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Advanced glycated albumin (AGE-albumin) impairs cholesterol efflux and contributes to inflammation in macrophages. The current study evaluated: (1) the persistence of the deleterious effect of AGE-albumin in cholesterol efflux and in inflammation, and (2) how metabolic control in diabetes mellitus (DM) contributes to attenuate the deleterious role of AGE-albumin in macrophage cholesterol homeostasis. Methods: AGE-albumin was produced in vitro or isolated from uncontrolled DM subjects' serum before (bGC) and after improved glycemic control (aGC). Albumin samples were incubated with bone marrow-derived macrophages and 14 C-cholesterol efflux or LPS- induced cytokine secretion were determined immediately, or after cell resting in culture media alone. The ABCA-1 degradation rate was determined after cell incubation with cycloheximide, and ABCA1 protein level by immunoblot. Oil Red O staining was used to assess intracellular lipid accumulation. Results: A persistent effect of AGE-albumin was observed in macrophages in terms of the secretion of inflammatory cytokines and reduced cholesterol efflux. HDL-mediated 14 C-cholesterol efflux was at least two times higher in macrophages treated with aCG-albumin as compared to bGC-albumin, and intracellular lipid content was significantly reduced in aGC-albumin-treated cells. As compared to bGC-albumin, the ABCA-1 protein content in whole cell bulk was 94% higher in aCG-albumin. A 20% increased ABCA-1 decay rate was observed in macrophages treated with albumin from poorly controlled DM. AGE-albumin has a persistent deleterious effect on macrophage lipid homeostasis and inflammation. The reduction of AGEs in albumin ameliorates cholesterol efflux.

Aerobic exercise training (AET) improves the reverse cholesterol transport (RCT) in cholesteryl ester transfer protein-transgenic (CETP-tg) mice. We aimed at investigating the role of AET in the expression of genes and proteins involved in lipid flux in the aorta and macrophages of CETP-tg mice. Three-month-old male mice were randomly divided into trained (T; treadmill 15 m/min; 30 min/day) and sedentary (S) groups. After 6 weeks, peritoneal macrophages and the aortic arch were obtained immediately (0 h) or 48 h after the last exercise session. mRNA was determined by RT-qPCR, protein levels by immunoblot and 14 C-cholesterol efflux determined in macrophages. AET did not change body weight, plasma cholesterol, triglycerides, glucose and CETP activity. In macrophages, at time 0 h, a higher expression of genes that encode PPAR gamma, ABCA-1 and a lower expression of MCP-1 and IL-10, was observed in T as compared to S. After 48 h, lower expressions of MCP-1 and PPAR gamma genes were observed in T mice. Increase in ABCA-1, SR-BI and IL-6 and decrease of LOX-1, MCP-1, TNF and IL-10 gene expression was observed in the aorta of T compared to S mice (0 h) and LOX-1 and MCP-1 remained diminished after 48 h. The protein level of MCP-1 and SR-BI in the aortic arch was unchanged in T animals after 48 h as compared to S, but LOX-1 was reduced confirming data of gene expression. The apo A-I and the HDL 2 mediated-cholesterol efflux (8 and 24 h) were not different between T and S animals. In the presence of CETP, AET positively influences gene expression in the arterial wall and macrophages of CETP-tg mice contributing to the RCT and prevention of atherosclerosis. These changes were perceptible immediately after the exercise session and were influenced by the presence of CETP although independent of changes in its activity. Reductions in gene and protein expression of LOX-1 were parallel and reflect the ability of exercise training in reducing the uptake of modified LDL by the arterial wall macrophages.

Background and Aims: Advanced glycation end products (AGEs) induce cellular oxidative/endoplasmic reticulum stress and inflammation. We investigated its underlying mechanisms for atherogenesis focusing on regulation of ABCA1 protein decay in macrophages., Methods: The ABCA1 decay rate was evaluated in macrophages after treatment with LXR agonist and by incubation with control (C) or AGE-albumin concomitant or not with cycloheximide, MG-132, ammonium chloride and calpain inhibitors were utilized to inhibit, respectively, proteasome, lysosome and ABCA1 proteolysis at cell surface. ABCA1 was determined by immunoblot and the protein decay rate calculated along time by the slope of the linear regression. Ubiquitination level was determined in ABCA1 immunoprecipitated from whole cell lysate or bulk cell membrane. AGE effect was also analyzed in THP-1 cells transfected with siRNA-RAGE. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/MS. One-way ANOVA and Student t test were utilized to compare results., Results: CML and PYR-albumin were higher in AGE-albumin as compared to C. AGE-albumin reduced ABCA1 in J774 and THP-1 macrophages (20-30%) and induced a higher ABCA1 ubiquitination and a faster protein decay rate that was dependent on the presence of AGE during the kinetics of measurement in the presence of cycloheximide. Proteasomal inhibition restored and lysosomal inhibition partially recovered ABCA1 in cells treated with AGE-albumin. Calpain inhibition was not able to rescue ABCA1. RAGE knockdown prevented the reduction in ABCA1 elicited by AGE., Conclusions: AGE-albumin diminishes ABCA1 by accelerating its degradation through the proteasomal and lysosomal systems. This may increase lipid accumulation in macrophages by diminishing cholesterol efflux via RAGE signaling contributing to atherosclerosis in diabetes mellitus., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Advanced glycation endproducts elicit inflammation. However, their role in adipocyte macrophage infiltration and in the development of insulin resistance, especially in the absence of the deleterious biochemical pathways that coexist in diabetes mellitus, remains unknown. We investigated the effect of chronic administration of advanced glycated albumin (AGE-albumin) in healthy rats, associated or not with N-acetylcysteine (NAC) treatment, on insulin sensitivity, adipose tissue transcriptome and macrophage infiltration and polarization. Methods: Male Wistar rats were intraperitoneally injected with control (C) or AGE-albumin alone, or, together with NAC in the drinking water. Biochemical parameters, lipid peroxidation, gene expression and protein contents were, respectively, determined by enzymatic techniques, reactive thiobarbituric acid substances, RT-qPCR and immunohistochemistry or immunoblot. Carboxymethyllysine (CML) and pyrraline (PYR) were determined by LC/mass spectrometry (LC-MS/MS) and ELISA. Results: CML and PYR were higher in AGE-albumin as compared to C. Food consumption, body weight, systolic blood pressure, plasma lipids, glucose, hepatic and renal function, adipose tissue relative weight and adipocyte number were similar among groups. In AGE-treated animals, insulin resistance, adipose macrophage infiltration and Col12a1 mRNA were increased with no changes in M1 and M2 phenotypes as compared to C-albumin-treated rats. Total GLUT4 content was reduced by AGE-albumin as compared to C-albumin. NAC improved insulin sensitivity, reduced urine TBARS, adipose macrophage number and Itgam and Mrc mRNA and increased Slc2a4 and Ppara . CD11b, CD206, Ager, Ddost, Cd36, Nfkb1, Il6, Tnf , Adipoq, Retn, Arg, and Il12 expressions were similar among groups. Conclusions: AGE-albumin sensitizes adipose tissue to inflammation due to macrophage infiltration and reduces GLUT4, contributing to insulin resistance in healthy rats. NAC antagonizes AGE-albumin and prevents insulin resistance. Therefore, it may be a useful tool in the prevention of AGE action on insulin resistance and long-term complications of DM.

Aims: Advanced glycated albumin (AGE-albumin) adversely impairs macrophage lipid homeostasis in vitro, which may be prevented by angiotensin receptor blockers. In vivo studies are inconclusive whether AGE-albumin itself plays important role in early-stage atherogenesis. We aimed at investigating how AGE-albumin by itself drives atherosclerosis development in dyslipidemic non-diabetic mice and if its effects are due to the activation of renin-angiotensin system in the arterial wall and the expression of genes and proteins involved in lipid flux., Methods and Results: Murine albumin glycation was induced by incubation with 10mM glycolaldehyde and C-albumin with PBS alone. Twelve-week-old-male apoE knockout mice were submitted to a daily IP injection of control (C) or AGE-albumin (2mg/mL) during 30days with or without losartan (LOS: 100mg/L; C+LOS and AGE+LOS). Aortic arch was removed, and gene expression was determined by RT-PCR and protein content by immunofluorescence. Plasma lipid and glucose levels were similar among groups. Systolic blood pressure was similarly reduced in both groups treated with LOS. In comparison to C-albumin, aortic lipid infiltration was 5.3 times increased by AGE-albumin, which was avoided by LOS. LOS prevented the enhancement induced by AGE-albumin in Ager, Tnf and Cybb mRNA levels but did not reduce Olr1. Nfkb and Agt mRNA levels were unchanged by AGE-albumin. LOS similarly reduced Agtr1a mRNA level in both C and AGE-albumin groups. In AGE-albumin-treated mice, immunofluorescence for carboxymethyl-lysine, 4-hydroxynonenal and RAGE was respectively, 4.8, 2.6 and 1.7 times enhanced in comparison to C-albumin. These increases were all avoided by LOS., Conclusions: AGE-albumin evokes a pre-stage of atherogenesis in dyslipidemic mice independently of the presence of diabetes mellitus or modulation in the RAS in part by the induction of lipid peroxidation and inflammation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Regular exercise prevents and regresses atherosclerosis by improving lipid metabolism and antioxidant defenses. Exercise ameliorates the reverse cholesterol transport (RCT), an antiatherogenic system that drives cholesterol from arterial macrophages to the liver for excretion into bile and feces. In this study we analyzed the role of aerobic exercise on the in vivo RCT and expression of genes and proteins involved in lipid flux and inflammation in peritoneal macrophages, aortic arch and liver from wild type mice., Methods: Twelve-week-old male mice were divided into sedentary and trained groups. Exercise training was performed in a treadmill (15 m/min, 30 min/day, 5 days/week). Plasma lipids were determined by enzymatic methods and lipoprotein profile by fast protein liquid chromatography. After intraperitoneal injection of J774-macrophages the RCT was assessed by measuring the recovery of (3)H-cholesterol in plasma, feces and liver. The expression of liver receptors was determined by immunoblot, macrophages and aortic mRNAs by qRT-PCR. (14)C-cholesterol efflux mediated by apo A-I and HDL2 and the uptake of (3)H-cholesteryl oleoyl ether ((3)H-COE)-acetylated-LDL were determined in macrophages isolated from sedentary and trained animals 48 h after the last exercise session., Results: Body weight, plasma lipids, lipoprotein profile, glucose and blood pressure were not modified by exercise training. A greater amount of (3)H-cholesterol was recovered in plasma (24 h and 48 h) and liver (48 h) from trained animals in comparison to sedentary. No difference was found in (3)H-cholesterol excreted in feces between trained and sedentary mice. The hepatic expression of scavenger receptor class B type I (SR-BI) and LDL receptor (B-E) was enhanced by exercise. We observed 2.8 and 1.7 fold rise, respectively, in LXR and Cyp7a mRNA in the liver of trained as compared to sedentary mice. Macrophage and aortic expression of genes involved in lipid efflux was not systematically changed by physical exercise. In agreement, (14)C-cholesterol efflux and uptake of (3)H-COE-acetylated-LDL by macrophages was similar between sedentary and trained animals., Conclusion: Aerobic exercise in vivo accelerates the traffic of cholesterol from macrophages to the liver contributing to prevention and regression of atherosclerosis, independently of changes in macrophage and aorta gene expression.

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2., (© 2014 Wiley Periodicals, Inc.)

Objective: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT)., Methods and Results: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL. Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL(2) (43% and 47%, respectively) and HDL(3) (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively)., Conclusions: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM., (Copyright © 2012 Elsevier B.V. All rights reserved.)

ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We analyzed the effect of a 6-week aerobic exercise training program on the in vivo macrophage reverse cholesterol transport (RCT) in human cholesteryl ester transfer protein (CETP) transgenic (CETP-tg) mice. Male CETP-tg mice were randomly assigned to a sedentary group or a carefully supervised exercise training group (treadmill 15 m/min, 30 min sessions, five sessions per week). The levels of plasma lipids were determined by enzymatic methods, and the lipoprotein profile was determined by fast protein liquid chromatography (FPLC). CETP activity was determined by measuring the transfer rate of ¹⁴C-cholesterol from HDL to apo-B containing lipoproteins, using plasma from CETP-tg mice as a source of CETP. The reverse cholesterol transport was determined in vivo by measuring the [³H]-cholesterol recovery in plasma and feces (24 and 48 h) and in the liver (48 h) following a peritoneal injection of [³H]-cholesterol labeled J774-macrophages into both sedentary and exercise trained mice. The protein levels of liver receptors were determined by immunoblot, and the mRNA levels for liver enzymes were measured using RT-PCR. Exercise training did not significantly affect the levels of plasma lipids or CETP activity. The HDL fraction assessed by FPLC was higher in exercise-trained compared to sedentary mice. In comparison to the sedentary group, a greater recovery of [³H]-cholesterol from the injected macrophages was found in the plasma, liver and feces of exercise-trained animals. The latter occurred even with a reduction in the liver CYP7A1 mRNA level in exercised trained animals. Exercise training increased the liver LDL receptor and ABCA-1 protein levels, although the SR-BI protein content was unchanged. The RCT benefit in CETP-tg mice elicited by exercise training helps to elucidate the role of exercise in the prevention of atherosclerosis in humans.