RESUMEN

Heme homeostasis is of vital importance to many biological processes associated with cell redox activity. However, the role of heme in the doxorubicin (DOX)-induced cardiotoxicity is still not clear. The aim of the present study was to test the hypothesis that heme is related to the DOX-induced oxidative stress and inhibition of heme expression may protect H9c2 cardiomyocytes against DOX-induced cardiotoxicity. For the evaluation of heme changing under doxorubicin treatment, H9c2 cells were treated with 0.5, 1, 2, and 4 mg/mL doxorubicin respectively. H9c2 cells were divided into 5 groups: Control group (cells were cultured without intervention), DOX group (cells were treated with 2 mg/mL doxorubicin for 6 h), Heme depletion+DOX group (cells were cultured with heme-depleted serum media, 0.5 mM succinylacetone and 2 mg/mL doxorubicin), Heme group (cells were treated with 30 µM heme), and Heme depletion+DOX+Heme group. Apoptotic cells were detected by flow cytometry with Annexin V-FITC/PI. The intracellular oxidant levels were measured by DCFH-DA fluorescence. The levels of heme were detected by ELISA. Doxorubicin significantly increased intracellular heme level from 5013 ± 187 ng/mL to the highest level of 11,720 ± 107 ng/mL, as well as the intracellular oxidants and cell apoptosis rate elevated by the increase of doxorubicin concentration. Heme depletion can significantly suppress the DOX-induced apoptosis from 39.8 ± 0.5% to 20.8 ± 0.5% (p < 0.001). Re-supplemented with exogenous heme partially but significantly restored the DOX-induced apoptosis. Heme plays an important role in doxorubicin toxicity-induced cardiomyocyte injury. By appropriate reduction in the accumulation of free heme in cardiomyocytes, doxorubicin-induced cardiotoxicity may be alleviated.

Asunto(s)

Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Hemo/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Animales , Línea Celular

RESUMEN

Perilipin-1 (Plin1) coats lipid droplets exclusively in adipocytes and regulates two principle functions of adipose tissue, triglyceride storage and hydrolysis, which are disrupted upon Plin1 deficiency. In the present study, we investigated the alterations in systemic metabolites and hormones, vascular function and adipose function in spontaneous hypertensive mice lacking perilipin-1 (Plin1-/-). Plin1-/- mice developed spontaneous hypertension without obvious alterations in systemic metabolites and hormones. Plin1 expressed only in adipose cells but not in vascular cells, so its ablation would have no direct effect in situ on blood vessels. Instead, Plin1-/- mice showed dysfunctions of perivascular adipose tissue (PVAT), a fat depot that anatomically surrounds systemic arteries and has an anticontractile effect. In Plin1-/- mice, aortic and mesenteric PVAT were reduced in mass and adipocyte derived relaxing factor secretion, but increased in basal lipolysis, angiotensin II secretion, macrophage infiltration and oxidative stress. Such multiple culprits impaired the anticontractile effect of PVAT to promote vasoconstriction of aortic and mesenteric arteries of Plin1-/- mice. Furthermore, arterial vessels of Plin1-/- mice showed increasing angiotensin II receptor type 1, monocyte chemotactic protein-1 and interlukin-6 expression, structural damage of endothelial and smooth muscle cells, along with impaired endothelium-dependent relaxation. Hypertension in Plin1-/- mice might occur as a deleterious consequence of PVAT dysfunction. This finding provides the direct evidence that links dysfunctional PVAT to vascular dysfunction and hypertension, particularly in pathophysiological states. This hypertensive mouse model might mimic and explain the hypertension occurring in patients with adipose tissue dysfunction, particularly with Plin1 mutations.

Asunto(s)

Tejido Adiposo/fisiopatología , Eliminación de Gen , Hipertensión/genética , Hipertensión/fisiopatología , Perilipina-1/genética , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo/irrigación sanguínea , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Hipertensión/patología , Inflamación/genética , Inflamación/patología , Inflamación/fisiopatología , Ratones , Ratones Noqueados , Vasoconstricción , Vasodilatación

RESUMEN

AIMS: The objective of this study is to determine the role of perilipin 1 (Plin1) in whole body or bone marrow-derived cells on atherogenesis. METHODS AND RESULTS: Accumulated evidence have indicated the role of Plin1 in atherosclerosis, however, these findings are controversial. In this study, we showed that Plin1 was assembled and colocalized with CD68 in macrophages in atherosclerotic plaques of ApoE-/- mice. We further found 39% reduction of plaque size in the aortic roots of Plin1 and ApoE double knockout (Plin1-/-ApoE-/-) females compared with ApoE-/- female littermates. In order to verify whether this reduction was macrophage-specific, the bone marrow cells from wild-type or Plin1 deficient mice (Plin1-/-) were transplanted into LDL receptor deficient mice (LDLR-/-). Mice receiving Plin1-/- bone marrow cells showed also 49% reduction in aortic atherosclerotic lesions compared with LDLR-/- mice received wild-type bone marrow cells. In vitro experiments showed that Plin1-/- macrophages had decreased protein expression of CD36 translocase and an enhanced cholesterol ester hydrolysis upon aggregated-LDL loading, with unaltered expression of many other regulators of cholesterol metabolism, such as cellular lipases, and Plin2 and 3. Given the fundamental role of Plin1 in protecting LD lipids from lipase hydrolysis, it is reasonably speculated that the assembly of Plin1 in microphages might function to reduce lipolysis and hence increase lipid retention in ApoE-/- plaques, but this pro-atherosclerotic property would be abrogated on inactivation of Plin1. CONCLUSION: Plin1 deficiency in bone marrow-derived cells may be responsible for reduced atherosclerotic lesions in the mice.

Asunto(s)

Aterosclerosis/genética , Células de la Médula Ósea/metabolismo , Proteínas Portadoras/genética , Fosfoproteínas/genética , Placa Aterosclerótica/genética , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células de la Médula Ósea/patología , Trasplante de Médula Ósea , Proteínas Portadoras/metabolismo , Ésteres del Colesterol/genética , Ésteres del Colesterol/metabolismo , Femenino , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Perilipina-1 , Perilipina-2 , Perilipina-3 , Fosfoproteínas/deficiencia , Fosfoproteínas/metabolismo , Placa Aterosclerótica/patología , Receptores de LDL/genética , Receptores de LDL/metabolismo

RESUMEN

Perilipin 1 (Plin1) localizes at the surface of lipid droplets to regulate triglyceride storage and hydrolysis in adipocytes. Plin1 defect leads to low adiposity in mice and partial lipodystrophy in human. This study investigated the roles of Plin1 in adipocyte differentiation. Plin1 null (-/-) mice showed plenty of multilocular adipocytes and small unilocular adipocytes in adipose tissue, along with lack of a subpopulation of adipose progenitor cells capable of in vivo adipogenesis and along with downregulation of adipogenic pathway. Before initiation of differentiation, adipose stromal-vascular cells (SVCs) from Plin1-/- mice already accumulated numerous tiny lipid droplets, which increased in number and size during the first 12-h induction but thereafter became disappeared at day 1 of differentiation. The adipogenic signaling was dysregulated despite protein level of PPARγ was near normal in Plin1-/- SVCs like in Plin1-/- adipose tissue. Heterozygous Plin1+/- SVCs were able to develop lipid droplets, with both the number and size more than in Plin1-/- SVCs but less than in Plin1+/+ SVCs, indicating that Plin1 haploinsufficiency accounts for attenuated adipogenesis. Aberrant lipid droplet growth and differentiation of Plin1-/- SVCs were rescued by adenoviral Plin1 expression and were ameliorated by enhanced or prolonged adipogenic stimulation. Our finding suggests that Plin1 plays an important role in adipocyte differentiation and provides an insight into the pathology of partial lipodystrophy in patients with Plin1 mutation.

Asunto(s)

Tejido Adiposo/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Adipogénesis/efectos de los fármacos , Tejido Adiposo/citología , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Metabolismo de los Lípidos/fisiología , Lipodistrofia/metabolismo , Lipodistrofia/patología , Lipólisis/fisiología , Ratones , Ratones Noqueados , Microscopía Fluorescente , PPAR gamma/genética , PPAR gamma/metabolismo , Perilipina-1 , Inhibidores de Fosfodiesterasa/farmacología , Transducción de Señal , Células Madre/citología , Células Madre/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo

RESUMEN

AIMS: Perilipin-1 (Plin1), exclusively located on the surface of lipid droplets in adipocytes, regulates the storage and hydrolysis of adipose triglycerides. Plin1 deficiency primarily causes low adiposity and aberrant lipolysis in rodents and humans. Here, we investigated whether adipose tissue dysfunction in perilipin-1 null (Plin1⁻/⁻) mice has maladaptive consequences for the heart and an association with hypertrophic cardiomyopathy. METHODS AND RESULTS: Perilipin-1 was expressed specifically in adipocytes but was undetectable in cardiomyocytes. Plin1⁻/⁻ mice were histologically lipodystrophic, with reduced body fat. Paradoxically, the adipocytes of Plin1⁻/⁻ mice, like those of obese and diabetic mammals, showed robust basal lipolysis and fatty acid efflux to the plasma. Such adipose tissue dysfunctions accounted for the ectopic lipid accumulation and enhanced fatty acid transport and oxidation in Plin1⁻/⁻ mouse hearts. Excessive fatty acid ß-oxidation and lipotoxicity induced excessive production of reactive oxygen species and oxidative stress because antioxidative capacity was reduced in cardiomyocytes, These malefactors injured the myocardial structure and function, as evidenced by disorganized myofilaments as well as irregular and swollen mitochondria with disrupted cristae. Finally, Plin1⁻/⁻ mice showed grossly visible cardiac hypertrophy, with progressively up-regulated expression of hypertrophy and dysfunction marker genes, leading to heart failure, particularly with left ventricular diastolic dysfunction at 20 weeks of age. CONCLUSIONS: Adipose tissue dysfunction may have deleterious effects on the heart and contribute to the development of hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy in Plin1⁻/⁻ mice with adipose tissue dysfunction may mimic and mechanistically explain the cardiomyopathies occurring in two typical adipose tissue disorders in humans, lipodystrophy and obesity.

Asunto(s)

Tejido Adiposo/fisiopatología , Cardiomiopatía Hipertrófica/etiología , Fosfoproteínas/deficiencia , Animales , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/fisiopatología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Lipólisis , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés Oxidativo , Perilipina-1 , Fosfoproteínas/genética , Fosfoproteínas/fisiología

RESUMEN

In obesity and diabetes, adipocytes show significant endoplasmic reticulum (ER) stress, which triggers a series of responses. This study aimed to investigate the lipolysis response to ER stress in rat adipocytes. Thapsigargin, tunicamycin, and brefeldin A, which induce ER stress through different pathways, efficiently activated a time-dependent lipolytic reaction. The lipolytic effect of ER stress occurred with elevated cAMP production and protein kinase A (PKA) activity. Inhibition of PKA reduced PKA phosphosubstrates and attenuated the lipolysis. Although both ERK1/2 and JNK are activated during ER stress, lipolysis is partially suppressed by inhibiting ERK1/2 but not JNK and p38 MAPK and PKC. Thus, ER stress induces lipolysis by activating cAMP/PKA and ERK1/2. In the downstream lipolytic cascade, phosphorylation of lipid droplet-associated protein perilipin was significantly promoted during ER stress but attenuated on PKA inhibition. Furthermore, ER stress stimuli did not alter the levels of hormone-sensitive lipase and adipose triglyceride lipase but caused Ser-563 and Ser-660 phosphorylation of hormone-sensitive lipase and moderately elevated its translocation from the cytosol to lipid droplets. Accompanying these changes, total activity of cellular lipases was promoted to confer the lipolysis. These findings suggest a novel pathway of the lipolysis response to ER stress in adipocytes. This lipolytic activation may be an adaptive response that regulates energy homeostasis but with sustained ER stress challenge could contribute to lipotoxicity, dyslipidemia, and insulin resistance because of persistently accelerated free fatty acid efflux from adipocytes to the bloodstream and other tissues.

Asunto(s)

Grasa Abdominal/metabolismo , Adipocitos/fisiología , Estrés del Retículo Endoplásmico/fisiología , Lipólisis/fisiología , Grasa Abdominal/citología , Adipocitos/citología , Animales , Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Diabetes Mellitus/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Resistencia a la Insulina/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipasa/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Obesidad/metabolismo , Perilipina-1 , Fosfoproteínas/metabolismo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Tapsigargina/farmacología , Tunicamicina/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo