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Lipid composition of caveolae and of surrounding plasma membrane in rat adipocytes
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Department of Clinical Neuroscience, Sahlgrenska University Hospital/Mölndal, Göteborg University, Mölndal, Sweden.
Department of Clinical Neuroscience, Sahlgrenska University Hospital/Mölndal, Göteborg University, Mölndal, Sweden.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Caveolae are invaginations of the plasma membrane that may arise from so called rafts in the presence of the structural protein caveolin. We have isolated caveolae from purified plasma membrane of primary rat adipocytes using ultrasonication to disrupt the membrane followed by density gradient ultracentrifugation. This caveolae fraction was further purified by adsorption to antibodies against caveolin. As a comparison we also isolated a detergent-insoluble fraction of the plasma membrane, utilizing the detergent insolubility of caveolae and rafts. Caveolae were strongly enriched in cholesterol and sphingomyelin, the concentration was 3.5 and 2.8-fold, respectively, higher in the caveolar membrane than in the surrounding plasma membrane. Phosphoacylglycerols were also concentrated in caveolae, while proteins were depleted compared to the surrounding plasma membrane. We have calculated that an average adipocyte caveola contains 18000 molecules of cholesterol, 6000 of sphingomyelin, 18000 of phosphoacylglycerol, 350 protein molecules, and about I 00 glycolipid molecules.

We analyzed for a range of glycolipids and especially gangliosides. Of these GM3 and GD3 are the most prevalent and both were enriched in caveolae, together with GM1 and GDla. GDlb and GTib were present in the plasma membrane at low levels, while GM2, GD2, GQ1b, sulphatide, and lactosylceramide sulphate were not detected. None of them were detected in caveolae. As a first comprehensive and quantitative analysis of purified caveolae from primary cells, our results provide a firm basis for the examination of caveolae formation using artificial membranes.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84464OAI: oai:DiVA.org:liu-84464DiVA: diva2:559411
Available from: 2012-10-09 Created: 2012-10-09 Last updated: 2013-09-10Bibliographically approved
In thesis
1. Caveolae in insulin signalling in human and rat adipocytes
Open this publication in new window or tab >>Caveolae in insulin signalling in human and rat adipocytes
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The pancreatic hormone insulin is a key hormone in maintenance of metabolic homeostasis but it also exerts control on gene expression and cell growth. This thesis presents results on fhe role of caveolae in insulin signalling in human and rat adipocytes. Caveolae are invaginations of the plasma membrane, characterised by the structural protein caveolin. Caveolae and caveolin have been implicated in a variety of functions, like uptake of molecular cargo into the cell, cholesterol transport and signal transduction. After isolation of caveolae and using electron microscopy on cell membranes, the insulin receptor was demonstrated to be localised in caveolae of human adipocytes. We also used biochemical and morphological methods to show that the glucose transporter GLUT4 was translocated to caveolae in response to insulin in rat adipocytes, indicating fhat the caveola is the locale for glucose uptake in adipocytes.

Adipocytes fhat were depleted of cholesterol using ß-cyclodextrin lacked caveolae invaginations. In cells fhus depleted of cholesterol and caveolae, fhe insulin receptor itself was not affected, but insulin signalling to metabolic control was inhibited. In rat adipocytes, insulin signalling to mitogenic control was not affected. In human fat cells, however, insulin's mitogenic signalling was dependent on caveolae/cholesterol. In contrast to other cells studied, including rat adipocytes, where the insulin receptor substrate (IRS-1) is mainly cytosolic, in human adipocytes IRS-1 was found in the plasma membrane and in caveolae. These results show the importance of choosing the relevant system to work with, since there are clear species differences.

We performed an analysis of the lipid composition of purified caveolae from rat adipocytes. As expected, cholesterol constitutes a major part of caveolae, but there is also an enrichment of sphingomyelin and the gangliosides GM1, GM3, GD3 and GD1a, while there is less protein, compared to the surrounding plasma membrane.

Taken together, caveolae appear as hnbs for insulin signalling. Caveolae seem necessary for fhe maintenance of metabolic signalling, like glucose uptake, and defects in caveolae may thus be the cause of insulin resistance.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 54 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 782
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25673 (URN)10049 (Local ID)91-7373-539-6 (ISBN)10049 (Archive number)10049 (OAI)
Public defence
2003-04-11, Berzeliussalen, Hälsouniversitet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-09Bibliographically approved

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Karlsson, MargaretaNyström, Fredrik H.Gustavsson, JohannaStrålfors, Peter

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