The Subcellular Distribution and Insulin: Responsiveness of Facilitative Glucose Transporters in Transfected Cells

Brant, Alison Mary (1994) The Subcellular Distribution and Insulin: Responsiveness of Facilitative Glucose Transporters in Transfected Cells. PhD thesis, University of Glasgow.

Abstract

Specific anti-peptide antibodies, raised against the C-terminal portion of mouse GLUT 3 were used to examine the tissue distribution of GLUT 3 protein in various mouse tissues. Previous studies have shown that in human tissues GLUT 3 mRNA is detectable in a wide range of tissues but that GLUT 3 protein levels are highest in the brain, with lower levels being detectable in heart, placenta and fat. Results from this investigation indicated that, in contrast to humans, the expression of GLUT 3 protein in mice was restricted to the brain and neurally derived cells only. An investigation to determine the levels of GLUT 3 protein within different rat brain regions was then carried out since in situ hybridisation analysis of GLUT 3 in the mouse brain had previously indicated that the expression of this isoform was not homogeneous throughout all regions of the brain. After demonstrating that anti- GLUT 3 antibodies were able to cross-react with rat GLUT 3 protein, an examination of GLUT 3 protein expression levels in individually dissected rat brain regions was carried out. The levels of GLUT 1 protein were also determined since this transporter constitutes the blood-brain barrier transport protein. Results indicated that when GLUT 1 and GLUT 3 levels were expressed relative to the levels measured in the frontal cortex, the trend was such that GLUT 1 levels increased, while GLUT 3 levels decreased towards the more posterior brain regions. It was therefore postulated that a low level of one transporter is compensated for by the expression of another. In addition, rat brain regions were screened for the expression of GLUT 2 and GLUT 4 protein. GLUT 4 expression was detected in three regions; the pituitary, hypothalamus, and cerebellum while GLUT 2 was detected in all brain regions analysed. The detection of these latter proteins may indicate that brain regions possess a "glucose-sensing" mechanism similar to the GLUT 2-expressing beta-cells of the pancreas, and that particular regions may be insulin-sensitive. GLUT 2 and GLUT 4 are expressed in insulin-responsive tissues, liver and fat respectively, yet only GLUT 4 undergoes translocation from an intracellular region to the plasma membrane following insulin stimulation. In an attempt to determine whether translocation is a process governed by the type of cell in which the glucose transporter protein is expressed or whether it is a property encoded in the protein sequence, GLUT 2 was expressed in an adipocyte cell environment (3T3-L1 cells) and GLUT 4 was expressed in a liver-type cell environment (H4IIe cells). Immunoblotting of cell lysates prepared from GLUT 2-transfected 3T3-L1 cells and GLUT 4-transfected H4IIe cells with anti-GLUT 2 and anti-GLUT 4 antibodies revealed that GLUT 4 and GLUT 2 proteins were expressed in the relevant cells. Results indicated that GLUT 4-expressing H4IIe cells exhibited a greater increase in 2-deoxyglucose uptake in response to insulin relative to non GLUT 4-expressing cells. However GLUT 4 translocation in response to insulin could not be conclusively demonstrated. GLUT 2-expressing 3T3-L1 adipocytes exhibited a significant increase in basal 2-deoxyglucose uptake relative to non GLUT 2-expressing cells, but an approximate 3 to 6-fold reduction in insulin-stimulated 2-deoxyglucose uptake. GLUT 2 protein was predominandy localised to the plasma membrane in both basal and insulin-stimulated cells, and did not exhibit significant translocation in response to insulin. This reduced insulin-stimulated glucose transport could not be explained by a down-regulation in GLUT 4 expression in these GLUT 2-expressing cells since GLUT 4 levels were similar in both GLUT 2 and non-GLUT 2 expressing adipocytes. The nature of the reduced sensitivity to insulin with regard to glucose transport in GLUT 2-expressing adipocytes thus remains to be elucidated.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Gwyn Gould
• Keywords: Biochemistry
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-75635
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 19:03
• Last Modified: 19 Nov 2019 19:03
• URI: https://theses.gla.ac.uk/id/eprint/75635