby increased ADMA levels may result form organ failure. Unfortunately, we could not distinguish whether impaired DDAH activity and increased ADMA levels were the cause or consequence of organ dysfunction. However, SDMA is mainly cleared via renal excretion and may serve as a marker for renal dysfunction in humans (28). Also in our model, plasma SDMA correlated well with plasma creatinine. Because SDMA levels in the tissues are not elevated, largely excluding an effect of our interventions on SDMA release, slightly elevated SDMA in plasma of HI/HG animals on d 7 should most probably be seen in the context of kidney dysfunction.

The limitations of our study need to be addressed. First, extrapolating from our animal model to the complex entity of human critical illness should be done with great caution. Second, protein content and the U/C ratio can be considered as valid global markers of catabolism because protein intake did not differ among groups, and kidney function remained stable the last days of the experiment (40). However, they are rough, not very specific and accurate markers of protein catabolism that are prone to several confounders. Most important, they can be influenced by organ dysfunction like, for example, renal failure. Therefore, a subtle effect of maintaining normoglycemia or glycemia-independent actions of insulin on ADMA release from protein breakdown cannot completely be excluded. The accurate assessment of protein turnover would require the use of stable isotopes. Third, because glucose intake inevitably differed among groups to obtain the independent manipulation of the blood glucose and insulin levels, we cannot exclude a contribution to our findings from the different caloric loads. However, because caloric intake in all groups remained in the range known as "normal" and physiological for rabbits (33), we can at least exclude effects of starvation or overfeeding. Finally, although not reaching statistical significance, animals of the HI/HG group seem to reveal worse results in almost all investigated parameters compared with the other groups. However, due to ethical reasons, the number of animals per group had to be limited and might not suffice to exclude completely a minor glycemia-independent effect of insulin during hyperglycemia.

In conclusion, in our in vivo model of prolonged critical illness, maintenance of normoglycemia and not glycemia-independent actions of insulin maintained physiological ADMA levels and arginine over ADMA ratios in plasma and tissues by preserving physiological DDAH activity.


   Acknowledgments
 
We thank Sigrid de Jong, Erik Van Herck, Sarah Vander Perre, Sarah Derde, Eric-Jan Ververs, Ivo Jans, Kristof Reyniers, Magda Mathys, and Pieter Wouters, as well as B. Braun, Melsungen, Germany, Tyco Healthcare, Mechelen, Belgium, Fresenius Kabi, Wilrijk, Belgium, and Baxter, Lessines, Belgium, for technical support.


   Footnotes
 
This work was supported by: Innovative Medizinische Forschung (EL 610304), Germany, and B. Braun Stiftung, Germany (to B.E.); postdoctoral fellowship of the Fund for Scientific Research (Fonds Wetenschappelijk Onderzoek) Flanders, Belgium (to L.L. and I.V.); doctoral fellowship of the Fonds Wetenschappelijk Onderzoek, Belgium (to Y.D.); and Fonds Wetenschappelijk Onderzoek (G.0144.00, G.0278.03, G.3C05.95N), an unrestricted Novo Nordisk grant, and the Research Council of the University of Leuven, Belgium (OT 03/56, GOA 2007/14) (to G.V.d.B.).

1 B.E. and M.C.R. contributed equally to this work. Back

Disclosure Statement: G.V.d.B. is the holder of an unrestricted grant from Novo Nordisk. B.E., M.C.R., P.A.M.v.L., Y.D., L.L., I.V., and T.T. have nothing to declare.

First Published Online February 21, 2008

Abbreviations: ADMA, Asymmetrical-dimethylarginine; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DDAH, dimethylarginine-dimethylaminohydrolase; HI/HG, high insulin levels and hyperglycemia; HI/NG, high insulin levels and normoglycemia; IIT, intensive insulin therapy; MA, methylarginine; NI/HG, normal insulin levels and hyperglycemia; NI/NG, normal insulin levels and normoglycemia; NO, nitric oxide; NOS, nitric oxide synthase; SDMA, symmetrical-dimethylarginine; U/C, urea to creatinine.

Received November 13, 2007.

Accepted for publication February 11, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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