Abstract-Apolipoprotein A-II (apoA-II), the second major high-density lipoprotein (HDL) apolipoprotein, has been linked to familial combined hyperlipidemia. Human apoA-II transgenic mice constitute an animal model for this proatherogenic disease. We studied the ability of human apoA-II transgenic mice HDL to protect against oxidative modification of apoB-containing lipoproteins. When challenged with an atherogenic diet, antigens related to low-density lipoprotein (LDL) oxidation were markedly increased in the aorta of 11.1 transgenic mice (high human apoA-II expressor). HDL from control mice and 11.1 transgenic mice were coincubated with autologous very LDL (VLDL) or LDL, or with human LDL under oxidative conditions. The degree of oxidative modification of apoB lipoproteins was then evaluated by measuring relative electrophoretic mobility, dichlorofluorescein fluorescence, 9-and 13-hydroxyoctadecadienoic acid content, and conjugated diene kinetics. In all these different approaches, and in contrast to control mice, HDL from 11.1 transgenic mice failed to protect LDL from oxidative modification. A decreased content of apoA-I, paraoxonase (PON1), and platelet-activated factor acetyl-hydrolase activities was found in HDL of 11.1 transgenic mice. Liver gene expression of these HDL-associated proteins did not differ from that of control mice. In contrast, incubation of isolated human apoA-II with control mouse plasma at 37°C decreased PON1 activity and displaced the enzyme from HDL. Thus, overexpression of human apoA-II in mice impairs the ability of HDL to protect apoB-containing lipoproteins from oxidation. Further, the displacement of PON1 by apoA-II could explain in part why PON1 is mostly found in HDL particles with apoA-I and without apoA-II, as well as the poor antiatherogenic properties of apoA-II-rich HDL.

The concentration of high density lipoproteins (HDL) is inversely related to the risk of atherosclerosis. The two major protein components of HDL are apolipoprotein (apo) A-I and apoA-II. To study the role of apoA-II in lipoprotein metabolism and atherosclerosis, we have developed three lines of C57BL/6 transgenic mice expressing human apoA-II (lines 25.3, 21.5, and 11.1). Northern blot experiments showed that human apoA-II mRNA was present only in the liver of transgenic mice. SDS-polyacrylamide gel electrophoresis and Western blot analysis demonstrated a 17.4-kDa human apoA-II in the HDL fraction of the plasma of transgenic mice. After 3 months on a regular chow, the plasma concentrations of human apoA-II were 21 +/- 4 mg/dl in the 25.3 line, 51 +/- 6 mg/dl in the 21.5 line, and 74 +/- 4 mg/dl in the 11.1 line. The concentration of cholesterol in plasma was significantly lower in transgenic mice than in control mice because of a decrease in HDL cholesterol that was greatest in the line that expressed the most apoA-II (23 mg/dl in the 11.1 line versus 63 mg/dl in control mice). There was also a reduction in the plasma concentration of mouse apoA-I (32 +/- 2, 56 +/- 9, 91 +/- 7, and 111 +/- 2 mg/dl for lines 11.1, 21.5, 25.3, and control mice, respectively) that was inversely correlated with the amount of human apoA-II expressed. Additional changes in plasma lipid/lipoprotein profile noted in line 11.1 that expressed the highest level of human apoA-II include elevated triglyceride, increased proportion of total plasma, and HDL free cholesterol and a marked (>10-fold) reduction in mouse apoA-II. Total endogenous plasma lecithin:cholesterol acyltransferase (LCAT) activity was reduced to a level directly correlated with the degree of increased plasma human apoA-II in the transgenic lines. LCAT activity toward exogenous substrate was, however, only slightly decreased. The biochemical changes in the 11.1 line, which is markedly deficient in plasma apoA-I, an activator for LCAT, are reminiscent of those in patients with partial LCAT deficiency. Feeding the transgenic mice a high fat, high cholesterol diet maintained the mouse apoA-I concentration at a normal level (69 +/- 14 mg/dl in line 11.1 compared with 71 +/- 6 mg/dl in nontransgenic controls) and prevented the appearance of HDL deficiency. All this happened in the presence of a persistently high plasma human apoA-II (96 +/- 14 mg/dl). Paradoxical HDL elevation by high fat diets has been observed in humans and is reproduced in human apoA-II overexpressing transgenic mice but not in control mice. Finally, HDL size and morphology varied substantially in the three transgenic lines, indicating the importance of apoA-II concentration in the modulation of HDL formation. The LCAT and HDL deficiencies observed in this study indicate that apoA-II plays a dynamic role in the regulation of plasma HDL metabolism.

Deletion of glycine N-methyltransferase (GNMT), the main gene involved in liver S-adenosylmethionine (SAM) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis in mice. To demonstrate that the excess of hepatic SAM is the main agent contributing to liver disease in GNMT knockout (KO) mice, we treated 1.5-month-old GNMT-KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N-methyltransferase. NAM administration markedly reduced hepatic SAM content, prevented DNA hypermethylation, and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis. More importantly, NAM treatment prevented the development of fatty liver and fibrosis in GNMT-KO mice. Because GNMT expression is down-regulated in patients with cirrhosis, and because some subjects with GNMT mutations have spontaneous liver disease, the clinical implications of the present findings are obvious, at least with respect to these latter individuals. Because NAM has been used for many years to treat a broad spectrum of diseases (including pellagra and diabetes) without significant side effects, it should be considered in subjects with GNMT mutations. Conclusion: The findings of this study indicate that the anomalous accumulation of SAM in GNMT-KO mice can be corrected by NAM treatment leading to the normalization of the expression of many genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis, as well as reversion of the appearance of the pathologic phenotype. (HEPATOLOGY 2010;52:105-114) Abbreviations: 5mC, 5-methyl-cytosine; a-SMA, a-smooth muscle actin; ADFP, adipose differentiation-related protein; COL1A1, pro-a1-collagen type I; CYP2E1, cytochrome P4502E1; CYP39A1, cytochrome P45039A1; CYP4A10, cytochrome P4504A10; CYP4A14, cytochrome P4504A14; CD36, fatty acid translocase CD36; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GNMT, glycine N-methyltransferase; GSH, glutathione; HCC, hepatocellular carcinoma; IL6, interleukin-6; iNOS, inducible nitric oxide synthase; JAK, Janus kinase; KO, knockout; MAT, methionine adenosyltransferase; NAM, nicotinamide; NNMT, nicotinamide N-methyltransferase; PARP, poly(ADP-ribose) polymerase; PCR, polymerase chain reaction; PPAR, peroxisome proliferator-activated receptor; RASSF1A, Ras-association domain family/tumor suppressor-1A; SAH, S-adenosylhomocysteine; SAM, Sadenosylmethionine; SEM, standard error of the mean; SIRT1, sirtuin-1; SOCS1, suppressor of cytokine signaling-1; STAT, signal transducer and activator of transcription; TIMP-1, TIMP tissue inhibitor of metalloproteinase-1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UCP2, uncoupling protein-2; WT, wild-type.From the

Objective-A high-saturated fatty acid-and cholesterol-containing (HFHC) diet is considered to be a major risk factor for cardiovascular disease. The present study aimed to determine the effects of this Western-type diet on high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT) from macrophages to feces. Methods and Results-Experiments were carried out in mice fed a low-fat, low-cholesterol diet, an HFHC diet, or an HFHC diet without added cholesterol (high-saturated fatty acid and low-cholesterol [HFLC]). The HFHC diet caused a significant increase in plasma cholesterol, HDL cholesterol, and liver cholesterol and enhanced macrophage-derived [ 3 H]cholesterol flux to feces by 3-to 4-fold. These effects were greatly reduced in mice fed the HFLC diet. This HFHC diet-mediated induction of RCT was sex independent and was not associated with obesity or insulin resistance. The HFHC diet caused 1.4-and 3-fold increases in [ 3 H]cholesterol efflux to plasma and HDL-derived [ 3 H]tracer fecal excretion, respectively. Unlike a low-fat, low-cholesterol and HFLC diets, the HFHC diet increased liver ABCG5/G8 expression. The effect of the HFHC diet on fecal macrophage-derived [ 3 H]cholesterol excretion was totally blunted in ABCG5/G8-deficient mice. Conclusion-Despite its deleterious effects on atherosclerosis, the HFHC diet promoted a sustained compensatory macrophage-to-feces RCT. Our data provide direct evidence of the crucial role of dietary cholesterol signaling through liver ABCG5/G8 upregulation in the HFHC diet-mediated induction of macrophage-specific RCT. ietary saturated fat intake has been associated with an increased risk of atherosclerotic cardiovascular disease and metabolic diseases, such as obesity and type 2 diabetes. 1,2 This effect is thought to be mediated by an increase in plasma cholesterol, mainly low-density lipoprotein cholesterol. 3 However, both dietary saturated fat and cholesterol intake are known to raise plasma high-density lipoprotein cholesterol (HDL-C) levels. 4 -8 Several epidemiological studies and 1 meta-analysis of 60 controlled trials showed a positive correlation between high saturated fat intake and HDL-C. 9 -11 In an attempt to determine the mechanism underlying this paradoxical observation, several studies reported that a low saturated fat and cholesterol intake reduced HDL-C levels by reducing the apolipoprotein A-I secretion rate. [12][13][14] However, other studies found this effect to be associated with decreased apolipoprotein A-I fractional catabolic rates. 15,16 Also, when dietary cholesterol was increased along with total and saturated fat, increases in large high-density lipoprotein (HDL) subpopulations and HDL apolipoprotein E amounts were observed. 7,17 Macrophage-specific reverse cholesterol transport (RCT) is thought to be one of the most important HDL-mediated cardioprotective mechanisms. 18 HDL plays a critical role in cholesterol efflux from macrophages, the first step in RCT. 18 However, despite the reported changes in HDL compositi...