Most studies of the effects of diets in apoE^−/− mice have focused tightly on development and progression of atherosclerotic plaques. However, the HC diet used here and in many other studies also induces inflammatory responses in the liver [25]–[28], raising the possibility that liver damage might result in elevated plasma levels of arginase I and thus reduce circulating arginine levels. Thus, plasma arginase activities were determined for three independent experiments in which C57BL6 and apoE^−/− mice were fed standard, HF or HC diets for 2 months as described in Methods. Because the HC diet differed from the standard chow diet with regard to both fat and cholesterol content, the HF diet was included to differentiate any effects of increased fat content alone. Whereas plasma arginase activities did not differ significantly for C57BL6 and apoE^−/− mice on the standard chow diet, there were strain-specific differences in responses to the HF and HC diets (Figure 1). Plasma arginase activities were increased equally in apoE^−/− mice on HF and HC diets. Although there was a trend toward increased plasma arginase activity in C57BL6 mice on the HF diet, it was not statistically significant. However, there was a dramatic increase in plasma arginase activity in C57BL6 mice fed the HC diet, to a level nearly twice as high as the elevated plasma arginase activity in apoE^−/− mice fed the HF or HC diet (Figure 1).

As significant diet- and strain-specific differences in plasma arginase activities were found, more detailed biochemical and molecular studies were performed for a representative experiment in which samples of multiple tissues also had been collected. All subsequent data are from that experiment. On all diets, the apoE^−/− mice had significantly higher plasma cholesterol levels as compared to C57BL/6J. Whereas the HF diet had no effect on plasma cholesterol, the HC diet induced marked increases in both strains (Figure 2). Plasma triglycerides were significantly reduced by the HC diet in both strains but were not affected by the HF diet in either strain. No changes in blood urea nitrogen were observed in any of the groups, indicating no loss of renal function (data not shown). However, plasma alanine aminotransferase (ALT) levels were significantly increased by the HC diet in both strains but by the HF diet only in the apoE^−/− mice (Figure 3). Elevated plasma ALT levels have been shown previously for C57BL/6J mice fed the HC diet [25], [26]. The elevated ALT levels are indicative of liver damage, consistent with previous studies showing liver inflammation and injury associated with high cholesterol-high fat diets in mice [25]–[30]. Notably, the profile of plasma ALT levels closely parallels the profile of plasma arginase activities in Figure 1. For example, plasma ALT and arginase activities are significantly correlated for apoE^−/− mice on the three diets (Figure 4), even though apoE^−/− mice on the HC diet differ significantly from apoE^−/− mice on the standard and HF diets with regard to arginase expression in nonhepatic tissues (discussed below). Because the liver is the greatest source of arginase in the body, the association between the increases in plasma arginase activities and ALT levels indicates that liver damage represents the principal source of increased arginase activity.

Plasma levels of arginine, ornithine and citrulline were not significantly affected by any diet in C57BL/6J mice (Table 1). In the apoE^−/− mice, however, a reduction in circulating arginine levels was found only with the HC diet. There was also a trend for reduced plasma arginine in apoE^−/− mice on the HF diet, but this did not reach statistical significance (p = 0.06; Table 1). Plasma ornithine was significantly increased only in the apoE^−/− mice on the HF diet. There were no diet- or strain-specific differences in plasma citrulline levels, suggesting no difference in activity of the intestinal-renal axis of citrulline/arginine metabolism [1]. Overall, there was no consistent pattern of dietary lipid effect on plasma levels of these individual amino acids.

Consideration of the plasma levels of these individual amino acids may fail to reveal significant changes in arginine metabolism because it overlooks the fact that they are metabolically interrelated [1]. Consequently, the ratio of plasma arginine to the sum of plasma ornithine plus citrulline has been developed as an indicator of global arginine bioavailability that is more sensitive to perturbations in arginine metabolism than are levels of the individual amino acids [23], [24]. Unlike the values of individual amino acids, this ratio clearly revealed effects of the diets: global arginine bioavailability was significantly reduced in C57BL6 fed the HC diet and apoE^−/− mice fed the HF or HC diet (Figure 5). The groups with reduced global arginine bioavailability also exhibited increases in plasma arginase activity and liver enzymes (Figures. 1 and 3).

In contrast to the changes in the plasma, arginase activities in tissues were increased only in the apoE^−/− mice fed the HC diet (Figure 6), the group with the highest plasma cholesterol levels. No increases were observed in apoE^−/− mice fed the HF diet or in any of the C57BL/6J groups (data not shown). Increased arginase activity was observed in the heart, lung, kidney and spleen of apoE^−/− mice fed the HC diet (Figure 6). The high levels of liver arginase activity did not differ significantly between strains and were not altered by any of the experimental conditions (e.g., apoE^−/− mice: 3584±507 nmol/mg/min; standard diet vs 3958±324 nmol/mg/min; HC diet), consistent with the fact that hepatic levels of arginase and the other urea cycle enzymes are responsive primarily to changes in the intake and catabolism of protein [31]–[33].

As arginase activity represents the sum of contributions by arginases I and II, levels of arginase mRNAs and protein were evaluated. Increased arginase activity was accompanied by increased arginase II mRNA in heart, lung, spleen and kidney of apoE^−/− mice fed the HC diet (Figure 7A). Only the heart had significantly increased arginase I mRNA. Increased arginase activity and arginase II mRNA in lung, spleen, and kidney were accompanied by increased levels of arginase II protein (Figure 7B and D). Arginase I protein was increased in extracts from spleen (Figure 7C and D) but was below the level of detection in Western blots of lung and kidney extracts. Thus, arginase II was primarily responsible for the overall arginase activity in lung and kidney whereas both isoforms contributed to overall activity in spleen.

Male C57BL/6J and B6.129P2-Apoe^tm1Unc (apoE^−/− mice) (Jackson Laboratory, Bar Harbor, ME) aged 6–7 weeks were used in this study. All mice were provided water ad libitum in ventilated cages in a controlled humidity and temperature environment with a 12hr light/dark cycle. Animal care and use procedures were conducted in accordance with the “PHS Policy on Human Care and Use of Laboratory Animals” and the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86–23, 1996). These procedures were approved by the National Institute for Occupational Safety and Health Institutional Animal Care and Use Committee (internal approved protocol ID 07-PS-M-014). In three independent experiments, different groups of mice were placed on one of the following diets for 2 months: 1) standard chow diet (Harlan 7913 irradiated NIH-31 6% rodent diet containing protein, 186 g/Kg; fat, 62.5 g/Kg; fiber, 45.3 g/Kg; metabolizable energy, 3.13 Kcal/g; 18% total kcal from fat; 0.053% cholesterol), 2) high fat and high cholesterol diet containing cholate (HC; TD88051 Harlan; 37.1% total kcal from fat; 1.25% cholesterol; 0.5% sodium cholate) [43] and 3) high fat diet (HF; D12451 Research Diets; 45% total kcal from fat; 0.20% cholesterol) [44]. Prior to sacrifice and collection of blood mice were fasted 16 hr. Following CO₂ asphyxiation blood was collected from vena cava followed by perfusion with cold PBS via the heart. For one group of experimental animals in which responses to all 3 diets were evaluated, tissues also were harvested, quick frozen in liquid nitrogen and stored at −80°C for arginase activity and RNA isolation.

In initial experiments we found that plasma from some samples of whole blood that had been placed on ice for a couple of hours prior to fractionation had remarkably low arginine (<20 µM) and high ornithine levels (>150 µM) that were not representative of values in plasma that was rapidly fractionated and frozen. Thus, EDTA blood samples were immediately centrifuged, and plasma was quick-frozen and stored at −80°C until used for analysis. Plasma levels of total cholesterol, triglycerides, blood urea nitrogen and ALT were analyzed (AniLytics Inc.). A complete plasma amino acid profile was determined by the Medical Genetics Laboratory at Baylor College of Medicine.

For arginase activity, tissue homogenates were prepared using the TissueLyser (Qiagen) in 0.1% Triton X-100 with 2 mM Pefabloc (Roche), 2 µg/ml pepstatin A (Sigma) and 10 µg/ml leupeptin (Sigma). Samples were placed on ice for 10 minutes then centrifuged at 12,000 g for 10 minutes and supernatants were used. Arginase activity in plasma and tissue homogenates was determined as the conversion of [¹⁴C-guanidino]-L-arginine (American Radiolabeled Chemicals or Moravek) to [¹⁴C]urea (2.5-hr incubation for plasma and all tissues except liver – 1/50 dilution for 30 min), which was then converted to ¹⁴CO₂ by urease and trapped as Na₂¹⁴CO₃ and counted on a scintillation counter as described previously [8]. Protein concentrations were determined by the BCA protein assay kit (Pierce) and used to calculate arginase specific activities. Plasma arginase activity was expressed as activity per volume plasma (nmol/ml/min).

Real-time RT-PCR was performed as previously described [45]. Briefly, RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer’s directions. One µg of total RNA was reverse transcribed using random hexamers (Applied Biosystems) and Superscript III (Invitrogen). Five µl of cDNA (in duplicate for each gene) was then used for mRNA determinations. Pre-designed Assays-on-Demand™ TaqMan^® gene expression assays (Applied Biosystems) for arginase I and arginase II were used. Ribosomal 18S RNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA were used as internal references for normalization. Relative gene expression was calculated using the comparative threshold method (2^−ΔΔCt) [46]. There was no difference in relative fold change compared to reference using either 18S rRNA or GAPDH mRNA.

Using methods described previously [45], Western blotting was performed on the tissue homogenates used for assays of arginase activity. Western blots were developed with antibody to arginase I (BD Transduction Laboratories #610708;1∶1000 dilution in 1% nonfat milk/1%BSA/Tris-buffered saline containing 0.1% Tween 20 (TBS-T)), antibody to arginase II (Santa Cruz Biotechnology #sc-20151; 1∶500 dilution in 5% nonfat milk in TBS-T) and secondary HRP-linked antibodies (1∶5000) from GE Healthcare. Following immunodetection of arginase, blots were stripped (Pierce Restore Plus Western Blot Stripping Buffer) and re-probed with antibody to GAPDH (Santa Cruz Biotechnology #sc-25778; 1∶2000 dilution in 5% milk/TBS-T) to verify equal protein loading. There were insufficient amounts of heart extract to analyze by Western blot.

All data are presented as mean ± standard error. Analyses were performed using JMP^® Statistical Discovery Software utilizing analysis of variance with a least squares means table generated by a student’s t-test post-hoc test. In data sets where there was no homogeneity of variance, values were log transformed prior to analysis. Student’s t-test was used only when two groups of equal sample size were compared. Differences were considered statistically significant at p<0.05.