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Central adipocyte functions in specialized caveolae
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Obesity and overweight is an increasing health problem in the world. Besides reducing quality of life obesity is a great risk factor for development of diseases such as type 2 diabetes, cardiovascular disease, and stroke. The adipose tissue is a flexible organ that grows in size if the body is provided with excess energy. Adipocytes store the excess energy in the form of triacylglycerol. The incorporation of fat is stimulated by insulin, while catecholamines promote hydrolysis of triacylglycerol. In the membrane of adipocytes there are cave-like invaginations, caveolae, where proteins involved in insulin signalling, such as the insulin receptor are located. The focus of this thesis is on the function, and thereby the importance, of adipocytes and caveolae in metabolism.

The lipid content of the plasma membrane and caveolae was analyzed and compared to lipid phases determined in model membranes. The amount of different membrane lipids indicated heterogeneity of the caveolae, since there were not enough of some specific molecules to be present in every caveola. Using density gradient ultra centrifugation three different subclasses of caveolae were identified. These subclasses were named according to their relative densities: very high density caveolae, high density caveolae and low density caveolae. There were clear differences between the classes with regard to the distribution of specific proteins in them. In low density caveolae proteins linked to cholesterol metabolism, scavenger receptor class B type I and CD36, were abundant. Proteins involved in fatty acid metabolism, fatty acid transporter protein 1 and 4, acyl CoA-syntethase, perilipin and hormone sensitive lipase, were localized to high density caveolae. Cholesteryl ester uptake from HDL particles and hydrolysis to cholesterol was found in low density caveolae and the uptake of fatty acids was restricted to high density caveolae. Remarkably, the fatty acids were not only taken up via high density caveolae, but also incorporated into triacylglycerol, diacylglycerol, and phosphatidylcholine. This indicates that the whole machinery for lipid synthesis is located in these caveolae.

In addition to being an energy store the adipose tissue is also an endocrine organ. Several of the secreted proteins affect the metabolism of the body, for instance adiponectin that affects insulin sensitivity. Here adiponectin was located at the plasma membrane to the caveolae, both by biochemical isolation of caveolae and by electron microscopy. Together with earlier findings this indicates that caveolae may be involved in the secretion of adiponectin.

In conclusion, caveolae are involved in central processes such as glucose metabolism and signalling. Herein it is shown that caveolae is involved in the metabolism of fatty acids and cholesterol and may also be involved in the secretion of the metabolic hormone adiponectin. The findings in this thesis together with previous research establish caveolae as metabolic platforms.

Place, publisher, year, edition, pages
Linköping: Institutionen för biomedicin och kirurgi , 2006. , 50 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 971
Keyword [en]
Obesity, Overweight, Diabetes, Stroke, Cholesterol metabolism, Fatty acids
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-8244ISBN: 91-85643-37-8 (print)OAI: oai:DiVA.org:liu-8244DiVA: diva2:23093
Public defence
2006-12-08, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Available from: 2007-02-01 Created: 2007-02-01 Last updated: 2012-10-24Bibliographically approved
List of papers
1. Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes
Open this publication in new window or tab >>Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes
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2004 (English)In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 271, no 10, 2028-2036 p.Article in journal (Refereed) Published
Abstract [en]

We have made a comprehensive and quantitative analysis of the lipid composition of caveolae from primary rat fat cells and compared the composition of plasma membrane inside and outside caveolae. We isolated caveolae from purified plasma membranes using ultrasonication in carbonate buffer to disrupt the membrane, or extraction with nonionic detergent, followed by density gradient ultracentrifugation. The carbonate-isolated caveolae fraction was further immunopurified using caveolin antibodies. Carbonate-isolated caveolae were enriched in cholesterol and sphingomyelin, and the concentration was three- and twofold higher, respectively, in caveolae compared to the surrounding plasma membrane. The concentration of glycerophospholipids was similar suggesting that glycerophospholipids constitute a constant core throughout the plasma membrane. The composition of detergent-insoluble fractions of the plasma membrane was very variable between preparations, but strongly enriched in sphingomyelin and depleted of glycerophospholipids compared to carbonate-isolated caveolae; indicating that detergent extraction is not a suitable technique for caveolae preparation. An average adipocyte caveola contained about 22 × 103 molecules of cholesterol, 7.5 × 103 of sphingomyelin and 23 × 103 of glycerophospholipid. The glycosphingolipid GD3 was highly enriched in caveolae, whereas GM3, GM1 and GD1a were present inside as well as outside the caveolae membrane. GD1b, GT1b, GM2, GQ1b, sulfatide and lactosylceramide sulfate were not detected in caveolae.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-22409 (URN)10.1111/j.1432-1033.2004.04117.x (DOI)1621 (Local ID)1621 (Archive number)1621 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes
Open this publication in new window or tab >>Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 1, 5-8 p.Article in journal (Refereed) Published
Abstract [en]

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20650 (URN)10.1074/jbc.C400429200 (DOI)15537657 (PubMedID)
Available from: 2009-09-16 Created: 2009-09-16 Last updated: 2017-12-13Bibliographically approved
3. Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes: segregation of specific proteins and functions
Open this publication in new window or tab >>Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes: segregation of specific proteins and functions
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2006 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 14, 3381-3392 p.Article in journal (Refereed) Published
Abstract [en]

Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm. They constitute sites of entry into the cell as well as platforms for cell signalling. We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy. We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells. These caveolae contained caveolin-1 and caveolin-2. Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4. This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol. A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4. Small amounts of these proteins were also detected in the high-density caveolae. In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae. The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae. There was no correlation between the caveolar contents of caveolin and cholesterol. The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake/metabolism. These findings demonstrate a segregation of functions in caveolae subclasses.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-35681 (URN)10.1111/j.1742-4658.2006.05345.x (DOI)28128 (Local ID)28128 (Archive number)28128 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
4. Localization of adiponectin at the plasma membrane in caveolae of human adipocytes
Open this publication in new window or tab >>Localization of adiponectin at the plasma membrane in caveolae of human adipocytes
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The hormone adiponectin is exclusively secreted by adipocytes and it regulates whole body energy homeostasis primarily by affecting liver and muscles. The circulating level of adiponectin inversely correlates with insulin resistance and type 2 diabetes. Little is known about the secretion of adiponectin by the adipocyte, but in caveolin-1 knockout mice the amount of secreted adiponectin is severely reduced. Here we show that in human adipocytes adiponectin was associated with the plasma membrane and to a specific class of high-density-caveolae. Substantial amounts of adiponectin were also found in the microsomal and cytosolic fractions of adipocytes, while very little was associated with mitochondria, nuclei, or the fat. Adiponectin in isolated caveolae was readily degraded by added trypsin, demonstrating that adiponectin was largely bound to the cytosolic face of the caveolae membrane. The findings indicate that caveolae may have a critical role in the secretion of adiponectin by human adipocytes.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84835 (URN)
Available from: 2012-10-24 Created: 2012-10-24 Last updated: 2012-10-24Bibliographically approved

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