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2.
Proc Natl Acad Sci U S A ; 114(11): 2958-2963, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28246329

RESUMO

How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin ß2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.


Assuntos
Membrana Basal Glomerular/metabolismo , Túbulos Renais/metabolismo , Substâncias Macromoleculares/metabolismo , Animais , Feminino , Membrana Basal Glomerular/ultraestrutura , Ouro , Humanos , Lactente , Recém-Nascido , Túbulos Renais/ultraestrutura , Túbulos Renais Proximais/metabolismo , Masculino , Nanopartículas Metálicas , Camundongos , Microscopia Confocal , Permeabilidade , Podócitos/metabolismo
3.
Diabetes ; 60(9): 2285-94, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21810592

RESUMO

OBJECTIVE: We investigated the differential roles of apolipoprotein E (apoE) isoforms in modulating diabetic dyslipidemia-a potential cause of the increased cardiovascular disease risk of patients with diabetes. RESEARCH DESIGN AND METHODS: Diabetes was induced using streptozotocin (STZ) in human apoE3 (E3) or human apoE4 (E4) mice deficient in the LDL receptor (LDLR(-/-)). RESULTS: Diabetic E3LDLR(-/-) and E4LDLR(-/-) mice have indistinguishable levels of plasma glucose and insulin. Despite this, diabetes increased VLDL triglycerides and LDL cholesterol in E4LDLR(-/-) mice twice as much as in E3LDLR(-/-) mice. Diabetic E4LDLR(-/-) mice had similar lipoprotein fractional catabolic rates compared with diabetic E3LDLR(-/-) mice but had larger hepatic fat stores and increased VLDL secretion. Diabetic E4LDLR(-/-) mice demonstrated a decreased reliance on lipid as an energy source based on indirect calorimetry. Lower phosphorylated acetyl-CoA carboxylase content and higher gene expression of fatty acid synthase in the liver indicated reduced fatty acid oxidation and increased fatty acid synthesis. E4LDLR(-/-) primary hepatocytes cultured in high glucose accumulated more intracellular lipid than E3LDLR(-/-) hepatocytes concomitant with a 60% reduction in fatty acid oxidation. Finally, the exaggerated dyslipidemia in diabetic E4LDLR(-/-) mice was accompanied by a dramatic increase in atherosclerosis. CONCLUSIONS: ApoE4 causes severe dyslipidemia and atherosclerosis independent of its interaction with LDLR in a model of STZ-induced diabetes. ApoE4-expressing livers have reduced fatty acid oxidation, which contributes to the accumulation of tissue and plasma lipids.


Assuntos
Apolipoproteína E4/metabolismo , Aterosclerose/metabolismo , Diabetes Mellitus Experimental/metabolismo , Dislipidemias/metabolismo , Receptores de LDL/metabolismo , Animais , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/genética , Aterosclerose/etiologia , Aterosclerose/genética , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Dislipidemias/etiologia , Dislipidemias/genética , Ácidos Graxos/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Receptores de LDL/genética
4.
FASEB J ; 24(10): 3809-18, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20501792

RESUMO

Thiazolidinediones (TZDs) are insulin sensitizers used to treat patients with insulin resistance. To assess potential gene-drug interactions, mice expressing human apolipoprotein E3 or E4 (APOE3 or APOE4) were fed a Western-type high-fat diet for 12 wk, at which time they developed similarly impaired glucose tolerance. Supplementing the diet with rosiglitazone (1.5 mg/g body weight) for an additional 4 wk improved plasma lipid profiles in both APOE3 and APOE4 mice. However, glucose tolerance improved only in APOE3 mice. Induction of adipogenesis and lipogenesis was severely blunted in adipose tissues, but not in the livers, of APOE4 mice. Consequently, lipids were channeled to the liver, causing marked steatosis in these mice. Impaired glucose tolerance was not a prerequisite for this adverse effect, and long-term treatment with rosiglitazone altered liver enzymes and caused hepatic fibrosis in APOE4 mice. Finally, TZDs failed to stimulate PPARγ activation and adipocyte differentiation in preadipocytes and embryonic fibroblasts isolated from APOE4 mice compared to those from APOE3 mice. We conclude that the effects of TZDs are APOE isoform dependent, and that the metabolic damage observed in APOE4 mice is rooted in an impaired activation of the adipogenic program in the adipose tissues expressing APOE4.


Assuntos
Tecido Adiposo/efeitos dos fármacos , Apolipoproteína E4/genética , Tiazolidinedionas/farmacologia , Tecido Adiposo/citologia , Animais , Peso Corporal/efeitos dos fármacos , Diferenciação Celular , Fígado Gorduroso/prevenção & controle , Humanos , Masculino , Camundongos , PPAR gama/metabolismo
5.
J Lipid Res ; 50 Suppl: S178-82, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19060252

RESUMO

Given the multiple differences between mice and men, it was once thought that mice could not be used to model atherosclerosis, principally a human disease. Apolipoprotein E-deficient (apoEKO) mice have convincingly changed this view, and the ability to model human-like plaques in these mice has provided scientists a platform to study multiple facets of atherogenesis and to explore potential therapeutic interventions. In addition to its well-established role in lipoprotein metabolism, recent observations of reduced adiposity and improved glucose homeostasis in apoEKO mice suggest that apoE may also play a key role in energy metabolism in peripheral organs, including adipose tissue. Finally, along with apoEKO mice, knockin mice expressing human apoE isoforms in place of endogenous mouse apoE have provided insights into how quantitative and qualitative genetic alterations interact with the environment in the pathogenesis of complex human diseases.


Assuntos
Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Aterosclerose/metabolismo , Síndrome Metabólica/metabolismo , Adipócitos/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Humanos , Síndrome Metabólica/genética , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
6.
Arterioscler Thromb Vasc Biol ; 28(10): 1745-52, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18617647

RESUMO

OBJECTIVE: To examine the effects of apoB100 structure, specifically a mutation in the LDLr binding region, on the production of LDL and development of atherosclerosis in vivo. METHODS AND RESULTS: Ldlr(-/-)Apobec1(-/-) mice lacking the LDLR and apoB editing enzyme accumulated LDL in plasma and developed severe atherosclerosis when they had wild-type apoB100. In marked contrast, in Ldlr(-/-)Apobec1(-/-) mice carrying the Apob100-beta mutation, in the 2 putative LDLR-binding domains of apoB prevented both LDL accumulation and atherosclerosis. Intestinal absorption of lipids and triglyceride secretion from the liver were not affected. However, the VLDL particles with apoB100-beta were larger in volume by about 70%, and carried approximately four times as much apoE per particle. ApoB100-beta synthesis rate in the primary hepatocytes was normal, but its intracellular degradation was enhanced. Additionally, mutant apoB100 VLDL cleared from the circulation more quickly in vivo through apoE-LRP-mediated mechanism than VLDL with wild-type apoB100. In contrast, uptake of the 2 VLDL by macrophages were not different. CONCLUSIONS: While conformational change to apoB100 during conversion of VLDL to LDL exposes LDLR binding domains and facilitates LDLR-mediated lipoprotein clearance, it may also inhibit LRP-mediated VLDL uptake and contribute to LDL accumulation in familial hypercholesterolemia.


Assuntos
Apolipoproteína B-100/metabolismo , Aterosclerose/prevenção & controle , Citidina Desaminase/metabolismo , Hiperlipidemias/prevenção & controle , Receptores de LDL/metabolismo , Desaminase APOBEC-1 , Animais , Apolipoproteína B-100/química , Apolipoproteína B-100/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Sítios de Ligação , Citidina Desaminase/deficiência , Citidina Desaminase/genética , Gorduras na Dieta , Modelos Animais de Doenças , Hepatócitos/metabolismo , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Tamanho da Partícula , Ligação Proteica , Conformação Proteica , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo
7.
J Lipid Res ; 48(2): 366-72, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17079794

RESUMO

Factors that regulate apolipoprotein E (apoE) secretion by macrophages will have important effects on vessel wall lipid flux and atherosclerosis. Macrophages express the LDL receptor, which binds apoE with high affinity and could thereby affect the net secretion of apoE from macrophages. In these studies, we demonstrate that treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA with simvastatin, to increase LDL receptor activity, reduces the secretion of apoE. To further examine the relationship between LDL receptor expression and apoE secretion from macrophages, mouse peritoneal macrophages (MPMs) were isolated from mice with constitutively high expression of human LDL receptor to increase overall LDL receptor expression by 2- to 3-fold. Cells with increased LDL receptor expression also showed reduced apoE secretion compared with MPMs with basal LDL receptor expression. The effect of changes in LDL receptor expression on apoE secretion was isoform-specific, with greater reduction of apoE4 compared with apoE3 secretion and no reduction of apoE2 secretion, paralleling the known affinity of each isoform for LDL receptor binding. The effect of the LDL receptor on apoE secretion for each isoform was further reflected in LDL receptor-dependent changes in apoE-mediated cholesterol efflux. These results establish a regulatory interaction between two branches of macrophage sterol homeostatic pathways that could facilitate a rapid response to changes in macrophage sterol content relative to need.


Assuntos
Apolipoproteínas E/metabolismo , Macrófagos/metabolismo , Receptores de LDL/metabolismo , Esteróis/metabolismo , Animais , Apolipoproteína E2/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Regulação da Expressão Gênica , Camundongos , Isoformas de Proteínas , Sinvastatina/farmacologia , Especificidade por Substrato
8.
Arterioscler Thromb Vasc Biol ; 24(1): 91-7, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12969990

RESUMO

OBJECTIVE: Increased expression of the low-density lipoprotein receptor (LDLR) is generally considered beneficial for reducing plasma cholesterol and atherosclerosis, and its downregulation has been thought to explain the association between apolipoprotein (apo) E4 and increased risk of coronary heart disease in humans. METHODS AND RESULTS: Contrary to this hypothesis, doubling Ldlr expression caused severe atherosclerosis with marked accumulation of cholesterol-rich, apoE-poor remnants in mice with human apoE4, but not apoE3, when the animals were fed a Western-type diet. The increased Ldlr expression enhanced in vivo clearance of exogenously introduced remnants in mice with apoE4 only when the remnants were already enriched with apoE4. The rates of nascent lipoprotein production were the same. The adverse effects of increased LDLR suggest a possibility that the receptor can trap apoE4, reducing its availability for the transfer to nascent lipoproteins needed for their rapid clearance, thereby increasing the production of apoE-poor remnants that are slowly cleared. The lower affinity for the LDLR of apoE3 compared with apoE4 could then explain why increased receptor expression had no adverse effects with apoE3. CONCLUSIONS: Our results emphasize the occurrence of important and unexpected interactions between APOE genotype, LDLR expression, and diet.


Assuntos
Apolipoproteínas E/fisiologia , Arteriosclerose/genética , Hipercolesterolemia/genética , Receptores de LDL/fisiologia , Animais , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/genética , Arteriosclerose/sangue , Cruzamentos Genéticos , Dieta Aterogênica , Gorduras na Dieta/efeitos adversos , Feminino , Expressão Gênica , Marcação de Genes , Humanos , Hipercolesterolemia/sangue , Lipoproteínas VLDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/fisiologia , Especificidade da Espécie , Transfecção
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