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Insulin-induced GLUT4 translocation to the plasma membrane is blunted in large compared with small primary fat cells isolated from the same individual
Linköping University, Department of Medicine and Health Sciences, Internal Medicine . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Medicine and Health Sciences, Internal Medicine . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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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.

Place, publisher, year, edition, pages
2007. Vol. 50, no 8, 1716-1722 p.
Keyword [en]
Adipocyte, GLUT4, Human, Insulin, Insulin receptor, Insulin resistance, IRS-1, Primary fat cell
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-14541DOI: 10.1007/s00125-007-0713-1OAI: oai:DiVA.org:liu-14541DiVA: diva2:23676
Available from: 2007-06-01 Created: 2007-06-01 Last updated: 2017-12-13Bibliographically approved
In thesis
1. On the importance of fat cell size, location and signaling in insulin resistance
Open this publication in new window or tab >>On the importance of fat cell size, location and signaling in insulin resistance
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Obesity has reached epidemic proportions worldwide and is associated with insulin resistance, type 2 diabetes and cardiovascular disease. During the past decades, substantial evidence has demonstrated that not only the amount of adipose tissue constitutes a major determinant in the development of metabolic disorders, but also the distribution. The visceral adipose tissue has shown to be stronger correlated with insulin resistance, type 2 diabetes and cardiovascular disease than the subcutaneous depot. When we measured the activity of the nuclear receptor PPARγ in visceral and subcutaneous adipocytes, we found considerably lower activity in fat cells obtained from the visceral portion. This finding provides additional evidence to the unfavorable consequences of visceral obesity. The common PPARγ polymorphism Pro12Ala was studied in type 2 diabetic patients. We found that men with the Ala isoform exhibited higher sagittal abdominal diameter, waist circumference and body weight compared with homozygotes for the Pro isoform. However, no differences in either gender with regard to blood pressure or markers of cardiovascular disease and organ damage could be observed.

In addition to an excessive visceral adipose tissue mass, obese subjects with enlarged adipocytes display an increased risk for developing metabolic disorders compared with individuals exhibiting smaller fat cells but a similar degree of adiposity. The insulin responsiveness in small and large adipocytes obtained from the same subject was examined. Upon insulin stimulation, we found approximately a 2 fold increase of GLUT4 at the plasma membrane in small adipocytes, whereas the large fat cells were refractory to insulin induced GLUT4 translocation. This finding demonstrates a causal relationship between the accumulation of large fat cells in obese subjects and reduced insulin responsiveness.

Caloric restriction in humans ameliorates insulin responsiveness in liver and muscle prior to any substantial weight loss. By combining gene expression profiles of adipose tissue and adipocytes from human subjects undergoing either caloric restriction or overfeeding, we identified genes regulated by changes in caloric intake independent of weight loss per se. We found several genes under the control of mTOR and SREBP1 as well as genes involved in β-oxidation, liberation of fatty acids and glyceroneogenesis to be regulated during the interventions. These genes may indicate pathways and mechanisms mediating the effects of nutrient deprivation and obesity on morbidity and mortality.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 51 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1123
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18466 (URN)978-91-7393-640-8 (ISBN)
Public defence
2009-06-03, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2009-05-28 Created: 2009-05-28 Last updated: 2013-09-10Bibliographically approved
2. A molecular approach to insulin signalling and caveolae in primary adipocytes
Open this publication in new window or tab >>A molecular approach to insulin signalling and caveolae in primary adipocytes
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
caveolae, insulin signalling, adipocytes
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-8960 (URN)91-85497-94-0 (ISBN)
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

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Franck, NiclasStenkula, Karin G.Lindström, TorbjörnStrålfors, PeterNyström, Fredrik H.Öst, Anita

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