Increased Low-Density Lipoprotein Oxidation and Impaired High-Density Lipoprotein Antioxidant Defense Are Associated With Increased Macrophage Homing and Atherosclerosis in Dyslipidemic Obese Mice

Abstract

Background-Obesity-associated dyslipidemia in humans is associated with increased low-density lipoprotein (LDL) oxidation. Mice with combined leptin and LDL receptor deficiency are obese and show severe dyslipidemia and insulin resistance. We investigated the association between oxidation of apolipoprotein B-containing lipoproteins, high-density lipoprotein (HDL) antioxidant defense, and atherosclerosis in these mice. Methods and Results-LDL receptor knockout (LDLR Ϫ/Ϫ ), leptin-deficient (ob/ob), double-mutant (LDLR Ϫ/Ϫ ;ob/ob), and C57BL6 mice were fed standard chow. Double-mutant mice had higher levels of non-HDL (PϽ0.001) and HDL (PϽ0.01) cholesterol and of triglycerides (PϽ0.001). They also had higher oxidative stress, evidenced by higher titers of autoantibodies against malondialdehyde-modified LDL (PϽ0.001). C57BL6 and ob/ob mice had no detectable lesions. Lesions covered 20% of total area of the thoracic abdominal aorta in double-mutant mice compared with 3.5% in LDLR Ϫ/Ϫ mice (PϽ0.01). Higher macrophage homing and accumulation of oxidized apolipoprotein B-100 -containing lipoproteins were associated with larger plaque volumes in the aortic root of double-mutant mice (PϽ0.01). The activity of the HDL-associated antioxidant enzymes paraoxonase and lecithin:cholesterol acyltransferase (LCAT) (ANOVA; PϽ0.0001 for both) was lower in double-mutant mice. Adenovirus-mediated LCAT gene transfer in double-mutant mice increased plasma LCAT activity by 64% (PϽ0.01) and reduced the titer of autoantibodies by 40% (PϽ0.01) and plaque volume in the aortic root by 42% (PϽ0.05) at 6 weeks. Conclusions-Dyslipidemia and insulin resistance in obese LDL receptor-deficient mice are associated with increased oxidative stress and impaired HDL-associated antioxidant defense, evidenced by decreased paraoxonase and LCAT activity. Transient LCAT overexpression was associated with a reduction of oxidative stress and atherosclerosis.

Stevioside inhibits atherosclerosis by improving insulin signaling and antioxidant defense in obese insulin-resistant mice

“…Previously, obesity in DKO mice was shown to be associated with increased oxidative stress due to a lack of antioxidant enzymes. 13 Therefore, we measured Sod3, because it was shown to have a protective role against hyperglycemia-induced reactive oxygen species (ROS). 22 Sod3 was decreased in placebo-treated DKO mice compared with lean control mice; stevioside normalized its expression from 0.64±0.28 to 1.06±0.47 (Po0.05).…”

“…DKO mice, on the C57BL6 background, were obtained as previously described. 11,13 All offspring were genotyped by PCR techniques. 14 Mice were treated with stevioside (n ¼ 14) or placebo (n ¼ 20) for 12 weeks starting at the age of 12 weeks.…”

“…Therefore, we measured the titer of autoantibodies against malondialdehyde (MDA)-modified LDL as a proxy for ox-LDL in mice, as described before. 13,17,18 Real-time reverse transcription-PCR analysis The levels of RNA expression in extracts of white visceral (IP) adipose tissue and aorta were measured by quantitative realtime reverse transcription-PCR. Total RNA was extracted with Trizol reagent (Invitrogen, Merelbeke, Belgium) and purified on RNeasy Mini kit columns (Qiagen, Venlo, The Netherlands).…”

“…RNA expression levels are thus indicated as arbitrary units ± s.d. [11][12][13] Atherosclerosis The extent of atherosclerosis was determined by analysis of ten 7-mm cross-sections of aortic root of 24-week placeboand stevioside-treated DKO mice. Lipids were stained with oil red O, ox-LDL with mAb4E6, 21 smooth muscle cells (SMC) with an a-actin-specific antibody (Dako, Heverlee, Belgium), macrophages with an antibody against mouse Mac-3 antigen (Pharmingen, Erembodegem, Belgium) and paraoxonase 1 (Pon1) with polyclonal antibodies from Santa Cruz Biotechnology (Tebu-bio, Boechout, Belgium).…”

“…These exhibit most of the metabolic syndrome components, which are associated with increased inflammation and oxidative stress, accelerated atherosclerosis and impaired cardiovascular function. [11][12][13] Weight loss 11 and treatment with statin 12 were associated with improved insulin sensitivity and decreased macrophage and ox-LDL accumulation in their aortic arch. These improvements were associated with increased expression of the antioxidant superoxide dismutases (Sods) in the aorta.…”