RESUMO
Type 2 diabetes and dyslipidemia are risk factors for cardiovascular disease. However, mechanisms by which hypertriglyceridemia influences atherogenesis remain unclear. We examined effects of dyslipidemic diabetic serum on macrophage lipid accumulation as a model of foam cell formation. Normal human macrophages were cultured in media supplemented with 10% serum from non-diabetic normolipidemic or non-diabetic hypercholesterolemic adults versus adults with Type 2 diabetes; diabetes and hypertriglyceridemia; or diabetes and hypercholesterolemia. Exposure to diabetic sera resulted in increased macrophage fatty acids (2-3 fold higher, both saturated and unsaturated). Macrophage expression of CD36, scavenger receptor A (SR-A) and stearoyl-CoA desaturase (SCD) was increased, most prominently in macrophages exposed to hypertriglyceridemic diabetic serum (twofold increase in CD36 and fourfold increase in SCD, p < 0.05). In these conditions, RNA inhibition of CD36 reduced macrophage free cholesterol (163.9 ± 10.5 vs. 221.9 ± 26.2 mmol free cholesterol/g protein, p = 0.04). RNA inhibition of SCD decreased macrophage fatty acid content, increased ABCA1 level and enhanced cholesterol efflux (18.0 ± 3.9 vs. 8.0 ± 0.8% at 48 h, p = 0.03). Diabetic dyslipidemia may contribute to accelerated atherosclerosis via alterations in macrophage lipid metabolism favoring foam cell formation. Increased expression of CD36 and SR-A would facilitate macrophage lipid uptake, while increased expression of SCD could block compensatory upregulation of ABCA1 and cholesterol efflux. Further studies are needed to clarify whether modulation of macrophage lipid metabolism might reduce progression of diabetic atherosclerosis.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Dislipidemias/sangue , Metabolismo dos Lipídeos , Macrófagos/enzimologia , Estearoil-CoA Dessaturase/metabolismo , Adulto , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Dislipidemias/complicações , Ácidos Graxos/metabolismo , Células Espumosas/metabolismo , Humanos , RNA Interferente Pequeno/genética , Receptores Depuradores Classe A/metabolismo , Estearoil-CoA Dessaturase/genéticaRESUMO
Macrophages in arterial walls accumulate lipids leading to the development of atherosclerotic plaques. However, mechanisms underlying macrophage lipid accumulation and foam cell formation are often studied without accounting for risk factors such as dyslipidemia. We investigated the effect of varying concentrations of triglyceride (TG) within physiological range on macrophage fatty acid (FA) accumulation and expression of cholesterol efflux proteins. Human monocytes were cultured in media supplemented with 10% sera containing low (0.7 mmol/L) to high (1.4 mmol/L) TG. The resulting macrophages were harvested after 10 days for analysis of FA content and composition and expression of genes involved in lipid metabolism. Exposure to higher TG and lower HDL concentrations in media increased macrophage lipid content. Macrophages exposed to higher TG had increased total FA content compared with controls (876 µg/mg protein vs. 652 µg/mg protein) and greater proportions of C16:0, C18:1 and C18:2. Macrophage expression of both ABCA1 and ABCG1 cholesterol efflux proteins were reduced when higher TG concentrations were present in the media. Expression of scavenger receptor CD36, involved in lipoprotein uptake, was also downregulated in macrophages exposed to higher TG. Culturing macrophages in conditions of higher versus lower TG influenced macrophage FA content and composition, and levels of regulatory proteins. Replicating in vitro levels of dyslipidemia encountered in vivo may provide an informative model for investigation of atherogenesis.
Assuntos
Dislipidemias/sangue , Ácidos Graxos/metabolismo , Macrófagos/química , Macrófagos/metabolismo , Soro/química , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células Cultivadas , Meios de Cultura/química , Ácidos Graxos/química , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/farmacologiaRESUMO
Lipid rafts are enriched in cholesterol and sphingomyelin and are isolated on the basis of insolubility in detergents, such as Brij 98 and Triton X-100. Recent work by Holm et al. has shown that rafts insoluble in Brig 98 can be found in human immunodeficiency virus type 1 (HIV-1) virus-like particles, although it is not known whether raft-like structures are present in authentic HIV-1 and it is unclear whether a virion-associated raft-like structure is required for HIV replication. Independently, it was previously reported that virion-associated cholesterol is critical for HIV-1 infectivity, although the specific requirement of virion cholesterol in HIV-1 was not examined. In the present study, we have demonstrated that infectious wild-type HIV-1 contains Brij 98 rafts but only minimal amounts of Triton X-100 rafts. To directly assess the functional requirement of virion-associated rafts and various features of cholesterol on HIV-1 replication, we replaced virion cholesterol with exogenous cholesterol analogues that have demonstrated either raft-promoting or -inhibiting capacity in model membranes. We observed that variable concentrations of exogenous analogues are required to replace a defined amount of virion-associated cholesterol, showing that structurally diverse cholesterol analogues have various affinities toward HIV-1. We found that replacement of 50% of virion cholesterol with these exogenous cholesterol analogues did not eliminate the presence of Brij 98 rafts in HIV-1. However, the infectivity levels of the lipid-modified HIV-1s directly correlate with the raft-promoting capacities of these cholesterol analogues. Our data provide the first direct assessment of virion-associated Brij 98 rafts in retroviral replication and illustrate the importance of the raft-promoting property of virion-associated cholesterol in HIV-1 replication.
Assuntos
Colesterol/fisiologia , HIV-1/química , HIV-1/fisiologia , Lipídeos de Membrana/fisiologia , Microdomínios da Membrana/fisiologia , Vírion/química , Centrifugação com Gradiente de Concentração , Humanos , Immunoblotting , Lipídeos/análise , Microdomínios da Membrana/química , Octoxinol , Óleos de Plantas , Polietilenoglicóis , Proteínas Virais/análise , Replicação ViralRESUMO
OBJECTIVES: This study investigated the effects of androgens on gene expression in male- and female-donor macrophages. BACKGROUND: Men have more severe coronary disease than women. Androgen exposure increases foam cell formation in male but not female macrophages, and male macrophages express >4-fold more androgen receptor messenger ribonucleic acid than female macrophages. Therefore, androgen exposure may have gender-specific and potentially pro-atherogenic effects in macrophages. METHODS: Utilizing complementary deoxyribonucleic acid arrays, we studied the effects of a pure androgen (dihydrotestosterone, 40 nmol/l) on human monocyte-derived macrophages from healthy male and female donors (n = 4 hybridizations; 2 men, 2 women). Differential expression of atherosclerosis-related genes was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) in five male and five female donors. Functional corroboration of foam cell formation-related findings was undertaken by experiments using (125)I-acetylated low-density lipoprotein (AcLDL). RESULTS: In male macrophages, androgen treatment produced differential up-regulation of 27 genes concentrated in five functional classes: 1) lipoprotein processing; 2) cell-surface adhesion; 3) extracellular signaling; 4) coagulation and fibrinolysis; and 5) transport protein genes. By contrast, none of 588 genes were up-regulated in female macrophages. By RT-PCR, we confirmed the gender-specific up-regulation of six of these atherosclerosis-related genes: acyl coenzyme A:cholesterol acyl transferase I, lysosomal acid lipase (LAL), caveolin-2, CD40, vascular endothelial growth factor-165 receptor, and tissue factor pathway inhibitor. Functionally, androgen-treated male macrophages showed increased rates of lysosomal AcLDL degradation, by 45% to 75% after 15 to 20 h of (125)I-AcLDL incubation (p = 0.001), consistent with increased LAL activity. CONCLUSIONS: Androgens increase expression of atherosclerosis-related genes in male but not female macrophages, with functional consequences. These findings may contribute to the male predisposition to atherosclerosis.