Effect of L-arginine supplement on liver regeneration after partial hepatectomy in rats

The result of extensive hepatectomy has been greatly advanced through operative techniques and perioperative management. That strategy has been maintained by the capacity for liver regeneration. Under normal conditions, the basal hepatocyte proliferation rate is low, and mitosis seldom occurs. However, after an event involving a substantial loss of hepatic tissue such as that found in severe viral hepatitis or a major hepatectomy, cell division leads to intense tissue growth until regeneration occurs, with the energetic and synthesis capacity being essentially maintained. This regenerative process is accompanied by compensatory cellular hyperplasia and hypertrophy, and is controlled and mediated by nutrients and growth factors. If liver regeneration after major hepatectomy could be enhanced artificially, the cases in which hepatectomy for liver cancers can be used would be extended, with improved patient prognoses.

Nitric oxide (NO) is known to be a multifunctional mediator, regulating blood pressure, gene expression, apoptosis, and mitogenesis in essentially every organ and tissue [1‐3]. NO is enzymatically synthesized from L-arginine (L-Arg) by three nitric oxide synthase (NOS) isoforms: neuronal (n)NOS, inducible (i)NOS and endothelial (e)NOS. eNOS is a crucial mediator of vascular tone and blood flow, and plays major roles in liver physiology and pathophysiology [4]. NO has been reported to reduce organ injury and enhance liver regeneration by experimentally modulating hepatic macrohaemodynamics [5].

Recently, there have been studies assessing whether delivery of a specific amino acid can improve patient outcome, as some amino acids are precursors of many important biologic compounds essential for a normal functioning of the human organism [6]. Furthermore, these supplements can be taken orally to provide effective clinical availability. There have been no reports describing whether or not L-Arg enhances liver regeneration after hepatectomy. In the present study, we evaluated the effect of oral administration of L-Arg on liver regeneration after hepatectomy in rats.

Amino acids are the essential building blocks of all mammalian cells. In addition to their role in protein synthesis, amino acids also play an important role as energy fuel, precursors of a variety of metabolites, and as signaling molecules. Recently, there has been interest in assessing whether delivery of specific amino acids can improve patient outcome under various pathological conditions [6]. Gln is the most abundant amino acid in human muscle and plasma. Apart from providing nitrogen for protein synthesis, Gln is a precursor for nucleic acids, nucleotides, and the major antioxidant glutathione [9]. Recent data also indicate that Gln functions as a signaling molecule [10], particularly under catabolic conditions. Yoshida et al.[11] reported beneficial effects of Gln on DNA and on the protein synthesis of remnant liver in the 70% rat hepatectomy model. Since then, similar findings have been reported by a number of investigators [12, 13]. In the present study, we evaluated the effects of L-Gln on liver regeneration, as measured by a restituted liver mass, the PCNA labeling index, and total DNA, and found basically equivalent results. Although Gln was administered intravenously in previous reports, we found oral administration of L-Gln to be similarly beneficial. Because oral supplementation of L-Gln is considered safer than intravenous infusion, this may be a better choice for clinical studies.

NO plays an important role in vasodilatation and in increasing blood flow by signaling smooth muscles to relax. A beneficial effect of NO on liver regeneration through an increase in hepatic arterial flow has been reported [5]. Studies have also shown that NO derived from eNOS regulates sinusoidal endothelial cell proliferation [14] and hepatocyte proliferation [4]. Carnovale et al.[15] showed that the hepatocyte iNOS level was increased after hepatectomy, and that NO derived from iNOS enhanced hepatic DNA synthesis, which had formerly been blocked by inhibiting iNOS expression. Observations of impaired liver regeneration and hepatocyte survival in iNOS knockout mice provide additional proof for the importance of NO-mediated signaling in liver regeneration [16]. It has also been shown that nitric oxide production contributes to hepatocyte proliferation in normal juvenile rats [17]. Although the mechanism of NO contribution to hepatocyte proliferation is not yet fully understood, it is known that NO derived from both eNOS and iNOS enhances liver regeneration.

Arg supplementation has been identified as advantageous in wound healing in experimental animals and humans [18, 19]. The beneficial effects of Arg supplementation on wound healing have been attributed to enhanced synthesis of NO by NOS. NO is synthesized by both parenchymal and nonparenchymal cells of the liver [20]. The aim of the present study was to clarify whether direct administration of L-Arg has any effect on liver regeneration. We found that increases in restituted liver mass enhanced by L-Arg and L-Gln supplementation were associated with increases in the quantity of total hepatic DNA. Both restituted liver mass and DNA quantity were significantly higher in the L-Arg and L-Gln groups than in the control, suggesting that the hepatic regeneration was accompanied by cell proliferation. However, the hepatic regeneration was not accompanied by increases in the total RNA quantity, suggesting that the rate of protein synthesis might be in steady state. L-Ala was used as the negative control to match the energy and amino acid supply to L-Arg or L-Gln [21]. The significant differences found between L-Arg/L-Gln and L-Ala in the present study also reveals the specific functions of L-Arg and L-Gln in improving liver regeneration beyond the functions of simple nutrients. One report has shown different effects of L-Arg and L-Gln on liver regeneration in rats receiving total parenteral nutrition after hepatectomy [22]. To reveal the different mechanisms between L-Arg and L-Gln, another study containing an experimental group given both L-Arg and L-Gln together might be useful. Further study is warranted.

In our study, we found that L-Arg seems to have a similar potential for liver regeneration to that of L-Gln. Both amino acids proved effective even when orally administered, which is likely to facilitate clinical use. In our study, we gave rats oral 10% L-Arg (1 ml/100 g body weight) before hepatectomy and also administered it after the operation by adding it to tap water at a concentration of 1%. Witte et al. [23] reported that although the doses of Arg (20–50 mg/100 g/day) must be considered pharmacological, there is a notable lack of toxic side-effects. In light of their knowledge, our doses were two to five times higher on the operation day and about twice as high afterwards. However, when excessive amino acids are administered to patients with an impaired liver function such as liver cirrhosis, the increased ammonia levels can result in hepatic encephalopathy. Therefore, both the potential risks and benefits of L-Gln or L-Arg must be considered before treatment.

In the present study, we found that both L-Arg and L-Gln enhanced liver regeneration after 70% rat hepatectomy. These results may contribute to the treatment of patients in need of major liver resection.