Nuclear Factor-Kappa-B Mediates the Advanced Glycation End Product-Induced Repression of Slc2a4 Gene Expression in 3T3-L1 Adipocytes.

Advanced glycated end products (AGEs) are cytotoxic compounds that are mainly increased in diabetes mellitus (DM), kidney failure, inflammation, and in response to the ingestion of AGE-rich diets. AGEs can also impair glycemic homeostasis by decreasing the expression of the Slc2a4 (solute carrier family 2 member 4) gene and its GLUT4 (solute carrier family 2, facilitated glucose transporter member 4) protein in muscle. However, the mechanisms underlying AGE's effect on adipocytes have not been demonstrated yet. This study investigated the effects of AGEs upon Slc2a4/GLUT4 expression in 3T3-L1 adipocytes, as well as the potential role of NFKB (nuclear factor NF-kappa-B) activity in the effects observed. Adipocytes were cultured in the presence of control albumin (CA) or advanced glycated albumin (GA) at concentrations of 0.4, 3.6, and 5.4 mg/mL for 24 h or 72 h. Slc2a4, Rela, and Nfkb1mRNAs were measured by RT-qPCR, GLUT4, IKKA/B, and p50/p65 NFKB subunits using Western blotting, and p50/p65 binding into the Slc2a4 promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. GA at 0.4 mg/mL increased Slc2a4/GLUT4 expression after 24 h and 72 h (from 50% to 100%), but at 5.4 mg/mL, Slc2a4/GLUT4 expression decreased at 72 h (by 50%). Rela and Nfkb1 expression increased after 24 h at all concentrations, but this effect was not observed at 72 h. Furthermore, 5.4 mg/mL of GA increased the p50/p65 nuclear content and binding into Slc2a4 at 72 h. In summary, this study reveals AGE-induced and NFKB-mediated repression of Slc2a4/GLUT4 expression. This can compromise the adipocyte glucose utilization, contributing not only to the worsening of glycemic control in DM subjects but also the impairment of glycemic homeostasis in non-DM subjects under the high intake of AGE-rich foods.

The Prolonged Activation of the p65 Subunit of the NF-Kappa-B Nuclear Factor Sustains the Persistent Effect of Advanced Glycation End Products on Inflammatory Sensitization in Macrophages.

Advanced glycation end products (AGEs) prime macrophages for lipopolysaccharide (LPS)-induced inflammation. We investigated the persistence of cellular AGE-sensitization to LPS, considering the nuclear content of p50 and p65 nuclear factor kappa B (NFKB) subunits and the expression of inflammatory genes. Macrophages treated with control (C) or AGE-albumin were rested for varying intervals in medium alone before being incubated with LPS. Comparisons were made using one-way ANOVA or Student t-test (n = 6). AGE-albumin primed macrophages for increased responsiveness to LPS, resulting in elevated levels of TNF, IL-6, and IL-1beta (1.5%, 9.4%, and 5.6%, respectively), compared to C-albumin. TNF, IL-6, and IL-1 beta secretion persisted for up to 24 h even after the removal of AGE-albumin (area under the curve greater by 1.6, 16, and 5.2 times, respectively). The expressions of Il6 and RelA were higher 8 h after albumin removal, and Il6 and Abca1 were higher 24 h after albumin removal. The nuclear content of p50 remained similar, but p65 showed a sustained increase (2.9 times) for up to 24 h in AGE-albumin-treated cells. The prolonged activation of the p65 subunit of NFKB contributes to the persistent effect of AGEs on macrophage inflammatory priming, which could be targeted for therapies to prevent complications based on the AGE-RAGE-NFKB axis.

Increased Expression of miR-223-3p and miR-375-3p and Anti-Inflammatory Activity in HDL of Newly Diagnosed Women in Advanced Stages of Breast Cancer.

The expression of inflammation-related miRs bound to high-density lipoproteins (HDLs), the anti-inflammatory activity of HDLs isolated from individuals with breast cancer, and controls were determined. Forty newly diagnosed women with breast cancer naïve of treatment and 10 control participants were included. Cholesterol-loaded bone-marrow-derived macrophages were incubated with HDL from both groups and challenged with lipopolysaccharide (LPS). Interleukin 6 (IL6) and tumor necrosis factor (TNF) in the medium were quantified. The miRs in HDLs were determined by RT-qPCR. Age, body mass index, menopausal status, plasma lipids, and HDL composition were similar between groups. The ability of HDL to inhibit IL6 and TNF production was higher in breast cancer compared to controls, especially in advanced stages of the disease. The miR-223-3p and 375-3p were higher in the HDLs of breast cancer independent of the histological type of the tumor and had a high discriminatory power between breast cancer and controls. The miR-375-3p was greater in the advanced stages of the disease and was inversely correlated with the secretion of inflammatory cytokines. Inflammation-related miRs and the anti-inflammatory role of HDLs may have a significant impact on breast cancer pathophysiology.

Dietary sodium restriction alters muscle lipidomics that relates to insulin resistance in mice.

A low-sodium (LS) diet has been shown to reduce blood pressure (BP) and the incidence of cardiovascular diseases. However, severe dietary sodium restriction promotes insulin resistance (IR) and dyslipidemia in animal models and humans. Thus, further clarification of the long-term consequences of LS is needed. Here, we investigated the effects of chronic LS on gastrocnemius gene and protein expression and lipidomics and its association with IR and plasma lipids in LDL receptor knockout mice. Three-month-old male mice were fed a normal sodium diet (NS; 0.5% Na; n = 12-19) or LS (0.06% Na; n = 14-20) over 90 days. Body mass (BM), BP, plasma total cholesterol, triacylglycerol (TG), glucose, hematocrit, and IR were evaluated. LS increased BM (9%), plasma TG (51%), blood glucose (19%), and IR (46%) when compared with the NS. RT-qPCR analysis revealed that genes involved in lipid uptake and oxidation were increased by the LS: Fabp3 (106%), Prkaa1 (46%), and Cpt1 (74%). Genes and proteins (assessed by Western blotting) involved in insulin signaling were not changed by the LS. Similarly, lipid species classically involved in muscle IR, such as diacylglycerols and ceramides detected by ultra-high-performance liquid chromatography coupled to mass spectrometry, were also unchanged by LS. Species of phosphatidylcholines (68%), phosphatidylinositol (90%), and free fatty acids (59%) increased while cardiolipins (41%) and acylcarnitines (9%) decreased in gastrocnemius in response to LS and were associated with glucose disposal rate. Together these results suggest that chronic LS alters glycerophospholipid and fatty acids species in gastrocnemius that may contribute to glucose and lipid homeostasis derangements in mice.

Discovery of (E)-4-styrylphenoxy-propanamide: A dual PPARα/γ partial agonist that regulates high-density lipoprotein-cholesterol levels, modulates adipogenesis, and improves glucose tolerance in diet-induced obese mice.

Peroxisome proliferator-activated receptors are promising therapeutic targets for metabolic diseases, including obesity, diabetes, and dyslipidemia. This study describes the design, synthesis and pharmacological evaluation of stilbene-based compounds as dual PPARα/γ partial agonists with potency in the nanomolar range. In vitro and in vivo assays revealed that the lead compound (E)-4-styrylphenoxy-propanamide (5b) removed 14C-cholesterol from the foam cells through apolipoprotein A-I and High-Density Lipoprotein-2. In the high-fat diet-induced obesity mouse model, the oral administration of compound 5b increased HDL levels, paraoxonase-1 activity, and insulin sensitivity, and decreased glucose levels. Moreover, the adipogenesis pathway and triglyceride accumulation slightly changed in the adipocyte cells upon treatment with compound 5b, without affecting the body weight and adipose tissue in obese mice. Compound 5b did not affect the plasma levels of hepatic and renal injury biomarkers. Thus, stilbene-based compound 5b is a promising prototype for developing novel candidates to treat dyslipidemia and diabetes.

Advanced Glycation End Products: A Sweet Flavor That Embitters Cardiovascular Disease.

Epidemiological studies demonstrate the role of early and intensive glycemic control in the prevention of micro and macrovascular disease in both type 1 and type 2 diabetes mellitus (DM). Hyperglycemia elicits several pathways related to the etiopathogenesis of cardiovascular disease (CVD), including the generation of advanced glycation end products (AGEs). In this review, we revisit the role played by AGEs in CVD based in clinical trials and experimental evidence. Mechanistic aspects concerning the recognition of AGEs by the advanced glycosylation end product-specific receptor (AGER) and its counterpart, the dolichyl-diphosphooligosaccharide-protein glycosyltransferase (DDOST) and soluble AGER are discussed. A special focus is offered to the AGE-elicited pathways that promote cholesterol accumulation in the arterial wall by enhanced oxidative stress, inflammation, endoplasmic reticulum stress and impairment in the reverse cholesterol transport (RCT).

Alcohol Use Disorder is Associated with Upregulation of MicroRNA-34a and MicroRNA-34c in Hippocampal Postmortem Tissue.

BACKGROUND: To investigate epigenetic mechanisms potentially involved in the cognitive decline associated with chronic alcohol intake, we evaluated the expressions of three micro-RNAs (miR-34a, -34b, and -34c) highly expressed in the hippocampus and involved in neuronal physiology and pathology. MiR-34a participates in functioning and survival of mature neurons; miR-34b is associated with Alzheimer-like disorders; and miR-34c is implicated in the memory impairment of Alzheimer disease in rodents and humans. METHODS: A total of 69 cases were selected from the Biobank for Aging Studies and categorized according to the absence (n = 50) or presence (n = 19) of alcohol use disorder (AUD). Cases presenting with neuropathological diagnoses of dementias were excluded. Total RNA was extracted from hippocampal paraffinized slices, complementary DNA was synthesized from miRs, and RT-qPCR was performed with TaqMan® assays. RESULTS: Higher expressions of miR-34a and miR-34c, but not of miR-34b, were found in the group with AUD in comparison with the group without AUD after adjustment for potential confounders (age, sex, body mass index, presence of hypertension, diabetes mellitus, smoking, and physical inactivity). CONCLUSIONS: Hippocampal upregulation of miR-34a and miR-34c may be involved in the cognitive decline associated with chronic alcohol consumption.

Comparing Data-Independent Acquisition and Parallel Reaction Monitoring in Their Abilities To Differentiate High-Density Lipoprotein Subclasses.

High-density lipoprotein (HDL) is a diverse group of particles with multiple cardioprotective functions. HDL proteome follows HDL particle complexity. Many proteins were described in HDL, but consistent quantification of HDL protein cargo is still a challenge. To address this issue, the aim of this work was to compare data-independent acquisition (DIA) and parallel reaction monitoring (PRM) methodologies in their abilities to differentiate HDL subclasses through their proteomes. To this end, we first evaluated the analytical performances of DIA and PRM using labeled peptides in pooled digested HDL as a biological matrix. Next, we compared the quantification capabilities of the two methodologies for 24 proteins found in HDL2 and HDL3 from 19 apparently healthy subjects. DIA and PRM exhibited comparable linearity, accuracy, and precision. Moreover, both methodologies worked equally well, differentiating HDL subclasses' proteomes with high precision. Our findings may help to understand HDL functional diversity.

Enrichment of apolipoprotein A-IV and apolipoprotein D in the HDL proteome is associated with HDL functions in diabetic kidney disease without dialysis.

BACKGROUND AND AIMS: Diabetic kidney disease (DKD) is associated with lipid derangements that worsen kidney function and enhance cardiovascular (CVD) risk. The management of dyslipidemia, hypertension and other traditional risk factors does not completely prevent CVD complications, bringing up the participation of nontraditional risk factors such as advanced glycation end products (AGEs), carbamoylation and changes in the HDL proteome and functionality. The HDL composition, proteome, chemical modification and functionality were analyzed in nondialysis subjects with DKD categorized according to the estimated glomerular filtration rate (eGFR) and urinary albumin excretion rate (AER). METHODS: Individuals with DKD were divided into eGFR> 60 mL/min/1.73 m2 plus AER stages A1 and A2 (n = 10) and eGFR< 60 plus A3 (n = 25) and matched by age with control subjects (eGFR> 60; n = 8). RESULTS: Targeted proteomic analyses quantified 28 proteins associated with HDL in all groups, although only 2 were more highly expressed in the eGFR< 60 + A3 group than in the controls: apolipoprotein D (apoD) and apoA-IV. HDL from the eGFR< 60 + A3 group presented higher levels of total AGEs (20%), pentosidine (6.3%) and carbamoylation (4.2 x) and a reduced ability to remove 14C-cholesterol from macrophages (33%) in comparison to HDL from controls. The antioxidant role of HDL (lag time for LDL oxidation) was similar among groups, but HDL from the eGFR< 60 + A3 group presented a greater ability to inhibit the secretion of IL-6 and TNF-alpha (95%) in LPS-elicited macrophages in comparison to the control group. CONCLUSION: The increase in apoD and apoA-IV could contribute to counteracting the HDL chemical modification by AGEs and carbamoylation, which contributes to HDL loss of function in well-established DKD.

Advanced Glycated apoA-IV Loses Its Ability to Prevent the LPS-Induced Reduction in Cholesterol Efflux-Related Gene Expression in Macrophages.

We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 (Il6), TNF-alpha (Tnf), IL-1 beta (Il1b), toll-like receptor 4 (Tlr4), tumor necrosis factor receptor-associated factor 6 (Traf6), Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/Stat3), nuclear factor kappa B (Nfkb), and AGE receptor 1 (Ddost) as well as IL-6 and TNF-alpha secretion. AGE-apoA-IV alone did not change cholesterol efflux or ABCA-1 levels but was unable to restore the LPS-induced reduction in expression of Abca1 and Abcg1. AGE-apoA-IV inhibited inflammation but lost its ability to counteract the LPS-induced changes in expression of genes involved in macrophage cholesterol efflux that may contribute to atherosclerosis.

Leukotriene Pathway Activation Associates with Poor Glycemic Control and with Cardiovascular Autonomic Neuropathy in Type 1 Diabetes.

BACKGROUND AND AIMS: Since hyperglycemia promotes inflammation by different pathways and inflammation participates in the development of chronic diabetes complications, we investigated the association between the leukotriene (LT) pathway and microvascular diabetes complications. METHODS AND RESULTS: Quantitative polymerase chain reaction was employed to quantify the expression of ALOX5 (encodes 5-lipoxygenase), LTB4R (encodes one of the LTB4 receptors), and MYD88 in peripheral blood mononuclear cells from 164 type 1 diabetes (T1D) individuals presenting or not diabetes kidney disease, retinopathy, peripheral neuropathy, and cardiovascular autonomic neuropathy (CAN); 26 nondiabetic subjects were included as controls. LTB4 plasmatic concentrations were also evaluated. The expression of LTB4R was significantly higher in T1D individuals than in controls. T1D individuals with microvascular complications presented lower MYD88 mRNA expression when compared to those without microvascular complications. Higher LTB4 concentrations were found in individuals with CAN versus without CAN. The observation of two distinct subgroups of T1D individuals in the correlation analyses motivated us to evaluate the characteristics of each one of these groups separately. The group presenting higher expression of ALOX5 and of LTB4R also presented higher values of HbA1C, of fructosamine, and of plasmatic LTB4. CONCLUSION: In the diabetes setting, the LT pathway is not only activated by hyperglycemia but is also modulated by the status of the autonomic nervous system.

RAGE Mediates Cholesterol Efflux Impairment in Macrophages Caused by Human Advanced Glycated Albumin.

We addressed the involvement of the receptor for advanced glycation end products (RAGE) in the impairment of the cellular cholesterol efflux elicited by glycated albumin. Albumin was isolated from type 1 (DM1) and type 2 (DM2) diabetes mellitus (HbA1c > 9%) and non-DM subjects (C). Moreover, albumin was glycated in vitro (AGE-albumin). Macrophages from Ager null and wild-type (WT) mice, or THP-1 transfected with siRNA-AGER, were treated with C, DM1, DM2, non-glycated or AGE-albumin. The cholesterol efflux was reduced in WT cells exposed to DM1 or DM2 albumin as compared to C, and the intracellular lipid content was increased. These events were not observed in Ager null cells, in which the cholesterol efflux and lipid staining were, respectively, higher and lower when compared to WT cells. In WT, Ager, Nox4 and Nfkb1, mRNA increased and Scd1 and Abcg1 diminished after treatment with DM1 and DM2 albumin. In Ager null cells treated with DM-albumin, Nox4, Scd1 and Nfkb1 were reduced and Jak2 and Abcg1 increased. In AGER-silenced THP-1, NOX4 and SCD1 mRNA were reduced and JAK2 and ABCG1 were increased even after treatment with AGE or DM-albumin. RAGE mediates the deleterious effects of AGE-albumin in macrophage cholesterol efflux.

Palmitate-induced Slc2a4/GLUT4 downregulation in L6 muscle cells: evidence of inflammatory and endoplasmic reticulum stress involvement.

BACKGROUND: Obesity is strongly associated to insulin resistance, inflammation, and elevated plasma free fatty acids, but the mechanisms behind this association are not fully comprehended. Evidences suggest that endoplasmic reticulum (ER) stress may play a role in this complex pathophysiology. The aim of the present study was to investigate the involvement of inflammation and ER stress in the modulation of glucose transporter GLUT4, encoded by Slc2a4 gene, in L6 skeletal muscle cells. METHODS: L6 cells were acutely (2 h) and chronically (6 and 12 h) exposed to palmitate, and the expression of several proteins involved in insulin resistance, ER stress and inflammation were analyzed. RESULTS: Chronic and acute palmitate exposure significantly reduced GLUT4 protein (~ 39%, P < 0.01) and its mRNA (18%, P < 0.01) expression. Only acute palmitate treatment increased GRP78 (28%, P < 0.05), PERK (98%, P < 0.01), eIF-2A (35%, P < 0.01), IRE1a (60%, P < 0.05) and TRAF2 (23%, P < 0.05) protein content, and PERK phosphorylation (106%, P < 0.001), but did not elicit eIF-2A, IKK phosphorylation or increased XBP1 nuclear content. Additionally, acute and chronic palmitate increased NFKB p65 nuclear content (~ 30%, P < 0.05) and NFKB binding activity to Slc2a4 gene promoter (~ 45%, P < 0.05). CONCLUSION: Different pathways are activated in acute and chronic palmitate induced-repression of Slc2a4/GLUT4 expression. This regulation involves activation of initial component of ER stress, such as the formation of a IRE1a-TRAF2-IKK complex, and converges to NFKB-induced repression of Slc2a4/GLUT4. These results link ER stress, inflammation and insulin resistance in L6 cells.

N-Acetyl Cysteine Attenuated the Deleterious Effects of Advanced Glycation End-Products on the Kidney of Non-Diabetic Rats.

AIM: To assess the renal effects of chronic exposure to advanced glycation end-products (AGEs) in the absence of diabetes and the potential impact of concomitant treatment with the antioxidant N-acetyl cysteine (NAC). METHODS: Wistar rats received intraperitoneally 20 mg/kg/day of albumin modified (AlbAGE) or not (AlbC) by advanced glycation for 12 weeks and oral NAC (600mg/L; AlbAGE+NAC and AlbC+NAC, respectively). Biochemical, urinary and renal morphological analyses; carboxymethyl-lysine (CML, an AGE), CD68 (macrophage infiltration), and 4-hydroxynonenal (4-HNE, marker of oxidative stress) immunostaining; intrarenal mRNA expression of genes belonging to pathways related to AGEs (Ager, Ddost, Nfkb1), renin-angiotensin system (Agt, Ren, Ace), fibrosis (Tgfb1, Col4a1), oxidative stress (Nox4, Txnip), and apoptosis (Bax, Bcl2); and reactive oxidative species (ROS) content were performed. RESULTS: AlbAGE significantly increased urine protein-to-creatinine ratio; glomerular area; renal CML content and macrophage infiltration; expression of Ager, Nfkb1, Agt, Ren, Tgfb1, Col4a1, Txnip, Bax/Bcl2 ratio; and 4-HNE and ROS contents. Some of these effects were attenuated by NAC concomitant treatment. CONCLUSION: Because AGEs are highly consumed in modern diets and implicated in the progression of different kidney diseases, NAC could be a therapeutic intervention to decrease renal damage, considering that long-term restriction of dietary AGEs is difficult to achieve in practice.

New PPARγ partial agonist improves obesity-induced metabolic alterations and atherosclerosis in LDLr(-/-) mice.

Peroxisome proliferator-activated receptor gamma (PPARγ) regulates multiple pathways involved in the pathogenesis of obesity and atherosclerosis. Here, we evaluated the therapeutic potential of GQ-177, a new thiazolidinedione, on diet-induced obesity and atherosclerosis. The intermolecular interaction between PPARγ and GQ-177 was examined by virtual docking and PPAR activation was determined by reporter gene assay identifying GQ-177 as a partial and selective PPARγ agonist. For the evaluation of biological activity of GQ-177, low-density lipoprotein receptor-deficient (LDLr(-/-)) C57/BL6 mice were fed either a high fat diabetogenic diet (diet-induced obesity), or a high fat atherogenic diet, and treated with vehicle, GQ-177 (20mg/kg/day), pioglitazone (20mg/kg/day, diet-induced obesity model) or rosiglitazone (15mg/kg/day, atherosclerosis model) for 28 days. In diet-induced obesity mice, GQ-177 improved insulin sensitivity and lipid profile, increased plasma adiponectin and GLUT4 mRNA in adipose tissue, without affecting body weight, food consumption, fat accumulation and bone density. Moreover, GQ-177 enhanced hepatic mRNA levels of proteins involved in lipid metabolism. In the atherosclerosis mice, GQ-177 inhibited atherosclerotic lesion progression, increased plasma HDL and mRNA levels of PPARγ and ATP-binding cassette A1 in atherosclerotic lesions. GQ-177 acts as a partial PPARγ agonist that improves obesity-associated insulin resistance and dyslipidemia with atheroprotective effects in LDLr(-/-) mice.

In type 2 diabetes mellitus glycated albumin alters macrophage gene expression impairing ABCA1-mediated cholesterol efflux.

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.

Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta.

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.

SEPTIC SHOCK: LPS TOLERANCE PROTECTS MITOCHONDRIAL BIOGENESIS AND RESPIRATION.

ABSTRACT: Mitochondrial dysfunction is a recognized feature of sepsis, characterized by ultrastructural damage, diminished oxidative phosphorylation, and depletion of mitochondrial antioxidant capacity observed in deceased septic patients. LPS tolerance induces a controlled response to sepsis. This study aimed to evaluate the function of tolerant mitochondria after cecal ligation and puncture (CLP)-induced sepsis. Mytochondrial oxygen consumption was determined using polarography. Extraction and quantification of RNA for the expression of Tfam, Nrf-1, and Ppargc-1α, and respiratory complex activity were measured. CLP-tolerant animals presented preserved respiratory rates of S3 and S4 and a ratio of respiratory control (RCR) compared to CLP-nontolerant animals with reduced oxidative phosphorylation and increased uncoupled respiration. Complex I Vmax was reduced in septic animals; however, CLP animals sustained normal Vmax. Mitochondrial biogenesis was preserved in CLP-tolerant animals compared to the CLP-nontolerant group, likely due to increased TFAM expression. LPS tolerance protected septic animals from mitochondrial dysfunction, favoring mitochondrial biogenesis and preserving mitochondrial respiration and respiratory complex I activity.

Proteomic Profiling of HDL in Newly Diagnosed Breast Cancer Based on Tumor Molecular Classification and Clinical Stage of Disease.

The association between high-density lipoprotein (HDL) cholesterol and breast cancer (BC) remains controversial due to the high complexity of the HDL particle and its functionality. The HDL proteome was determined in newly diagnosed BC classified according to the molecular type [luminal A or B (LA or LB), HER2, and triple-negative (TN)] and clinical stage of the disease. Women (n = 141) aged between 18 and 80 years with BC, treatment-naïve, and healthy women [n = 103; control group (CT)], matched by age and body mass index, were included. Data-independent acquisition (DIA) proteomics was performed in isolated HDL (D = 1.063-1.21 g/mL). Results: Paraoxonase1, carnosine dipeptidase1, immunoglobulin mMu heavy chain constant region (IGHM), apoA-4, and transthyretin were reduced, and serum amyloid A2 and tetranectin were higher in BC compared to CT. In TNBC, apoA-1, apoA-2, apoC-2, and apoC-4 were reduced compared to LA, LB, and HER2, and apoA-4 compared to LA and HER2. ComplementC3, lambda immunoglobulin2/3, serpin3, IGHM, complement9, alpha2 lysine rich-glycoprotein1, and complement4B were higher in TNBC in comparison to all other types; complement factor B and vitamin D-binding protein were in contrast to LA and HER2, and plasminogen compared to LA and LB. In grouped stages III + IV, tetranectin and alpha2-macroglobulin were reduced, and haptoglobin-related protein; lecithin cholesterol acyltransferase, serum amyloid A1, and IGHM were increased compared to stages I + II. Conclusions: A differential proteomic profile of HDL in BC based on tumor molecular classification and the clinical stage of the disease may contribute to a better understanding of the association of HDL with BC pathophysiology, treatment, and outcomes.

Advanced glycated albumin impairs HDL anti-inflammatory activity and primes macrophages for inflammatory response that reduces reverse cholesterol transport.

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.