Glutamine and ornithine alpha-ketoglutarate supplementation on malate dehydrogenases expression in hepatectomized rats1

PURPOSE: To evaluate the relative gene expression (RGE) of cytosolic (MDH1) and mitochondrial (MDH2) malate dehydrogenases enzymes in partially hepatectomized rats after glutamine (GLN) or ornithine alpha-ketoglutarate (OKG) suplementation. METHODS: One-hundred and eight male Wistar rats were randomly distributed into six groups (n=18): CCaL, GLNL and OKGL and fed calcium caseinate (CCa), GLN and OKG, 0.5g/Kg by gavage, 30 minutes before laparotomy. CCaH, GLNH and OKGH groups were likewise fed 30 minutes before 70% partial hepatectomy. Blood and liver samples were collected three, seven and 14 days after laparotomy/hepatectomy for quantification of MDH1/MDH2 enzymes using the real-time polymerase chain reaction (PCR) methodology. Relative enzymes expression was calculated by the 2T method using the threshold cycle (CT) value for normalization. RESULTS: MDH1/MDH2 RGE was not different in hepatectomized rats treated with OKG compared to rats treated with CCa. However, MDH1/MDH2 RGE was greater on days 3 (321:1/26.48:1) and 7 (2.12:1/2.48:1) while MDH2 RGE was greater on day 14 (7.79:1) in hepatectomized rats treated with GLN compared to control animals. CONCLUSION: Glutamine has beneficial effects in liver regeneration in rats by promoting an up-regulation of the MDH1 and MDH2 relative gene expression.


Introduction
Potential mechanisms and pathways of liver regeneration have been extensively investigated in recent reviews 1,2 . Holecek 3 studied the effects of carbohydrates, lipids, and amino acids on partial hepatectomy and concluded that administration of a standard amino acid mixture without energy substrate has an inhibitory effect on liver regeneration. Birkhahn et al. 4 studies demonstrated that the inhibitory effect of glucose on liver regeneration can be diminished by the simultaneous administration of aminoacids and other substances.
Experimental studies have shown that ornithine alpha-ketoglutarate (OKG) supplementation of enteral feeding significantly shortens wound healing time in severe burn patients 5 .
It has been reported that OKG stimulates the production on insulin and growth hormone promoting intracellular aminoacid transport and protein synthesis 6 .
Glutamine (GLN) is the most abundant amino acid in the circulation and the main source of nitrogen for various metabolic processes including purine and pyrimidine synthesis necessary for DNA replication and cellular proliferation 7,8 . Yoshida et al. 8 (Table 1).

Statistical analyses
To ensure the appropriateness of parametric testing, all data were examined for normality, using Kolmorogov-Smirnov test (with Dalal-Wilkins Lilliefor P Value). Data (expressed as mean ± standard deviation) were examined for significance using ANOVA followed by Tukey multiple comparisons test. GraphPad Prism 4.0 (GraphPad Software, San Diego, California, USA, www.graphpad.com) was used for statistical analysis and graphics design. Values of p<0.05 were accepted as statistically significant.

MDH1 and MDH2 relative gene expression (RGE) was
calculated by 2-ΔΔCt method using the Ct value for normalization 15 .
No differences were found in MDH1 RGE in rats submitted to laparotomy only when comparing the animals treated with CCa, GLN or OKG. Also, no significant differences were found in MDH2 expression in rats submitted to 70% hepatectomy and treated with OKG. However, these differences were greater in animals treated with GLN after three, seven and 14 days posthepatectomy compared with rats treated with CCa. Calculated values for days three, seven and 14 are presented in Tables 2 and 3, Figure 1.

Analysis of data from real-time quantitative PCR
experiments may be carried out using different methods. The two most commonly used methods are absolute quantification and relative quantification. The first method (absolute quantification) determines the input copy number, usually by relating the PCR signal to a standard curve. The second method (relative quantification) relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control 15  According to Livak and Schimittgen 15 , the endpoint of real-time PCR analysis is the threshold cycle of C t . Considering that C t is determined from a log-linear plot of the PCR signal versus the cycle number, the value obtained is an exponential and not a linear term. Therefore, any statistical presentation using the raw CT values should be avoided. Based on the assumptions, the 2 -C t calculation was used here for results analyses.
Liver regeneration mechanism has been extensively studied. Hepatic cells are normally quiescent. However, after partial hepatectomy approximately 95% of hepatic cells rapidly re-enter the cell cycle. In the rat liver, the rate of DNA synthesis in hepatocytes begins to increase after about 12 hours and peaks around 24 hours. Non-parenchymal tissues have a later start for DNA synthesis.       18 . Most of the increase in liver mass has occurred by three days after partial hepatectomy and mass restoration is complete in 5-7 days 19 .
In order to maintain the metabolic homeostasis during regeneration the liver undergoes an adaptive response to regulate the differentiated functions of the hepatocyte. This is accomplished by the interplay between different sets of transcription factors, induced by the regenerative response 20 . Liver regeneration requires large amounts of energy to take place. One of the mechanisms involved in supplying energy to the hepatocyte is the autophagy seen in acute liver injury. However, hyperactivation of autophagy induces cell death, and the necrosis rate is actually a predictor of liver failure 21 .
The malate/aspartate shuttle is the major pathway by which cytosolic reducing equivalents from NADH can enter the mitochondria and be oxidized 22 . MDH1 and MDH2 enzymes play important roles in the Krebs cycle for energy production through aerobic respiration 9 . The malate-aspartate shuttle translocates electrons produced during glycolysis across the semipermeable inner membrane of the mitochondrion to the electron transport chain via NADH to generate ATP. The shuttle system is required since the mitochondrial inner membrane is impermeable to NADH.
To circumvent this, malate carries the reducing equivalents across the membrane 23 .
This study has demonstrated that the use of GLN in partially hepatectomized rats promotes an over expression of MDH1 and MDH2 enzymes during liver regeneration. The explanation for the these findings is the same already duly proven in previous study 23