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Introduction: Unlike white adipose tissue depots, bone marrow adipose tissue (BMAT) expands during caloric restriction (CR). Although mechanisms for BMAT expansion remain unclear, prior research suggested an intermediary role for increased circulating glucocorticoids. Methods: In this study, we utilized a recently described mouse model (BMAd-Cre) to exclusively target bone marrow adipocytes (BMAds) for elimination of the glucocorticoid receptor (GR) (i.e. Nr3c1) whilst maintaining GR expression in other adipose depots. Results: Mice lacking GR in BMAds (BMAd-Nr3c1 -/-) and control mice (BMAd-Nr3c1 +/+) were fed ad libitum or placed on a 30% CR diet for six weeks. On a normal chow diet, tibiae of female BMAd-Nr3c1-/- mice had slightly elevated proximal trabecular metaphyseal bone volume fraction and thickness. Both control and BMAd-Nr3c1-/- mice had increased circulating glucocorticoids and elevated numbers of BMAds in the proximal tibia following CR. However, no significant differences in trabecular and cortical bone were observed, and quantification with osmium tetroxide and µCT revealed no difference in BMAT accumulation between control or BMAd-Nr3c1 -/- mice. Differences in BMAd size were not observed between BMAd-Nr3c1-/- and control mice. Interestingly, BMAd-Nr3c1-/- mice had decreased circulating white blood cell counts 4 h into the light cycle. Discussion: In conclusion, our data suggest that eliminating GR from BMAd has minor effects on bone and hematopoiesis, and does not impair BMAT accumulation during CR.
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Adipocitos , Adiposidad , Médula Ósea , Restricción Calórica , Hematopoyesis , Receptores de Glucocorticoides , Animales , Receptores de Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/deficiencia , Ratones , Adipocitos/metabolismo , Adiposidad/fisiología , Femenino , Médula Ósea/metabolismo , Ratones Noqueados , Huesos/metabolismo , Ratones Endogámicos C57BL , Tejido Adiposo/metabolismo , Masculino , Errores Innatos del MetabolismoRESUMEN
The brain augments glucose production during fasting, but the mechanisms are poorly understood. Here, we show that Cckbr-expressing neurons in the ventromedial hypothalamic nucleus (VMNCckbr cells) prevent low blood glucose during fasting through sympathetic nervous system (SNS)-mediated augmentation of adipose tissue lipolysis and substrate release. Activating VMNCckbr neurons mobilized gluconeogenic substrates without altering glycogenolysis or gluconeogenic enzyme expression. Silencing these cells (CckbrTetTox animals) reduced fasting blood glucose, impaired lipolysis, and decreased circulating glycerol (but not other gluconeogenic substrates) despite normal insulin, counterregulatory hormones, liver glycogen, and liver gluconeogenic gene expression. Furthermore, ß3-adrenergic adipose tissue stimulation in CckbrTetTox animals restored lipolysis and blood glucose. Hence, VMNCckbr neurons impact blood glucose not by controlling islet or liver physiology, but rather by mobilizing gluconeogenic substrates. These findings establish a central role for hypothalamic and SNS signaling during normal glucose homeostasis and highlight the importance of gluconeogenic substrate mobilization during physiologic fasting.
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OBJECTIVE: Exposure of adipocytes to 'cool' temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (Scd1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. The goal of this study is to further investigate the roles of Scd in adipocytes. METHOD: In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological Scd1 inhibition to dissect the enzyme's function in adipocyte physiology. RESULTS: Our study reveals that production of monounsaturated lipids by Scd1 is necessary for fusion of autophagosomes to lysosomes and that with a Scd1-deficiency, autophagosomes accumulate. In addition, Scd1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of Scd1-deficient adipocytes. CONCLUSION: This study demonstrates the indispensable role of Scd1 in adipocyte survival, with its inhibition in vivo triggering autophagy-dependent cell death and its depletion in vivo leading to the loss of bone marrow adipocytes.
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Adipocitos , Autofagia , Ácidos Grasos Monoinsaturados , Ratones Noqueados , Estearoil-CoA Desaturasa , Estearoil-CoA Desaturasa/metabolismo , Estearoil-CoA Desaturasa/genética , Animales , Ratones , Adipocitos/metabolismo , Ácidos Grasos Monoinsaturados/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Ratones Endogámicos C57BL , Lisosomas/metabolismo , Supervivencia Celular , Células 3T3-L1 , Masculino , Metabolismo de los Lípidos , Autofagosomas/metabolismoRESUMEN
Exposure of adipocytes to 'cool' temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (SCD1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological SCD1 inhibition, to investigate further the roles of SCD1 in adipocytes. Our study reveals that production of monounsaturated lipids by SCD1 is necessary for fusion of autophagosomes to lysosomes and that with a SCD1-deficiency, autophagosomes accumulate. In addition, SCD1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of SCD1-deficient adipocytes. Taken together, our results demonstrate that in vitro inhibition of SCD1 in adipocytes leads to autophagy-dependent cell death, and in vivo depletion leads to loss of bone marrow adipocytes.
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To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd-Pnpla2-/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.
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Médula Ósea , Lipólisis , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Médula Ósea/metabolismo , Lipasa/metabolismo , Lipólisis/genética , Masculino , RatonesRESUMEN
OBJECTIVE: Tamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity. METHODS: Adipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity. RESULTS: Tamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass. CONCLUSIONS: These results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.
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Doxiciclina , Transactivadores , Animales , Antibacterianos , Doxiciclina/farmacología , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Transgénicos , Recombinación Genética , Elementos de Respuesta , Tamoxifeno/farmacología , Transactivadores/metabolismoRESUMEN
CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.
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Tejido Adiposo Pardo/metabolismo , Sistemas CRISPR-Cas , Animales , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Ratones , Ratones Noqueados , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Mechanisms by which autosomal recessive mutations in Lmna cause familial partial lipodystrophy type 2 (FPLD2) are poorly understood. To investigate the function of lamin A/C in adipose tissue, we created mice with an adipocyte-specific loss of Lmna (Lmna ADKO). Although Lmna ADKO mice develop and maintain adipose tissues in early postnatal life, they show a striking and progressive loss of white and brown adipose tissues as they approach sexual maturity. Lmna ADKO mice exhibit surprisingly mild metabolic dysfunction on a chow diet, but on a high-fat diet they share many characteristics of FPLD2 including hyperglycemia, hepatic steatosis, hyperinsulinemia, and almost undetectable circulating adiponectin and leptin. Whereas Lmna ADKO mice have reduced regulated and constitutive bone marrow adipose tissue with a concomitant increase in cortical bone, FPLD2 patients have reduced bone mass and bone mineral density compared with controls. In cell culture models of Lmna deficiency, mesenchymal precursors undergo adipogenesis without impairment, whereas fully differentiated adipocytes have increased lipolytic responses to adrenergic stimuli. Lmna ADKO mice faithfully reproduce many characteristics of FPLD2 and thus provide a unique animal model to investigate mechanisms underlying Lmna-dependent loss of adipose tissues.
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Adipocitos/metabolismo , Adipogénesis/fisiología , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Densidad Ósea/fisiología , Modelos Animales de Enfermedad , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/metabolismo , Ratones , Ratones NoqueadosRESUMEN
OBJECTIVE: Obesity is a key risk factor for many secondary chronic illnesses, including type 2 diabetes and cardiovascular disease. Canonical Wnt/ß-catenin signaling is established as an important endogenous inhibitor of adipogenesis. This pathway is operative in mature adipocytes; however, its roles in this context remain unclear due to complexities of Wnt signaling and differences in experimental models. In this study, we used novel cultured cell and mouse models to investigate functional roles of Wnts secreted from adipocytes. METHODS: We generated adipocyte-specific Wntless (Wls) knockout mice and cultured cell models to investigate molecular and metabolic consequences of disrupting Wnt secretion from mature adipocytes. To characterize Wls-deficient cultured adipocytes, we evaluated the expression of Wnt target and lipogenic genes and the downstream functional effects on carbohydrate and lipid metabolism. We also investigated the impact of adipocyte-specific Wls deletion on adipose tissues and global glucose metabolism in mice fed normal chow or high-fat diets. RESULTS: Many aspects of the Wnt signaling apparatus are expressed and operative in mature adipocytes, including the Wnt chaperone Wntless. Deletion of Wntless in cultured adipocytes results in the inhibition of de novo lipogenesis and lipid monounsaturation, likely through repression of Srebf1 (SREBP1c) and Mlxipl (ChREBP) and impaired cleavage of immature SREBP1c into its active form. Adipocyte-specific Wls knockout mice (Wls-/-) have lipogenic gene expression in adipose tissues and isolated adipocytes similar to that of controls when fed a normal chow diet. However, closer investigation reveals that a subset of Wnts and downstream signaling targets are upregulated within stromal-vascular cells of Wls-/- mice, suggesting that adipose tissues defend loss of Wnt secretion from adipocytes. Interestingly, this compensation is lost with long-term high-fat diet challenges. Thus, after six months of a high-fat diet, Wls-/- mice are characterized by decreased adipocyte lipogenic gene expression, reduced visceral adiposity, and improved glucose homeostasis. CONCLUSIONS: Taken together, these studies demonstrate that adipocyte-derived Wnts regulate de novo lipogenesis and lipid desaturation and coordinate the expression of lipogenic genes in adipose tissues. In addition, we report that Wnt signaling within adipose tissues is defended, such that a loss of Wnt secretion from adipocytes is sensed and compensated for by neighboring stromal-vascular cells. With chronic overnutrition, this compensatory mechanism is lost, revealing that Wls-/- mice are resistant to diet-induced obesity, adipocyte hypertrophy, and metabolic dysfunction.
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Adipocitos/metabolismo , Regulación de la Expresión Génica , Lipogénesis/genética , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Biomarcadores , Células Cultivadas , Dieta/efectos adversos , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Glucosa/metabolismo , Inmunohistoquímica , Insulina/metabolismo , Metabolismo de los Lípidos/genética , Enfermedades Metabólicas/diagnóstico , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/genética , Vía de Señalización WntRESUMEN
The cell plasma membrane is a highly dynamic organelle governing a wide range of cellular activities including ion transport, secretion, cell division, growth, and development. The fundamental process involved in the addition of new membranes to pre-existing plasma membranes, however, is unclear. Here, we report, using biophysical, morphological, biochemical, and molecular dynamic simulations, the selective incorporation of proteins and lipids from the cytosol into the cell plasma membrane dictated by membrane stretch and composition. Stretching of the cell membrane as a consequence of volume increase following incubation in a hypotonic solution and results in the incorporation of cytosolic proteins and lipids into the existing plasma membrane. Molecular dynamic simulations further confirm that increased membrane stretch results in the rapid insertion of lipids into the existing plasma membrane. Similarly, depletion of cholesterol from the cell plasma membrane selectively alters the incorporation of lipids into the membrane.
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Proteínas Sanguíneas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Citosol/metabolismo , Eritrocitos/metabolismo , Insulinoma/metabolismo , Lípidos de la Membrana/metabolismo , Animales , Ratones , Simulación de Dinámica Molecular , Neoplasias Pancreáticas/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
Bariatric surgeries are integral to the management of obesity and its metabolic complications. However, these surgeries cause bone loss and increase fracture risk through poorly understood mechanisms. In a mouse model, vertical sleeve gastrectomy (VSG) caused trabecular and cortical bone loss that was independent of sex, body weight, and diet, and this loss was characterized by impaired osteoid mineralization and bone formation. VSG had a profound effect on the bone marrow niche, with rapid loss of marrow adipose tissue, and expansion of myeloid cellularity, leading to increased circulating neutrophils. Following VSG, circulating granulocyte-colony stimulating factor (G-CSF) was increased in mice, and was transiently elevated in a longitudinal study of humans. Elevation of G-CSF was found to recapitulate many effects of VSG on bone and the marrow niche. In addition to stimulatory effects of G-CSF on myelopoiesis, endogenous G-CSF suppressed development of marrow adipocytes and hindered accrual of peak cortical and trabecular bone. Effects of VSG on induction of neutrophils and depletion of marrow adiposity were reduced in mice deficient for G-CSF; however, bone mass was not influenced. Although not a primary mechanism for bone loss with VSG, G-CSF plays an intermediary role for effects of VSG on the bone marrow niche.
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Adipocitos/metabolismo , Células de la Médula Ósea/metabolismo , Resorción Ósea/sangre , Gastroplastia , Factor Estimulante de Colonias de Granulocitos/sangre , Obesidad/sangre , Complicaciones Posoperatorias/sangre , Adipocitos/patología , Adolescente , Adulto , Animales , Médula Ósea/patología , Células de la Médula Ósea/patología , Resorción Ósea/etiología , Resorción Ósea/genética , Resorción Ósea/patología , Femenino , Gastrectomía , Humanos , Estudios Longitudinales , Ratones , Ratones Noqueados , Obesidad/genética , Obesidad/patología , Obesidad/cirugía , Complicaciones Posoperatorias/genética , Complicaciones Posoperatorias/patologíaRESUMEN
Current approaches in regenerative medicine to develop human skeletal muscle replicating native tissue for engrafts and high-throughput drug screening and gene therapy are still in their infancy and have not proven to recapitulate the behavior and regulatory processes present in endogenous skeletal muscle tissue. This stems at least in part from the lack of a comprehensive understanding of the emergent properties of in vitro skeletal muscle growth and development. To address this gap in our current knowledge, we have developed a stretchable micropatterned 3D human skeletal muscle platform that recapitulates organized and parallel growth of muscle cells and fibers as opposed to the randomly oriented cells growth on a 2D glass surface. Mass spectrometry of the muscle cells growing on the 3D platform express key myogenic proteins such as myoferlin for myoblast fusion required in the formation of muscle tissue, and proteins involved in mitochondrial health and biogenesis, in contrast to cells growing on 2D glass surface. These results demonstrate that the engineered human muscle cells grown on the 3D platform holds great promise to further establish the emergent properties of in vitro skeletal muscle growth and development for a wide range of biomedical applications.
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A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5-6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion.
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Fluorescencia , Insulinoma/metabolismo , Insulinoma/patología , Fusión de Membrana , Microscopía de Fuerza Atómica , Imagen Óptica , Animales , Concentración de Iones de Hidrógeno , Simulación de Dinámica MolecularRESUMEN
In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.
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Plaquetas/química , Proteoma/aislamiento & purificación , Proteínas R-SNARE/aislamiento & purificación , Vesículas Secretoras/química , Proteína 3 de Membrana Asociada a Vesículas/aislamiento & purificación , Plaquetas/metabolismo , Células Cultivadas , Fraccionamiento Químico/métodos , Humanos , Inmunoprecipitación/métodos , Microscopía de Fuerza Atómica , Anotación de Secuencia Molecular , Presión Osmótica , Proteoma/metabolismo , Proteínas R-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteína 3 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.
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Lípidos de la Membrana/química , Vesículas Sinápticas/química , Sinaptosomas/química , Animales , Química Encefálica , Membrana Celular/química , Espectrometría de Masas , RatasRESUMEN
Efficient drug delivery is critical to therapy. Using electron microscopy, X-ray, and light microscopy, we have characterized functionalized superparamagnetic iron oxide (SPIO) nanoparticles, and determined their ability for rapid entry and release of the cancer drug doxorubicin in human pancreatic cancer cells. Dextran-coated SPIO nanoparticle ferrofluid, functionalized with the red-autofluorescing doxorubicin and the green-fluorescent dye fluorescein isothiocyanate as a reporter, enables tracking the intracellular nanoparticle transport and drug release. This engineered nanoparticle enables a >20 fold rapid entry and release of the drug in human pancreatic cancer cells, holding therapeutic potential as an advanced drug delivery and imaging platform. The low extracellular pH of most tumors precluding the entry of a number of weakly basic drugs such as doxorubicin, conferring drug resistance, can now be overcome.
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Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas de Magnetita/química , Nanopartículas/metabolismo , Antibióticos Antineoplásicos/metabolismo , Línea Celular Tumoral , Doxorrubicina/metabolismo , Compuestos Férricos/química , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescencia , Humanos , Nanopartículas de Magnetita/estadística & datos numéricos , Nanopartículas/química , Neoplasias Pancreáticas/tratamiento farmacológicoRESUMEN
Cup-shaped secretory portals at the cell plasma membrane called porosomes mediate the precision release of intravesicular material from cells. Membrane-bound secretory vesicles transiently dock and fuse at the base of porosomes facing the cytosol to expel pressurized intravesicular contents from the cell during secretion. The structure, isolation, composition, and functional reconstitution of the neuronal porosome complex have greatly progressed, providing a molecular understanding of its function in health and disease. Neuronal porosomes are 15 nm cup-shaped lipoprotein structures composed of nearly 40 proteins, compared to the 120 nm nuclear pore complex composed of >500 protein molecules. Membrane proteins compose the porosome complex, making it practically impossible to solve its atomic structure. However, atomic force microscopy and small-angle X-ray solution scattering studies have provided three-dimensional structural details of the native neuronal porosome at sub-nanometer resolution, providing insights into the molecular mechanism of its function. The participation of several porosome proteins previously implicated in neurotransmission and neurological disorders, further attest to the crosstalk between porosome proteins and their coordinated involvement in release of neurotransmitter at the synapse.
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Estructuras de la Membrana Celular/metabolismo , Estructuras de la Membrana Celular/ultraestructura , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Proteínas de la Membrana/ultraestructura , Neuronas/fisiología , Transmisión Sináptica , Animales , Estructuras de la Membrana Celular/química , Humanos , Sustancias Macromoleculares/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Microscopía de Fuerza Atómica , Modelos Biológicos , Conformación Proteica , Dispersión del Ángulo PequeñoRESUMEN
Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intravesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, GH-secreting cells of the pituitary, and insulin-secreting pancreatic ß-cells. Functional reconstitution of porosomes into artificial lipid membranes, have also been accomplished. Earlier studies on mouse insulin-secreting Min6 cells report 100-nm porosome complexes composed of nearly 30 proteins. In the current study, porosomes have been functionally reconstituted for the first time in live cells. Isolated Min6 porosomes reconstituted into live Min6 cells demonstrate augmented levels of porosome proteins and a consequent increase in the potency and efficacy of glucose-stimulated insulin release. Elevated glucose-stimulated insulin secretion 48 hours after reconstitution, reflects on the remarkable stability and viability of reconstituted porosomes, documenting the functional reconstitution of native porosomes in live cells. These results, establish a new paradigm in porosome-mediated insulin secretion in ß-cells.
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Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Microdominios de Membrana/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismo , Animales , Línea Celular Tumoral , Hiperglucemia/sangre , Hiperglucemia/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/ultraestructura , Microdominios de Membrana/enzimología , Microdominios de Membrana/ultraestructura , Ratones , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Estabilidad Proteica , Transporte de Proteínas , Dispersión del Ángulo Pequeño , Tasa de Secreción , Proteína 25 Asociada a Sinaptosomas/aislamiento & purificación , Difracción de Rayos XRESUMEN
Porosomes are secretory portals located at the cell plasma membrane involved in the regulated release of intravesicular contents from cells. Porosomes have been immunoisolated from a number of cells including the exocrine pancreas and neurons, biochemically characterized, and functionally reconstituted into an artificial lipid membrane. In the current study, the proteome of the porosome complex in mouse insulinoma Min6 cells was determined, demonstrating among other proteins, the presence of 30 core proteins including the heat shock protein Hsp90. Half maximal inhibition of Hsp90 using the specific inhibitor 17-demethoxy-17-(2-prophenylamino) geldanamycin, results in the loss of proteins, including the calcium-transporting ATPase type 2C and the potassium channel subfamily K member 2 from the Min6 porosome. This loss of porosome proteins is reflected in the observed inhibition of glucose stimulated insulin release from Min6 cells exposed to the Hsp90 specific inhibitor. Results from the study implicate Hsp90 in the assembly and function of the porosome complex. BIOLOGICAL SIGNIFICANCE: In the present study, the porosome proteome in the insulin-secreting mouse ß-cell line Min6 has been determined. Nearly 30 core proteins including the heat shock protein Hsp90 are found to compose the Min6 porosome complex. Results from the study implicate Hsp90 in the assembly of the Min6 porosome. These new findings will facilitate understanding of the porosome assembly and its function in insulin secretion.