Spatiotemporal analysis of endocytosis and membrane distribution of fluorescent sterols in living cells

Udgivelsesdato: 2008-NovDistribution and dynamics of cholesterol in the plasma membrane as well as internalization pathways for sterol from the cell surface are of great cell biological interest. Here, UV-sensitive wide field microscopy of the intrinsically fluorescent sterols, dehydroergosterol (DHE) and cholestatrienol (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other regions of the cell surface, and endocytosis contributed by 62% to total sterol uptake in J774 cells. DHE co-localized with fluorescent transferrin (Tf) in vesicles right after onset of endocytosis and in deepened surface patches of energy depleted cells. Surface caveolae labeled with GFP-tagged caveolin were not particularly enriched in DHE or CTL. Some sterol co-localized with internalized caveolin suggesting that caveolar endocytosis contributes to vesicular sterol uptake. These findings demonstrate that plasma membrane sterol is internalized by several endocytic pathways. Sterol endocytosis does not require formation of microscopically resolvable sterol clusters or enrichment of sterol in surface caveolae

Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

International audienceIn the present study, we microinjected fluorescently labelled liver bovine ACBP (FACI-50), into HeLa and bovine mammary gland epithelial (BMGE) cell lines to characterize the localization and dynamics of ACBP in living cells. Results showed that ACBP targeted to the endoplasmic reticulum (ER) and Golgi in a ligand-binding dependent manner. A variant Y28F/K32A-FACI-50, which is unable to bind acyl-CoA, did no longer show association with ER and became segregated from Golgi, as analysed by intensity correlation calculations. Depletion of fatty acids from cells by addition of fatty acid free BSA (FAFBSA) significantly decreased FACI-50 association with Golgi, while fatty acid overloading increased Golgi-association, strongly supporting that ACBP associates with Golgi in a ligand-dependent manner. Fluorescence recovery after photobleaching (FRAP) showed that the fatty acid induced targeting of FACI-50 to Golgi resulted in a 5-fold reduction in FACI-50 mobility. We suggest that ACBP is targeted to ER and Golgi in a ligand-binding dependent manner in living cells, and propose that ACBP may be involved in vesicular trafficking

The functional analysis of rare genetic variants in a familial neurodegenerative spectrum disorder

Whole genome sequencing can enable an unbiased approach to the diagnosis and to the understanding of genetic aetiology of neurodegenerative diseases, which are a clinically and genetically heterogeneous group of disorders. Two brothers, originally diagnosed with primary progressive multiple sclerosis (PPMS), displayed an atypical disease progression over a 30-year period with a clinical picture of paresis, ataxia, mental impairment and dysarthria. Late stage fasciculations, absence of sensory symptoms and a follow-up magnetic resonance imaging (MRI) scan showing severe atrophy of the cerebrum, cerebellum and brainstem, but no white matter lesions, called the original diagnosis into question. Given the familial nature and the rarity of this very similar clinical profile, we hypothesised that a rare variant could be contributing to disease. Whole genome sequencing identified three rare variants segregating with disease: a homozygous non-frameshift deletion in the fatty acid 2-hydroxylase (FA2H) gene, an X-bound non-synonymous single nucleotide variant in the adaptor protein-1 complex subunit sigma-2 (AP1S2) gene, and a homozygous frameshift deletion in the mixed lineage kinase domain-like protein (MLKL) gene. Lipidomic analysis using liquid chromatography-tandem mass spectrometry of FA2H variant-expressing cell lines demonstrated no detrimental impact of the deletion in FA2H on protein function. Bioinformatic and computational structural analysis suggested the variant in AP1S2 to be benign. In contrast, the frameshift deletion in MLKL prevents expression of MLKL protein as demonstrated in patient-derived primary cells. The absence of MLKL was shown ex vivo to cause complete deficiency in necroptosis, a recently identified form of programmed cell death that is involved in a spectrum of neurodegenerative diseases. This thesis provides the first clinical case report implicating MLKL and necroptosis deficiency in a neurodegenerative spectrum disorder. The results suggest that current strategies to pursue MLKL inhibition for human therapy should be approached with caution

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 mu M) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action

Proof of the geometric Langlands conjecture III: compatibility with parabolic induction

We establish the compatibility of the Langlands functor with the operations of Eisenstein series constant term, and deduce that the Langlands functor induces an equivalence on Eisenstein-generated subcategories

Completeness and positive predictive value of registration of upper limb embolectomy in the Danish National Vascular Registry

Ljubica V Andersen1, Leif S Mortensen2, Jes S Lindholt3, Ole Faergeman4, Eskild W Henneberg3, Lars Frost51Department of Pharmacology, Odense University Hospital, Denmark; 2UNI-C, The Danish IT Centre for Education and Research, Aarhus, Denmark; 3Vascular Research Unit, Department of Vascular Surgery, Viborg Hospital, Denmark; 4Department of Cardiology and Internal Medicine, Aarhus University Hospital, Denmark; 5Department of Medicine, Silkeborg Hospital, DenmarkObjective: To evaluate completeness and positive predictive value of the Danish National Vascular Registry regarding registration of the surgical procedures: embolectomy of brachial, ulnar, or radial artery. Study design and settings: The study was based on first-time embolectomies in the brachial, ulnar, or radial artery performed in Denmark from January 1, 1990 to December 31, 2002. The data were primarily retrieved from the Danish National Vascular Registry and secondarily from the Danish National Registry of Patients. Medical records were retrieved using a standardized form.Results: In total, 1433 incident cases of first-time embolectomy were found in both registries. The positive predictive value of the registration was 97.5% (95% confidence interval [CI]; 96.4–98.4). The degree of completeness was 86.5% (95% CI; 84.3–88.5). For the registration period from 1990 till 1996 the degree of completeness was 78.2% (95% CI; 74.4–81.7), and from 1997 till 2002 it was 93.8% (95% CI; 91.6–95.7). Conclusion: The completeness and positive predictive value of registration of embolectomy in the upper limb in the Danish National Vascular Registry was 86.5% and 97.5%, respectively. This registry can be a valuable tool for epidemiological research and quality-monitoring. Keywords: positive predictive value, completeness, capture-recapture method, validity, embolectomy, registration

Phosphoproteomic analysis of cells treated with longevity-related autophagy inducers

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Obstáculos para a implementação governamental de dietas saudáveis com base científica e como superá-los

The Bellagio Report on Healthy Agriculture, Healthy Nutrition, Healthy People was science-based but policy-oriented. The role and amount of healthy and unhealthy nutrients in foods that may underlie the epidemics of non-communicable diseases worldwide was extensively discussed, with attention to the relative content of omega-3 and omega-6 fatty acids, and also sugar, particularly fructose. The report concludes that sugar consumption, especially in the form of high energy fructose in soft drinks, poses a major and insidious health threat, and most diets are defficient in omega-3 fatty acids and too high in omega-6 fatty acids. The meeting established that: governments must elevate, as a matter of urgency, Nutrition to a national priority; access to a healthy diet should be considered a human right; and the lead responsibility for Nutrition should be placed in Ministries of Health rather than Agriculture so that the health requirements drive agricultural priorities, not vice versa.O Relatório de Bellagio sobre Agricultura Saudável, Alimentação Saudável, Populações Saudáveis foi baseado em ciência, mas politicamente orientado. Foram amplamente discutidos o papel e a quantidade de nutrientes saudáveis e não saudáveis em alimentos que podem estar subjacentes à epidemia de doenças não transmissíveis em todo o mundo, com atenção ao conteúdo relativo de ácidos graxos ômega-3 e ômega-6, e também de açúcar, especialmente frutose. O relatório conclui que o consumo de açúcar, principalmente na forma de frutose de alta energia em refrigerantes, representa uma grande e insidiosa ameaça à saúde, e a maioria das dietas é deficiente em ácidos graxos ômega-3 e muito rica em ômega-6 ácidos graxos. A reunião estabeleceu que: os governos devem elevar, como uma questão de urgência, a Nutrição para uma posição de prioridade nacional; o acesso a uma dieta saudável deve ser considerado um direito humano; e a responsabilidade principal sobre a Nutrição deve ser colocada nas mãos do Ministério da Saúde, em vez do da Agricultura, para que os requisitos de saúde conduzam as prioridades agrícolas, e não vice-versa

Global warming: is weight loss a solution?

The current climate change has been most likely caused by the increased greenhouse gas emissions. We have looked at the major greenhouse gas, carbon dioxide (CO2), and estimated the reduction in the CO2 emissions that would occur with the theoretical global weight loss. The calculations were based on our previous weight loss study, investigating the effects of a low-carbohydrate diet on body weight, body composition and resting metabolic rate of obese volunteers with type 2 diabetes. At 6 months we observed decreases in weight, fat mass, fat free mass and CO2 production. We estimated that a 10 kg weight loss of all obese and overweight people would result in a decrease of 49.560 Mt of CO2 per year, which would equal to 0.2 % of the CO2 emitted globally in 2007. This reduction could help meet the CO2 emission reduction targets and unquestionably would be of a great benefit to the global health

Propionate reinforces epithelial identity and reduces aggressiveness of lung carcinoma

The epithelial-to-mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT-focused integrative functional genomic approach and identified an inverse association between short-chain fatty acids (propionate and butanoate) and EMT in non-small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell-to-cell contact and cell adhesion, while reducing the aggressive and chemo-resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.</p