RESUMEN
BACKGROUND: Individuals with type 1 diabetes (T1D) generally have normal or even higher HDL (high-density lipoprotein)-cholesterol levels than people without diabetes yet are at increased risk for atherosclerotic cardiovascular disease (CVD). Human HDL is a complex mixture of particles that can vary in cholesterol content by >2-fold. To investigate if specific HDL subspecies contribute to the increased atherosclerosis associated with T1D, we created mouse models of T1D that exhibit human-like HDL subspecies. We also measured HDL subspecies and their association with incident CVD in a cohort of people with T1D. METHODS: We generated LDL receptor-deficient (Ldlr-/-) mouse models of T1D expressing human APOA1 (apolipoprotein A1). Ldlr-/-APOA1Tg mice exhibited the main human HDL subspecies. We also generated Ldlr-/-APOA1Tg T1D mice expressing CETP (cholesteryl ester transfer protein), which had lower concentrations of large HDL subspecies versus mice not expressing CETP. HDL particle concentrations and sizes and proteins involved in lipoprotein metabolism were measured by calibrated differential ion mobility analysis and targeted mass spectrometry in the mouse models of T1D and in a cohort of individuals with T1D. Endothelial transcytosis was analyzed by total internal reflection fluorescence microscopy. RESULTS: Diabetic Ldlr-/-APOA1Tg mice were severely hyperglycemic and hyperlipidemic and had markedly elevated plasma APOB levels versus nondiabetic littermates but were protected from the proatherogenic effects of diabetes. Diabetic Ldlr-/-APOA1Tg mice expressing CETP lost the atheroprotective effect and had increased lesion necrotic core areas and APOB accumulation, despite having lower plasma APOB levels. The detrimental effects of low concentrations of larger HDL particles in diabetic mice expressing CETP were not explained by reduced cholesterol efflux. Instead, large HDL was more effective than small HDL in preventing endothelial transcytosis of LDL mediated by scavenger receptor class B type 1. Finally, in humans with T1D, increased concentrations of larger HDL particles relative to APOB100 negatively predicted incident CVD independently of HDL-cholesterol levels. CONCLUSIONS: Our results suggest that the balance between APOB lipoproteins and the larger HDL subspecies contributes to atherosclerosis progression and incident CVD in the setting of T1D and that larger HDLs exert atheroprotective effects on endothelial cells rather than by promoting macrophage cholesterol efflux.
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Apolipoproteína A-I , Aterosclerosis , Diabetes Mellitus Tipo 1 , Receptores de LDL , Adulto , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Apolipoproteína A-I/sangre , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/sangre , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/sangre , Aterosclerosis/patología , Proteínas de Transferencia de Ésteres de Colesterol/genética , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol/sangre , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/sangre , Modelos Animales de Enfermedad , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de LDL/genética , Receptores de LDL/deficiencia , Receptores de LDL/metabolismoRESUMEN
BACKGROUND: Cholesterol efflux capacity (CEC) predicts cardiovascular disease independently of high-density lipoprotein (HDL) cholesterol levels. Isolated small HDL particles are potent promoters of macrophage CEC by the ABCA1 (ATP-binding cassette transporter A1) pathway, but the underlying mechanisms are unclear. METHODS: We used model system studies of reconstituted HDL and plasma from control and lecithin-cholesterol acyltransferase (LCAT)-deficient subjects to investigate the relationships among the sizes of HDL particles, the structure of APOA1 (apolipoprotein A1) in the different particles, and the CECs of plasma and isolated HDLs. RESULTS: We quantified macrophage and ABCA1 CEC of 4 distinct sizes of reconstituted HDL. CEC increased as particle size decreased. Tandem mass spectrometric analysis of chemically cross-linked peptides and molecular dynamics simulations of APOA1, the major protein of HDL, indicated that the mobility of C-terminus of that protein was markedly higher and flipped off the surface in the smallest particles. To explore the physiological relevance of the model system studies, we isolated HDL from LCAT-deficient subjects, whose small HDLs (like reconstituted HDLs) are discoidal and composed of APOA1, cholesterol, and phospholipid. Despite their very low plasma levels of HDL particles, these subjects had normal CEC. In both the LCAT-deficient subjects and control subjects, the CEC of isolated extra-small HDL (a mixture of extra-small and small HDL by calibrated ion mobility analysis) was 3- to 5-fold greater than that of the larger sizes of isolated HDL. Incubating LCAT-deficient plasma and control plasma with human LCAT converted extra-small and small HDL particles into larger particles, and it markedly inhibited CEC. CONCLUSIONS: We present a mechanism for the enhanced CEC of small HDLs. In smaller particles, the C-termini of the 2 antiparallel molecules of APOA1 are "flipped" off the lipid surface of HDL. This extended conformation allows them to engage with ABCA1. In contrast, the C-termini of larger HDLs are unable to interact productively with ABCA1 because they form a helical bundle that strongly adheres to the lipid on the particle. Enhanced CEC, as seen with the smaller particles, predicts decreased cardiovascular disease risk. Thus, extra-small and small HDLs may be key mediators and indicators of the cardioprotective effects of HDL.
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Apolipoproteína A-I , Enfermedades Cardiovasculares , Humanos , Apolipoproteína A-I/metabolismo , Enfermedades Cardiovasculares/metabolismo , Lipoproteínas HDL/metabolismo , Colesterol , Transportador 1 de Casete de Unión a ATP/genética , Transportador 1 de Casete de Unión a ATP/metabolismo , Macrófagos/metabolismo , HDL-ColesterolRESUMEN
PURPOSE OF REVIEW: Lipid droplets are increasingly recognized as distinct intracellular organelles that have functions exclusive to the storage of energetic lipids. Lipid droplets modulate macrophage inflammatory phenotype, control the availability of energy for muscle function, store excess lipid, sequester toxic lipids, modulate mitochondrial activity, and allow transfer of fatty acids between tissues. RECENT FINDINGS: There have been several major advances in our understanding of the formation, dissolution, and function of this organelle during the past two years. These include new information on movement and partition of amphipathic proteins between the cytosol and lipid droplet surface, molecular determinants of lipid droplet formation, and pathways leading to lipid droplet hydrophobic lipid formation. Rapid advances in mitochondrial biology have also begun to define differences in their function and partnering with lipid droplets to modulate lipid storage versus oxidation. SUMMARY: This relationship of lipid droplets biology and cellular function provides new understanding of an important cellular organelle that influences muscle function, adipose lipid storage, and diseases of lipotoxicity.
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Lipasa , Gotas Lipídicas , Humanos , Ácidos Grasos , Macrófagos , MitocondriasRESUMEN
Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3-/-) whereupon we observed â¼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3-/- cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3-/- cells; rather ANGPTL3-/- cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3-/- cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3-/-;LDLR-/- cells rescued the deficient LDL clearance of LDLR-/- cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.
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Proteína 3 Similar a la Angiopoyetina , Metabolismo de los Lípidos , Proteínas Similares a la Angiopoyetina/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Metabolismo de los Lípidos/genética , Lipoproteínas/metabolismo , Hígado/metabolismo , Triglicéridos/metabolismoRESUMEN
The positive relationship between increased levels of circulating triglycerides and cardiovascular events has been observed for decades. Driven by genetic cohort studies, inhibitors of APOC3 (apolipoprotein C3) and ANGPTL (angiopoietin-like protein) 3 that reduce circulating triglycerides are poised to enter clinical practice. We will review the biology of how inhibition of these 2 proteins affects circulating lipoproteins as well as the current state of clinical development of monoclonal antibodies, antisense oligonucleotides, and silencing RNAs targeting APOC3 and ANGPTL3.
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Proteína 3 Similar a la Angiopoyetina , Dislipidemias , Humanos , Proteínas Similares a la Angiopoyetina/genética , Apolipoproteína C-III , Triglicéridos/metabolismo , Dislipidemias/tratamiento farmacológicoRESUMEN
During obesity, tissue macrophages increase in number and become proinflammatory, thereby contributing to metabolic dysfunction. Lipoprotein lipase (LPL), which hydrolyzes triglyceride in lipoproteins, is secreted by macrophages. However, the role of macrophage-derived LPL in adipose tissue remodeling and lipoprotein metabolism is largely unknown. To clarify these issues, we crossed leptin-deficient Lepob/ob mice with mice lacking the Lpl gene in myeloid cells (Lplm-/m-) to generate Lplm-/m-;Lepob/ob mice. We found the weight of perigonadal white adipose tissue (WAT) was increased in Lplm-/m-;Lepob/ob mice compared with Lepob/ob mice due to substantial accumulation of both adipose tissue macrophages and collagen that surrounded necrotic adipocytes. In the fibrotic epidydimal WAT of Lplm-/m-;Lepob/ob mice, we observed an increase in collagen VI and high mobility group box 1, while α-smooth muscle cell actin, a marker of myofibroblasts, was almost undetectable, suggesting that the adipocytes were the major source of the collagens. Furthermore, the adipose tissue macrophages from Lplm-/m-;Lepob/ob mice showed increased expression of genes related to fibrosis and inflammation. In addition, we determined Lplm-/m-;Lepob/ob mice were more hypertriglyceridemic than Lepob/ob mice. Lplm-/m-;Lepob/ob mice also showed slower weight gain than Lepob/ob mice, which was primarily due to reduced food intake. In conclusion, we discovered that the loss of myeloid Lpl led to extensive fibrosis of perigonadal WAT and hypertriglyceridemia. In addition to illustrating an important role of macrophage LPL in regulation of circulating triglyceride levels, these data show that macrophage LPL protects against fibrosis in obese adipose tissues.
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Tejido Adiposo Blanco , Colágeno Tipo IV , Hipertrigliceridemia , Lipoproteína Lipasa , Obesidad , Actinas/metabolismo , Tejido Adiposo Blanco/patología , Animales , Colágeno Tipo IV/metabolismo , Fibrosis , Hipertrigliceridemia/genética , Hipertrigliceridemia/patología , Leptina/deficiencia , Leptina/genética , Lipoproteína Lipasa/genética , Lipoproteínas/metabolismo , Ratones , Ratones Obesos , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Triglicéridos/sangreRESUMEN
Lipid uptake and metabolism are central to the function of organs such as heart, skeletal muscle, and adipose tissue. Although most heart energy derives from fatty acids (FAs), excess lipid accumulation can cause cardiomyopathy. Similarly, high delivery of cholesterol can initiate coronary artery atherosclerosis. Hearts and arteries-unlike liver and adrenals-have nonfenestrated capillaries and lipid accumulation in both health and disease requires lipid movement from the circulation across the endothelial barrier. This review summarizes recent in vitro and in vivo findings on the importance of endothelial cell receptors and uptake pathways in regulating FAs and cholesterol uptake in normal physiology and cardiovascular disease. We highlight clinical and experimental data on the roles of ECs in lipid supply to tissues, heart, and arterial wall in particular, and how this affects organ metabolism and function. Models of FA uptake into ECs suggest that receptor-mediated uptake predominates at low FA concentrations, such as during fasting, whereas FA uptake during lipolysis of chylomicrons may involve paracellular movement. Similarly, in the setting of an intact arterial endothelial layer, recent and historic data support a role for receptor-mediated processes in the movement of lipoproteins into the subarterial space. We conclude with thoughts on the need to better understand endothelial lipid transfer for fuller comprehension of the pathophysiology of hyperlipidemia, and lipotoxic diseases such as some forms of cardiomyopathy and atherosclerosis.
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Colesterol/metabolismo , Células Endoteliales/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Trastornos del Metabolismo de los Lípidos/metabolismo , Transcitosis , Animales , Antígenos CD36/metabolismo , Quilomicrones/metabolismo , Humanos , Trastornos del Metabolismo de los Lípidos/patología , Lipólisis , Tamaño de la PartículaRESUMEN
[Figure: see text].
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Aterosclerosis/metabolismo , HDL-Colesterol/metabolismo , Hipertrigliceridemia/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Células Cultivadas , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Humanos , Hipertrigliceridemia/genética , Lipoproteína Lipasa/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.
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Cardiomiopatías Diabéticas/metabolismo , Proteína Forkhead Box O1/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , PPAR alfa/metabolismo , Anciano , Animales , Línea Celular , Cardiomiopatías Diabéticas/genética , Cardiomiopatías Diabéticas/patología , Modelos Animales de Enfermedad , Femenino , Proteína Forkhead Box O1/genética , Regulación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Miocitos Cardíacos/patología , PPAR alfa/genética , Transcripción GenéticaRESUMEN
Lipid accumulation in nonadipose tissues can cause lipotoxicity, leading to cell death and severe organ dysfunction. Adipose triglyceride lipase (ATGL) deficiency causes human neutral lipid storage disease and leads to cardiomyopathy; ATGL deficiency has no current treatment. One possible approach to alleviate this disorder has been to alter the diet and reduce the supply of dietary lipids and, hence, myocardial lipid uptake. However, in this study, when we supplied cardiac Atgl KO mice a low- or high-fat diet, we found that heart lipid accumulation, heart dysfunction, and death were not altered. We next deleted lipid uptake pathways in the ATGL-deficient mice through the generation of double KO mice also deficient in either cardiac lipoprotein lipase or cluster of differentiation 36, which is involved in an lipoprotein lipase-independent pathway for FA uptake in the heart. We show that neither deletion ameliorated ATGL-deficient heart dysfunction. Similarly, we determined that non-lipid-containing media did not prevent lipid accumulation by cultured myocytes; rather, the cells switched to increased de novo FA synthesis. Thus, we conclude that pathological storage of lipids in ATGL deficiency cannot be corrected by reducing heart lipid uptake.
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Aciltransferasas , Cardiomiopatías , Lipoproteína Lipasa , Animales , Humanos , Ratones , Tejido Adiposo/metabolismo , Cardiomiopatías/metabolismo , Lipasa/metabolismo , Lipoproteína Lipasa/genética , Lipoproteína Lipasa/metabolismo , Ratones Noqueados , Miocardio/metabolismo , Triglicéridos/metabolismo , Aciltransferasas/deficiencia , Aciltransferasas/genéticaRESUMEN
The LDL receptor-related protein 1 (LRP1) partakes in metabolic and signaling events regulated in a tissue-specific manner. The function of LRP1 in airways has not been studied. We aimed to study the function of LRP1 in smoke-induced disease. We found that bronchial epithelium of patients with chronic obstructive pulmonary disease and airway epithelium of mice exposed to smoke had increased LRP1 expression. We then knocked out LRP1 in human bronchial epithelial cells in vitro and in airway epithelial club cells in mice. In vitro, LRP1 knockdown decreased cell migration and increased transforming growth factor ß activation. Tamoxifen-inducible airway-specific LRP1 knockout mice (club Lrp1-/-) induced after complete lung development had increased inflammation in the bronchoalveolar space and lung parenchyma at baseline. After 6 months of smoke exposure, club Lrp1-/- mice showed a combined restrictive and obstructive phenotype, with lower compliance, inspiratory capacity, and forced expiratory volume0.05/forced vital capacity than WT smoke-exposed mice. This was associated with increased values of Ashcroft fibrotic index. Proteomic analysis of room air exposed-club Lrp1-/- mice showed significantly decreased levels of proteins involved in cytoskeleton signaling and xenobiotic detoxification as well as decreased levels of glutathione. The proteome fingerprint created by smoke eclipsed many of the original differences, but club Lrp1-/- mice continued to have decreased lung glutathione levels and increased protein oxidative damage and airway cell proliferation. Therefore, LRP1 deficiency leads to greater lung inflammation and damage and exacerbates smoke-induced lung disease.
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Remodelación de las Vías Aéreas (Respiratorias) , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Estrés Oxidativo , Humo , Animales , Epitelio/metabolismo , Glutatión/metabolismo , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Pulmón/metabolismo , Ratones , Proteómica , Humo/efectos adversosRESUMEN
BACKGROUND: We previously showed that cardiomyocyte KrÏppel-like factor (KLF) 5 regulates cardiac fatty acid oxidation. As heart failure has been associated with altered fatty acid oxidation, we investigated the role of cardiomyocyte KLF5 in lipid metabolism and pathophysiology of ischemic heart failure. METHODS: Using real-time polymerase chain reaction and Western blot, we investigated the KLF5 expression changes in a myocardial infarction (MI) mouse model and heart tissue from patients with ischemic heart failure. Using 2D echocardiography, we evaluated the effect of KLF5 inhibition after MI using pharmacological KLF5 inhibitor ML264 and mice with cardiomyocyte-specific KLF5 deletion (αMHC [α-myosin heavy chain]-KLF5-/-). We identified the involvement of KLF5 in regulating lipid metabolism and ceramide accumulation after MI using liquid chromatography-tandem mass spectrometry, and Western blot and real-time polymerase chain reaction analysis of ceramide metabolism-related genes. We lastly evaluated the effect of cardiomyocyte-specific KLF5 overexpression (αMHC-rtTA [reverse tetracycline-controlled transactivator]-KLF5) on cardiac function and ceramide metabolism, and rescued the phenotype using myriocin to inhibit ceramide biosynthesis. RESULTS: KLF5 mRNA and protein levels were higher in human ischemic heart failure samples and in rodent models at 24 hours, 2 weeks, and 4 weeks post-permanent left coronary artery ligation. αMHC-KLF5-/- mice and mice treated with ML264 had higher ejection fraction and lower ventricular volume and heart weight after MI. Lipidomic analysis showed that αMHC-KLF5-/- mice with MI had lower myocardial ceramide levels compared with littermate control mice with MI, although basal ceramide content of αMHC-KLF5-/- mice was not different in control mice. KLF5 ablation suppressed the expression of SPTLC1 and SPTLC2 (serine palmitoyltransferase [SPT] long-chain base subunit ()1 2, respectively), which regulate de novo ceramide biosynthesis. We confirmed our previous findings that myocardial SPTLC1 and SPTLC2 levels are increased in heart failure patients. Consistently, αMHC-rtTA-KLF5 mice showed increased SPTLC1 and SPTLC2 expression, higher myocardial ceramide levels, and systolic dysfunction beginning 2 weeks after KLF5 induction. Treatment of αMHC-rtTA-KLF5 mice with myriocin that inhibits SPT, suppressed myocardial ceramide levels and alleviated systolic dysfunction. CONCLUSIONS: KLF5 is induced during the development of ischemic heart failure in humans and mice and stimulates ceramide biosynthesis. Genetic or pharmacological inhibition of KLF5 in mice with MI prevents ceramide accumulation, alleviates eccentric remodeling, and increases ejection fraction. Thus, KLF5 emerges as a novel therapeutic target for the treatment of ischemic heart failure.
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Cardiomiopatías/fisiopatología , Ceramidas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Miocitos Cardíacos/metabolismo , Remodelación Ventricular/fisiología , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , RatonesRESUMEN
The development of potent cholesterol-reducing medications in the last decade of the twentieth century has altered the approach to prevention and treatment of cardiovascular disease (CVD). Initial experience with statins, and more recently with the addition of PCSK9 inhibitors, has proven that human CVD, like that in animal models, can be halted and regressed. Available clinical data show that the lower the achieved level of low-density lipoprotein cholesterol, the greater the regression of disease. Investigative studies are now aimed to understand those factors that both accelerate and impede this healing process. Some of these are likely to be modifiable, and the future of atherosclerotic CVD treatment is likely to be early screening, use of measures to repair atherosclerotic arteries, and prevention of most CVD events.
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Aterosclerosis/metabolismo , Placa Aterosclerótica/metabolismo , Animales , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/prevención & control , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/prevención & control , Colesterol/metabolismo , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Quilomicrones/metabolismo , Angiografía por Tomografía Computarizada , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Macrófagos/metabolismo , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Ratones Noqueados para ApoE , Inhibidores de PCSK9 , Placa Aterosclerótica/diagnóstico por imagen , Placa Aterosclerótica/tratamiento farmacológico , Placa Aterosclerótica/prevención & control , Conejos , Ratas , Receptores de LDL/genética , Receptores de LDL/metabolismo , Inducción de Remisión , Triglicéridos/metabolismo , Ultrasonografía IntervencionalRESUMEN
RATIONALE: Treatment efficacy for diabetes mellitus is largely determined by assessment of HbA1c (glycated hemoglobin A1c) levels, which poorly reflects direct glucose variation. People with prediabetes and diabetes mellitus spend >50% of their time outside the optimal glucose range. These glucose variations, termed transient intermittent hyperglycemia (TIH), appear to be an independent risk factor for cardiovascular disease, but the pathological basis for this association is unclear. OBJECTIVE: To determine whether TIH per se promotes myelopoiesis to produce more monocytes and consequently adversely affects atherosclerosis. METHODS AND RESULTS: To create a mouse model of TIH, we administered 4 bolus doses of glucose at 2-hour intervals intraperitoneally once to WT (wild type) or once weekly to atherosclerotic prone mice. TIH accelerated atherogenesis without an increase in plasma cholesterol, seen in traditional models of diabetes mellitus. TIH promoted myelopoiesis in the bone marrow, resulting in increased circulating monocytes, particularly the inflammatory Ly6-Chi subset, and neutrophils. Hematopoietic-restricted deletion of S100a9, S100a8, or its cognate receptor Rage prevented monocytosis. Mechanistically, glucose uptake via GLUT (glucose transporter)-1 and enhanced glycolysis in neutrophils promoted the production of S100A8/A9. Myeloid-restricted deletion of Slc2a1 (GLUT-1) or pharmacological inhibition of S100A8/A9 reduced TIH-induced myelopoiesis and atherosclerosis. CONCLUSIONS: Together, these data provide a mechanism as to how TIH, prevalent in people with impaired glucose metabolism, contributes to cardiovascular disease. These findings provide a rationale for continual glucose control in these patients and may also suggest that strategies aimed at targeting the S100A8/A9-RAGE (receptor for advanced glycation end products) axis could represent a viable approach to protect the vulnerable blood vessels in diabetes mellitus. Graphic Abstract: A graphic abstract is available for this article.
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Aterosclerosis/etiología , Glucemia/metabolismo , Hiperglucemia/complicaciones , Monocitos/metabolismo , Mielopoyesis , Neutrófilos/metabolismo , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Biomarcadores/sangre , Calgranulina A/genética , Calgranulina A/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Glucólisis , Hiperglucemia/sangre , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Monocitos/patología , Neutrófilos/patología , Placa Aterosclerótica , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transducción de SeñalRESUMEN
Objective: To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage. Approach and Results: Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans. Conclusions: Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.
Asunto(s)
Adipocitos/metabolismo , Aterosclerosis/metabolismo , HDL-Colesterol/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microdominios de Membrana/metabolismo , Obesidad/metabolismo , Receptores Depuradores de Clase B/metabolismo , Grasa Subcutánea/metabolismo , Adipocitos/patología , Adipogénesis , Adiposidad , Adulto , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Células CHO , Caveolina 1/metabolismo , Cricetulus , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Metabolismo Energético , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Microdominios de Membrana/genética , Microdominios de Membrana/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Obesidad/genética , Obesidad/patología , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores Depuradores de Clase B/genética , Grasa Subcutánea/patologíaRESUMEN
Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluorescence method to measure lipase activity in real time that does not require the separation of substrates from products. We developed this method using adipose triglyceride lipase (ATGL) and lipoprotein lipase (LpL) as model lipases. We first incubated a source of ATGL or LpL with substrate vesicles containing nitrobenzoxadiazole (NBD)-labeled TAG, then measured increases in NBD fluorescence, and calculated enzyme activities. Incorporation of NBD-TAG into phosphatidylcholine (PC) vesicles resulted in some hydrolysis; however, incorporation of phosphatidylinositol into these NBD-TAG/PC vesicles and increasing the ratio of NBD-TAG to PC greatly enhanced substrate hydrolysis. This assay was also useful in measuring the activity of pancreatic lipase and hormone-sensitive lipase. Next, we tested several small-molecule lipase inhibitors and found that orlistat inhibits all lipases, indicating that it is a pan-lipase inhibitor. In short, we describe a simple, rapid, fluorescence-based triacylglycerol hydrolysis assay to assess four major TAG hydrolases: intracellular ATGL and hormone-sensitive lipase, LpL localized at the extracellular endothelium, and pancreatic lipase present in the intestinal lumen. The major advantages of this method are its speed, simplicity, and elimination of product isolation. This assay is potentially applicable to a wide range of lipases, is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and can be used for diagnostic purposes.
Asunto(s)
Aciltransferasas/metabolismo , Fluorescencia , Lipoproteína Lipasa/metabolismo , Animales , Células COS , Chlorocebus aethiops , Ensayos Analíticos de Alto Rendimiento , Hidrólisis , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
PURPOSE OF REVIEW: To discuss the recent developments in structure, function and physiology of lipoprotein lipase (LpL) and the regulators of LpL, which are being targeted for therapy. RECENT FINDINGS: Recent studies have revealed the long elusive crystal structure of LpL and its interaction with glycosylphosphatidylinositol anchored high-density lipoprotein binding protein 1 (GPIHBP1). New light has been shed on LpL being active as a monomer, which brings into questions previous thinking that LpL inhibitors like angiopoietin-like 4 (ANGPTL4) and stabilizers like LMF1 work on disrupting or maintaining LpL in dimer form. There is increasing pharmaceutical interest in developing targets to block LpL inhibitors like ANGPTL3. Other approaches to reducing circulating triglyceride levels have been using an apoC2 mimetic and reducing apoC3. SUMMARY: Lipolysis of triglyceride-rich lipoproteins by LpL is a central event in lipid metabolism, releasing fatty acids for uptake by tissues and generating low-density lipoprotein and expanding high-density lipoprotein. Recent mechanistic insights into the structure and function of LpL have added to our understanding of triglyceride metabolism. This has also led to heightened interest in targeting its posttranslational regulators, which can be the next generation of lipid-lowering agents used to prevent hypertriglyceridemic pancreatitis and, hopefully, cardiovascular disease.
Asunto(s)
Proteínas Similares a la Angiopoyetina/genética , Apolipoproteína C-II/genética , Lipoproteína Lipasa/genética , Receptores de Lipoproteína/genética , Proteína 3 Similar a la Angiopoyetina , Proteína 4 Similar a la Angiopoyetina/genética , Animales , Apolipoproteína C-II/uso terapéutico , Humanos , Lipólisis/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Triglicéridos/genética , Triglicéridos/metabolismoRESUMEN
BACKGROUND: Despite robust cholesterol lowering, cardiovascular disease risk remains increased in patients with diabetes mellitus. Consistent with this, diabetes mellitus impairs atherosclerosis regression after cholesterol lowering in humans and mice. In mice, this is attributed in part to hyperglycemia-induced monocytosis, which increases monocyte entry into plaques despite cholesterol lowering. In addition, diabetes mellitus skews plaque macrophages toward an atherogenic inflammatory M1 phenotype instead of toward the atherosclerosis-resolving M2 state typical with cholesterol lowering. Functional high-density lipoprotein (HDL), typically low in patients with diabetes mellitus, reduces monocyte precursor proliferation in murine bone marrow and has anti-inflammatory effects on human and murine macrophages. Our study aimed to test whether raising functional HDL levels in diabetic mice prevents monocytosis, reduces the quantity and inflammation of plaque macrophages, and enhances atherosclerosis regression after cholesterol lowering. METHODS: Aortic arches containing plaques developed in Ldlr-/- mice were transplanted into either wild-type, diabetic wild-type, or diabetic mice transgenic for human apolipoprotein AI, which have elevated functional HDL. Recipient mice all had low levels of low-density lipoprotein cholesterol to promote plaque regression. After 2 weeks, plaques in recipient mouse aortic grafts were examined. RESULTS: Diabetic wild-type mice had impaired atherosclerosis regression, which was normalized by raising HDL levels. This benefit was linked to suppressed hyperglycemia-driven myelopoiesis, monocytosis, and neutrophilia. Increased HDL improved cholesterol efflux from bone marrow progenitors, suppressing their proliferation and monocyte and neutrophil production capacity. In addition to reducing circulating monocytes available for recruitment into plaques, in the diabetic milieu, HDL suppressed the general recruitability of monocytes to inflammatory sites and promoted plaque macrophage polarization to the M2, atherosclerosis-resolving state. There was also a decrease in plaque neutrophil extracellular traps, which are atherogenic and increased by diabetes mellitus. CONCLUSIONS: Raising apolipoprotein AI and functional levels of HDL promotes multiple favorable changes in the production of monocytes and neutrophils and in the inflammatory environment of atherosclerotic plaques of diabetic mice after cholesterol lowering and may represent a novel approach to reduce cardiovascular disease risk in people with diabetes mellitus.
Asunto(s)
Apolipoproteína A-I/genética , Aterosclerosis/patología , Diabetes Mellitus Experimental/patología , Animales , Apolipoproteína A-I/metabolismo , Aterosclerosis/complicaciones , Aterosclerosis/tratamiento farmacológico , Colesterol/metabolismo , HDL-Colesterol/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/complicaciones , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Leucocitosis , Lipoproteínas HDL/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/citología , Células Mieloides/metabolismo , Mielopoyesis , Activación Neutrófila , Receptores de LDL/deficiencia , Receptores de LDL/genéticaRESUMEN
BACKGROUND: A protein that is expressed on capillary endothelial cells, called GPIHBP1 (glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1), binds lipoprotein lipase and shuttles it to its site of action in the capillary lumen. A deficiency in GPIHBP1 prevents lipoprotein lipase from reaching the capillary lumen. Patients with GPIHBP1 deficiency have low plasma levels of lipoprotein lipase, impaired intravascular hydrolysis of triglycerides, and severe hypertriglyceridemia (chylomicronemia). During the characterization of a monoclonal antibody-based immunoassay for GPIHBP1, we encountered two plasma samples (both from patients with chylomicronemia) that contained an interfering substance that made it impossible to measure GPIHBP1. That finding raised the possibility that those samples might contain GPIHBP1 autoantibodies. METHODS: Using a combination of immunoassays, Western blot analyses, and immunocytochemical studies, we tested the two plasma samples (as well as samples from other patients with chylomicronemia) for the presence of GPIHBP1 autoantibodies. We also tested the ability of GPIHBP1 autoantibodies to block the binding of lipoprotein lipase to GPIHBP1. RESULTS: We identified GPIHBP1 autoantibodies in six patients with chylomicronemia and found that these autoantibodies blocked the binding of lipoprotein lipase to GPIHBP1. As in patients with GPIHBP1 deficiency, those with GPIHBP1 autoantibodies had low plasma levels of lipoprotein lipase. Three of the six patients had systemic lupus erythematosus. One of these patients who had GPIHBP1 autoantibodies delivered a baby with plasma containing maternal GPIHBP1 autoantibodies; the infant had severe but transient chylomicronemia. Two of the patients with chylomicronemia and GPIHBP1 autoantibodies had a response to treatment with immunosuppressive agents. CONCLUSIONS: In six patients with chylomicronemia, GPIHBP1 autoantibodies blocked the ability of GPIHBP1 to bind and transport lipoprotein lipase, thereby interfering with lipoprotein lipase-mediated processing of triglyceride-rich lipoproteins and causing severe hypertriglyceridemia. (Funded by the National Heart, Lung, and Blood Institute and the Leducq Foundation.).
Asunto(s)
Autoanticuerpos/sangre , Hiperlipoproteinemia Tipo I/inmunología , Lipoproteína Lipasa/metabolismo , Receptores de Lipoproteína/inmunología , Adulto , Autoanticuerpos/fisiología , Femenino , Humanos , Hiperlipoproteinemia Tipo I/sangre , Inmunoensayo , Lipólisis , Lipoproteína Lipasa/sangre , Masculino , Persona de Mediana Edad , Unión Proteica , Transporte de Proteínas , Receptores de Lipoproteína/metabolismoRESUMEN
RATIONALE: Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and methods that block liver lipoprotein secretion. OBJECTIVE: We sought to develop a more direct or time-effective method to create and then reverse hypercholesterolemia and atherosclerosis via transient knockdown of the hepatic LDLR (low-density lipoprotein receptor) followed by its rapid restoration. METHODS AND RESULTS: We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild-type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels because of recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, that is, regression of inflammatory cell content, after a period of 2 to 3 weeks. CONCLUSIONS: We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. Although cholesterol reduction decreased early en face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.