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A molecular approach to insulin signalling and caveolae in primary adipocytes
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear.

Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell.

The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects.

The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane.

In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

Place, publisher, year, edition, pages
Institutionen för biomedicin och kirurgi , 2007. , 63 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 977
Keyword [en]
caveolae, insulin signalling, adipocytes
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:liu:diva-8960ISBN: 91-85497-94-0 (print)OAI: oai:DiVA.org:liu-8960DiVA: diva2:23677
Public defence
2007-01-19, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2013-09-10
List of papers
1. Expression of a mutant IRS inhibits metabolic and mitogenic signalling of insulin in human adipocytes
Open this publication in new window or tab >>Expression of a mutant IRS inhibits metabolic and mitogenic signalling of insulin in human adipocytes
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2004 (English)In: Molecular and Cellular Endocrinology, ISSN 0303-7207, Vol. 221, no 1-2, 1-8 p.Article in journal (Refereed) Published
Abstract [en]

Adipose tissue is a primary target of insulin, but knowledge about insulin signalling in human adipocytes is limited. We developed an electroporation technique for transfection of primary human adipocytes with a transfection efficiency of 15% ± 5 (mean ± S.D.). Human adipocytes were co-transfected with a mutant of IRS-3 (all four potential PI3-kinase binding motifs mutated: IRS-3F4) and HA-tagged protein kinase B (HA-PKB/Akt). HA-PKB/Akt was immunoprecipitated from cell lysates with anti-HA antibodies, resolved with SDS-PAGE, and immunoblotted with phospho-specific antibodies. We found that IRS-3F4 blocked insulin stimulation of HA-PKB/Akt phosphorylation and in further analyses also translocation of recombinant HA-tagged glucose transporter to the plasma membrane. IRS-3F4 also blocked insulin-induced activation of the transcription factor Elk-1. Our results demonstrate the critical importance of IRS for metabolic as well as mitogenic signalling by insulin. This method for transfection of primary human adipocytes will be useful for studying insulin signalling in human adipocytes with molecular biological techniques.

Keyword
Insulin, Transfection, Human, Adipocytes, Protein kinase B, Elk-1
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14538 (URN)10.1016/j.mce.2004.04.011 (DOI)000222854100001 ()
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2013-10-22Bibliographically approved
2. Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes
Open this publication in new window or tab >>Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes
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2003 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 14, no 10, 3967-3976 p.Article in journal (Refereed) Published
Abstract [en]

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14539 (URN)10.1091/mbc.E03-01-0050 (DOI)
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2017-12-13
3. Human, but not rat, IRS1 targets to the plasma membrane in both human and rat primary adipocytes
Open this publication in new window or tab >>Human, but not rat, IRS1 targets to the plasma membrane in both human and rat primary adipocytes
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2007 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 363, no 3, 840-845 p.Article in journal (Refereed) Published
Abstract [en]

Adipocytes are primary targets for insulin control of metabolism. The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS1), which acts as a docking protein for downstream signal mediators. In the absence of insulin stimulation, IRS1 in rat adipocytes is intracellular but in human adipocytes IRS1 is constitutively targeted to the plasma membrane. Stimulation of adipocytes with insulin increased the amount of IRS1 at the plasma membrane 2-fold in human adipocytes, but >10-fold in rat adipocytes, with the same final amount of IRS1 at the plasma membrane in cells from both species. Cross-transfection of rat adipocytes with human IRS1, or human adipocytes with rat IRS1, demonstrated that the species difference was due to the IRS1 protein and not the cellular milieus or posttranslational modifications. Chimeric IRS1, consisting of the conserved N-terminus of rat IRS1 with the variable C-terminal of human IRS1, did not target the plasma membrane, indicating that subtle sequence differences direct human IRS1 to the plasma membrane.

Keyword
Insulin receptor substrate; Human; Rat; Insulin; Plasma membrane; Signaling; Transfection; Caveolae
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14540 (URN)10.1016/j.bbrc.2007.09.065 (DOI)
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2017-12-13
4. Insulin-induced GLUT4 translocation to the plasma membrane is blunted in large compared with small primary fat cells isolated from the same individual
Open this publication in new window or tab >>Insulin-induced GLUT4 translocation to the plasma membrane is blunted in large compared with small primary fat cells isolated from the same individual
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2007 (English)In: Diabetologia, ISSN 0012-186X, E-ISSN 1432-0428, Vol. 50, no 8, 1716-1722 p.Article in journal (Refereed) Published
Abstract [en]

Aims/hypothesis: Several studies have suggested that large fat cells are less responsive to insulin than small fat cells. However, in these studies, large fat cells from obese individuals were compared with smaller fat cells from leaner participants, in effect making it impossible to draw conclusions about whether there is a causal relationship between fat cell size and insulin sensitivity. We hypothesised that small fat cells might be more insulin-responsive than large adipocytes when obtained from the same individual.

Materials and methods: We developed a method of sorting isolated primary human fat cells by using nylon filters of two different pore sizes. The cells were stained to visualise DNA, which allowed discrimination from artefacts such as lipid droplets. The sorted cells were left to recover overnight, since we had previously demonstrated that this is necessary for correct assessment of insulin response.

Results: We found similar amounts of the insulin receptor (IR), IRS-1 and GLUT4 when we compared small and large adipocytes from the same volunteer by immunoblotting experiments using the same total cell volume from both cell populations. Activation of IR, IRS-1 and Akt1 (also known as protein kinase B) by insulin was similar in the two cell populations. However, immunofluorescence confocal microscopy of plasma membrane sheets did not reveal any increase in the amount of GLUT4 in the plasma membrane following insulin stimulation in the large fat cells, whereas we saw a twofold increase in the amount of GLUT4 in the small fat cells.

Conclusions/interpretation: Our results support a causal relationship between the accumulation of large fat cells in obese individuals and reduced insulin responsiveness.

Keyword
Adipocyte, GLUT4, Human, Insulin, Insulin receptor, Insulin resistance, IRS-1, Primary fat cell
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14541 (URN)10.1007/s00125-007-0713-1 (DOI)
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2017-12-13Bibliographically approved

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