Abstract
Insulin and its receptor are widely expressed in a variety of tissues throughout the body including liver, adipose tissue, liver and brain. The insulin receptor is expressed as two functionally distinct isoforms, differentiated by a single 12 amino acid exon. The two receptor isoforms, designated IR/A and IR/B, are expressed in a highly tissue and cell specific manner and relative proportions of the different isoforms vary during development, aging and disease states. The high degree of similarity between the two isoforms has prevented detailed studies as differentiation of the two isoforms by traditional immunological methods cannot be achieved. We describe here a new in situ RT-PCR/ FISH assay that allows for the visualization of IR/A and IR/B in tissue along with tissue specific markers. We used this new method to show for the first time that IR/A and IR/B are both expressed in neurons in the adult human brain. Thus, we present a method that enables the investigation of IR/A and IR/B insulin receptor isoform expression in situ in various tissues.
Highlights
Signaling through insulin receptor occurs through the phosphoinositol-3 kinase (PI3K), the specific PI3K utilized by each isoform determines downstream signaling cascades[6]
We describe here the development of an in situ RT-PCR/ FISH assay for the differential detection of IR/A and IR/B in cultured cells and in human tissue that, by coupling with immunohistochemistry, allows the specific localization of IR/A and IR/B expression to specific cell types for the first time
We first analyzed the expression of insulin receptor isoforms in two brain-derived cell lines using the isoform specific primers for quantitative PCR
Summary
Signaling through insulin receptor occurs through the phosphoinositol-3 kinase (PI3K), the specific PI3K utilized by each isoform determines downstream signaling cascades[6]. We describe here the development of an in situ RT-PCR/ FISH assay for the differential detection of IR/A and IR/B in cultured cells and in human tissue that, by coupling with immunohistochemistry, allows the specific localization of IR/A and IR/B expression to specific cell types for the first time. This technique will be useful in the study of specific IR isoform expression in a variety of human tissues such as the brain, liver and pancreas to understand their role in development and disease
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