Arginase Activities and Global Arginine Bioavailability in Wild-Type and ApoE-Deficient Mice: Responses to High Fat and High Cholesterol Diets

Abstract Increased catabolism of arginine by arginase is increasingly viewed as an important pathophysiological factor in cardiovascular disease, including atherosclerosis induced by high cholesterol diets. Whereas previous studies have focused primarily on effects of high cholesterol diets on arginase expression and arginine metabolism in specific blood vessels, there is no information regarding the impact of lipid diets on arginase activity or arginine bioavailability at a systemic level. We, therefore, evaluated the effects of high fat (HF) and high fat-high cholesterol (HC) diets on arginase activity in plasma and tissues and on global arginine bioavailability (defined as the ratio of plasma arginine to ornithine + citrulline) in apoE 2/2 and wild-type C57BL/6J mice. HC and HF diets led to reduced global arginine bioavailability in both strains. The HC diet resulted in significantly elevated plasma arginase in both strains, but the HF diet increased plasma arginase only in apoE 2/2 mice. Elevated plasma arginase activity correlated closely with increased alanine aminotransferase levels, indicating that liver damage was primarily responsible for elevated plasma arginase. The HC diet, which promotes atherogenesis, also resulted in increased arginase activity and expression of the type II isozyme of arginase in multiple tissues of apoE 2/2 mice only. These results raise the possibility that systemic changes in arginase activity and global arginine bioavailability may be contributing factors in the initiation and/or progression of cardiovascular disease

Arginase activities and global arginine bioavailability in wild-type and ApoE-deficient mice: Responses to high fat and high cholesterol diets

Increased catabolism of arginine by arginase is increasingly viewed as an important pathophysiological factor in cardiovascular disease, including atherosclerosis induced by high cholesterol diets. Whereas previous studies have focused primarily on effects of high cholesterol diets on arginase expression and arginine metabolism in specific blood vessels, there is no information regarding the impact of lipid diets on arginase activity or arginine bioavailability at a systemic level. We, therefore, evaluated the effects of high fat (HF) and high fat-high cholesterol (HC) diets on arginase activity in plasma and tissues and on global arginine bioavailability (defined as the ratio of plasma arginine to ornithine + citrulline) in apoE-/- and wild-type C57BL/6J mice. HC and HF diets led to reduced global arginine bioavailability in both strains. The HC diet resulted in significantly elevated plasma arginase in both strains, but the HF diet increased plasma arginase only in apoE-/- mice. Elevated plasma arginase activity correlated closely with increased alanine aminotransferase levels, indicating that liver damage was primarily responsible for elevated plasma arginase. The HC diet, which promotes atherogenesis, also resulted in increased arginase activity and expression of the type II isozyme of arginase in multiple tissues of apoE-/- mice only. These results raise the possibility that systemic changes in arginase activity and global arginine bioavailability may be contributing factors in the initiation and/or progression of cardiovascular disease

Induction of arginase mRNA and protein in apoE^−/− mice on HC diet.

<p>(A) Effect of HC diet on arginase I and II mRNAs in heart, lung, spleen and kidney. mRNA levels are expressed relative to the levels in apoE^−/− mice on standard diet (arbitrarily set to 1.0 for each tissue and indicated by dotted line). Values are means ± SE for n = 5–7 in each group. *p<0.05 vs standard diet. (B) Effect of HC diet on arginase II protein abundance in lung, spleen and kidney. For each tissue, the upper panel represents arginase II and the lower panel GAPDH. Western blots of extracts from tissues of 5 representative animals on each diet are shown. Amounts of protein loaded in each lane were 10 µg (lung and kidney) or 25 µg (spleen). An extract of C57BL/6J whole kidney (20 µg for lung and kidney blots, 3 µg for spleen blot) was used in the first lane of each blot as positive control (+CT) for arginase II. Twenty µg protein from kidney of the arginase II knockout mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015253#pone.0015253-Shi1" target="_blank">[47]</a> was included in the second lane of the kidney blot in order to establish identity of the lowest band as arginase II. (C) Effect of HC diet on arginase I protein abundance in spleen. The upper panel represents arginase I and the lower panel GAPDH. The Western blot represents extracts from spleen (50 µg each lane) of 5 representative animals on each diet and 100 ng of C57BL/6J liver extract (+CT) as positive control. (D) Densitometry of Western blots, represented in arbitrary units (Arginase/GAPDH), was analyzed by Image Quant 5.2 Software. *p<0.01 vs standard diet. Molecular weights are indicated by the following symbols: solid triangles, 39 kDa; solid diamonds, 37 kDa; open triangles, 37 kDa.</p

Induction of tissue arginase activities in apoE^−/− mice on HC diet.

<p>Values are means ± SE for n = 6 in each group. *p<0.05 vs standard diet.</p

HF and HC diets reduce global arginine bioavailability.

<p>Values are means ± SE for n = 3–4 in each group. Abbreviations: ARG, arginine; ORN, ornithine; CIT, citrulline. Symbols are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015253#pone-0015253-g002" target="_blank">Figure 2</a> legend.</p

Effect of HF and HC diets on plasma levels of ALT.

<p>Values are means ± SE for n = 5 mice in each group. Symbols are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015253#pone-0015253-g002" target="_blank">Figure 2</a> legend.</p