Martine Auclair

Estrogens inhibit copper and cell-mediated modification of low density lipoprotein

The effects of estrogens on LDL modification by copper ions, U 937 monocyte-like cells or endothelial cells was studied by determination of the lipid peroxidation product content and measurement of the relative electrophoretic mobility. The presence of estradiol, estriol and estrone inhibited LDL oxidation in a dose-dependent manner in the range of concentrations from 5 to 50 microM. In the case of oxidation by Cu2+, the decreasing order of efficiency was: estradiol, estriol, estrone. In monocyte-induced oxidation, the protective effect of estrogens was more marked, and the order of efficiency was the same, except that estrone was as active as estriol. Pretreatment of monocyte cells with estrogens also inhibited the subsequent modification of LDL by these cells, tested in the absence of the hormones. Testosterone had no effect in all the studied systems. Furthermore, the degradation by J774 macrophage like cells of LDL modified either by Cu2+ or monocytes was markedly reduced when modification has been performed in the presence of estrogens. Since oxidative modification of LDL is believed to be involved in the appearance of atherosclerotic plaques, this effect of estrogens might be related to their protective action against atherosclerosis.

Perilipin Deficiency and Autosomal Dominant Partial Lipodystrophy

Perilipin is the most abundant adipocyte-specific protein that coats lipid droplets, and it is required for optimal lipid incorporation and release from the droplet. We identified two heterozygous frameshift mutations in the perilipin gene (PLIN1) in three families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes. Subcutaneous fat from the patients was characterized by smaller-than-normal adipocytes, macrophage infiltration, and fibrosis. In contrast to wild-type perilipin, mutant forms of the protein failed to increase triglyceride accumulation when expressed heterologously in preadipocytes. These findings define a novel dominant form of inherited lipodystrophy and highlight the serious metabolic consequences of a primary defect in the formation of lipid droplets in adipose tissue.

The HIV-1 nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro.

Objectives: Nucleoside analogues are suspected of playing a role in peripheral fat loss in patients during long-term treatment with antiretroviral drugs. Design and methods: We compared the long-term effects of stavudine (10 μM), zidovudine (1 μM), didanosine (10 μM), abacavir (4 μM), lamivudine (10 μM), and tenofovir (1 μM), near their maximum concentration values, on the differentiation, lipid accumulation, survival and mitochondrial function of differentiating 3T3-F442A and differentiated 3T3-L1 adipocytes. Results: None of the nucleoside reverse transcriptase inhibitors (NRTI) markedly altered the differentiation of 3T3-F442A cells, as shown by the unmodified percentage of cells with lipid droplets on day 7 and the expression of the early differentiation markers CCAAT/enhancer binding protein (C/EBP) β (on day 2) and sterol regulatory element-binding protein. However, stavudine and zidovudine altered the lipid phenotype, decreasing the lipid content and expression of markers involved in lipid metabolism, namely C/EBPα, peroxisome proliferator-activated receptor γ, adipocyte lipid binding protein 2, fatty acid synthase and acetyl-coenzyme A carboxylase. Stavudine and zidovudine, contrary to the other NRTI, drove 5–10% of 3T3-F442A cells towards apoptosis, and reduced the lipid content and survival of differentiated 3T3-L1 adipocytes. Stavudine and zidovudine increased mitochondrial mass by two to fourfold, and lowered the mitochondrial membrane potential (JC-1 stain) as did zalcitabine (0.2 μM). Co-treatment with zidovudine plus lamivudine, or zidovudine plus lamivudine and abacavir, did not increase the effect of zidovudine on cell viability or apoptosis. Conclusion: The thymidine analogues stavudine and zidovudine decreased lipid content, mitochondrial activity, and adipocyte survival in vitro.

Malondialdehyde modification and copper-induced autooxidation of high-density lipoprotein decrease cholesterol efflux from human cultured fibroblasts

Malondialdehyde modification and copper ion-induced autooxidation of the apo E-free HDL3 fraction of high-density lipoproteins were studied with respect to physico-chemical characteristics and physiological properties of the lipoprotein. Cu(2+)-oxidized HDL was much less modified than MDA-treated HDL, in terms of electrophoretic mobility, lipid peroxidation product content, Lys and Trp amino acid residue level and polymerization of apo A-I. With [3H]cholesteryl linoleate-labeled LDL, an inhibition of cholesterol efflux was observed in the presence of modified HDL, with a more marked effect with MDA-modified HDL. Competition studies with iodinated native HDL demonstrated a decreased binding of modified HDL to cell surface receptors. The decrease in cholesterol intracellular content, determined either by the isotopic equilibrium method or by the enzymatic cholesterol oxidase technic, was less marked in the presence of modified HDL than in the presence of native HDL. MDA-modified HDL was the less effective in decreasing cellular cholesterol content. It is thus suggested that malondialdehyde-induced alteration of HDL, or HDL peroxidation, if occurring in vivo, could contribute to the progress of atherogenesis by decreasing cholesterol efflux from peripheral tissues.

Tumor necrosis factor enhances low density lipoprotein oxidative modification by monocytes and endothelial cells.

The effect of tumor necrosis factor on the oxidative modification of LDL by U937 human monocytes or murine endothelial cells was studied by determination of the lipid peroxidation product content and the electrophoretic mobility of the particle. In the range of concentrations from 2.5 to 10 , the cytokine induced a dose‐dependent increase in cellular‐induced oxidation of LDL. This effect was accompanied by a stimulation of LDL degradation by J774 macrophage‐like cells. Concurrently, the TNF‐treated cells secreted superoxide anion with a higher rate. Since LDL oxidation is believed to be an inportant feature in the formation of the atherosclerotic plaque, the described effects of TNF might be of importance in long‐term exposure to this cytokine during inflammation.

Copper- and malondialdehyde-induced modification of high density lipoprotein and parallel loss of lecithin cholesterol acyltransferase activation

Incubation of high density lipoproteins (HDL) with 0.1-10 microM copper ions resulted in a decrease in tryptophan residues and a moderate diminution of lysine residues. Polymerization of apolipoprotein AI (apo A-I) was only observed for the highest concentration of Cu2+. A dose-dependent loss in lecithin cholesterol acyl-transferase (LCAT) activity was noted. Following incubation with 10 mM malondialdehyde, the physicochemical properties of HDL were more pronouncedly affected, in terms of lipid peroxidation products, relative electrophoretic mobility and percentages of intact tryptophan and lysine residues. Polymerization of apo A-I occurred after 40 min incubation, and a time-dependent loss of LCAT activation was noted. Since the deficiency in LCAT activation was observed in relatively mild conditions, when no perturbation of the physico-chemical properties of the particle could be shown, the determination of LCAT activity appears to be a sensitive test for HDL discrete modification.

Premature Senescence of Vascular Cells Is Induced by HIV Protease Inhibitors. Implication of Prelamin A and Reversion by Statin

Objective—To determine whether and how protease inhibitors (PIs) could affect vascular aging. Methods and Results—HIV therapy with PIs is associated with an increased risk of premature cardiovascular disease. The effect of ritonavir and a combination of lopinavir and ritonavir (for 30 days) on senescence, oxidative stress, and inflammation was evaluated in human coronary artery endothelial cells (HCAECs). These HCAECs were either cotreated or not cotreated with pravastatin or farnesyl transferase inhibitor (FTI)-277 or with 2 antioxidants (manganese [III] tetrakis [4-benzoic acid] porphyrin [MnTBAP] and N-acetyl cysteine). Senescence markers were evaluated in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients under PI treatment. PIs induced senescence markers, prelamin A accumulation, oxidative stress, and inflammation in HCAECs. Senescence markers and prelamin A were also observed in PBMCs from HIV-infected patients under ritonavir-boosted PIs. Pravastatin, FTI-277, and antioxidants improved PI adverse effects in HCAECs. Senescence markers were lower in PBMCs from PI-treated patients cotreated with statins. Conclusion—PIs triggered premature senescence in endothelial cells by a mechanism involving prelamin A accumulation. Accordingly, circulating cells from HIV-infected patients receiving PI therapy expressed senescence markers and prelamin A. Statin was associated with improved senescence in endothelial cells and patient PBMCs. Thus, PIs might promote vascular senescence in HIV-infected patients; and statins might exert beneficial effects in these patients.

HIV protease inhibitors activate the adipocyte renin angiotensin system.

BACKGROUND HIV-infected patients under antiretroviral therapy that includes HIV protease inhibitors (PIs) are prone to develop a complex metabolic syndrome including insulin resistance, lipodystrophy and hypertension. Whether hypertension and cardiovascular events could result from the adipocyte renin angiotensin system (RAS) overactivation has never been investigated. METHODS Primary human adipocytes and 3T3-F442A murine adipocytes were incubated with lopinavir or atazanavir boosted with ritonavir, with or without the angiotensin II type-1 receptor (AT1R) blockers (ARBs), irbesartan or telmisartan, and the peroxysome proliferator-activated receptor-gamma (PPAR-gamma) regulators, rosiglitazone and GW9662. Adipose RAS activation and adipocyte functions were evaluated. RESULTS The ritonavir-boosted PIs activated the adipose RAS in human and murine adipocytes as shown by the overexpression of AT1R protein, angiotensinogen messenger RNA and the amplified effect of angiotensin II on extracellular signal-regulated kinase 1/2 activity. ARBs prevented the PI effect on RAS activation (AT1R overexpression and signalling) and adipocyte functions (dedifferentiation, insulin resistance, oxidative stress and inflammation). Consistent with a role of PPAR-gamma signalling in PI-induced RAS activation, the PPAR-gamma agonist (rosiglitazone) normalized PI-induced AT1R overexpression and adipocyte dysfunction. Conversely, the PPAR-gamma antagonist (GW9662) induced AT1R overexpression and reduced the beneficial effect of telmisartan on PI toxicity. CONCLUSIONS We report that two frequently prescribed PI combinations could activate the adipose RAS in cultured cells, in part through a PPAR-gamma-dependant signalling pathway. Our data suggest a role for the adipose RAS in the development of hypertension in HIV-infected patients under PI treatment, and point out the potential use of ARBs to decrease PI adverse effects.

Effects of ritonavir-boosted darunavir, atazanavir and lopinavir on adipose functions and insulin sensitivity in murine and human adipocytes.

BACKGROUND Ritonavir-boosted protease inhibitors (PIs) could adversely affect metabolism and adipose tissue to different extents, depending on the molecule. Using drugs with minimal adverse metabolic effects is an important consideration in at-risk HIV-infected patients. In vitro adipocyte models can be useful for comparing the effects of different PIs. METHODS We compared the effects of darunavir, darunavir/ritonavir, atazanavir/ritonavir and lopinavir/ritonavir in murine and human adipocytes on differentiation, mitochondrial function, reactive oxygen species (ROS) production and insulin sensitivity. RESULTS In human and murine adipocytes, differentiation evaluated by lipid content and protein expression of adipogenic markers, mitochondrial function evaluated by aggregation of the cationic dye JC-1 and by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide lysis, and mitochondrial mass evaluated by MitoTracker fluorescence and the expression of mitochondrial proteins were unaffected by darunavir, mildly affected by darunavir/ritonavir and further altered by atazanavir/ritonavir and lopinavir/ritonavir. ROS production was unaltered by darunavir and darunavir/ritonavir but was increased by lopinavir/ritonavir and atazanavir/ritonavir. Regarding insulin sensitivity, darunavir and darunavir/ritonavir had no significant effect on insulin activation of protein kinase B (Akt/PKB) and MAP kinase and of glucose transport, whereas lopinavir/ritonavir and atazanavir/ritonavir partly impaired the effect of insulin. The effect of atazanavir/ritonavir was generally milder than that of lopinavir/ritonavir. CONCLUSIONS The various PIs differentially modified adipocyte functions. Darunavir alone did not affect adipocyte functions and only modestly altered differentiation and mitochondrial function when associated with ritonavir. Lopinavir/ritonavir adversely affected differentiation and lipid content, mitochondrial function, ROS production and insulin sensitivity, and the effect of atazanavir/ritonavir was intermediate. Thus, in vitro, darunavir/ritonavir presented a safer metabolic profile on adipocytes than atazanavir/ritonavir and lopinavir/ritonavir.

Peroxisome Proliferator-Activated Receptor-γ Mutations Responsible for Lipodystrophy With Severe Hypertension Activate the Cellular Renin–Angiotensin System

Objective—Inactivating peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) mutations lead to a syndrome of familial partial lipodystrophy (FPLD3) associated with early-onset severe hypertension. PPAR&ggr; can repress the vascular renin–angiotensin system (RAS) and angiotensin II receptor 1 expression. We evaluated the relationships between PPAR&ggr; inactivation and cellular RAS using FPLD3 patients’ cells and human vascular smooth muscle cells expressing mutant or wild-type PPAR&ggr;. Approach and Results—We identified 2 novel PPARG mutations, R165T and L339X, located in the DNA and ligand-binding domains of PPAR&ggr;, respectively in 4 patients from 2 FPLD3 families. In cultured skin fibroblasts and peripheral blood mononuclear cells from the 4 patients and healthy controls, we compared markers of RAS activation, oxidative stress, and inflammation, and tested the effect of modulators of PPAR&ggr; and angiotensin II receptor 1. We studied the impact of the 2 mutations on the transcriptional activity of PPAR&ggr; and on the vascular RAS in transfected human vascular smooth muscle cells. Systemic RAS was not altered in patients. However, RAS markers were overexpressed in patients’ fibroblasts and peripheral blood mononuclear cells, as in vascular cells expressing mutant PPAR&ggr;. Angiotensin II–mediated mitogen-activated protein kinase activity increased in patients’ fibroblasts, consistent with RAS constitutive activation. Patients’ cells also displayed oxidative stress and inflammation. PPAR&ggr; activation and angiotensin II receptor 1 mRNA silencing reversed RAS overactivation, oxidative stress, and inflammation, arguing for a role of angiotensin II receptor 1 in these processes. Conclusions—Two novel FPLD3-linked PPARG mutations are associated with a defective transrepression of cellular RAS leading to cellular dysfunction, which might contribute to the specific FPLD3-linked severe hypertension.