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…
Contents
INTRODUCTION
Insulin action and resistance
Adipose tissue
Visceral and subcutaneous fat tissues
Peroxisome proliferator activated receptor-γ
Regulation of whole body insulin-sensitivity
Importance of the size of adipocytes
Insulin signalling pathways and signal mediators
Insulin receptor
Insulin receptor substrate
PI3-kinase
Cbl/CAP complex
Growth factor receptor-binding protein
Glucose transporter
Signalling through caveolae
Caveolae
Importance of caveolae for insulin signalling
Caveolin
Distribution and importance of caveolin in caveolae
Recombinant expression
Molecular biology in the study of signal transduction
Fluorescent tags
Transfection techniques
Electroporation
Recombinant expression in adipocytes
SPECIFIC AIMS OF THE THESIS
MATERIAL AND METHODS
Materials
IRS constructs
Isolation of adipocytes
Cell culture and transfection of AG-01518
Separation of small and large cells
Electroporation
Measurement of Elk-1 phosphorylation
Sample preparations for SDS-PAGE and immunoblotting
SDS-PAGE and immunoblotting
Fluorescence microscopy
Immunofluorescence confocal microscopy of plasma membrane sheets
Electron microscopy of plasma membrane sheets
Electron microscopy of intact cells
Immunofluorescence deconvolution microscopy
RESULTS AND DISCUSSION
PAPER I
Results
Optimization
Insulin sensitivity
Importance of intact IRS function for insulin-induced signalling
Discussion
PAPER II
Results
Openings of caveolae
Localization of caveolin in caveolae
Recombinantly expressed caveolin-1 and -2
Caveolin distribution in human fibroblasts
Discussion
PAPER III
Results
Insulin-regulated IRS1 distribution
Human IRS1 in rat cells and rat IRS1 in human cells
PI3-kinase sensitive translocation
IRS1 chimer
Discussion
PAPER IV
Results
Method for separation
Insulin sensitivity in small and large cells
Amount of insulin receptor, IRS1 and caveolin-1
Insulin-induced activation of Akt
Discussion
CONCLUSIONS
ACKNOWLEDGEMENTS
REFERENCES
Author: Stenkula, Karin
Source: Linköping University
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