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Title: Interactions between telomerase reverse transcriptase (TERT) and insulin signalling in muscle
Author: Shaheen, Fozia
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2010
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Ageing is a well established risk factor for the development of the metabolic syndrome and type 2 diabetes (T2DM). It is well-recognised that there is an increase in insulin resistance with age, and disturbances in insulin signalling have been shown to affect both insulin sensitivity and lifespan. A decrease in mean telomere length provides a marker for biological age at the cellular level. Accelerated telomere shortening in both human and animal models has been documented in conditions associated with insulin resistance, including T2DM. Maintenance of telomere length is dependent on the activity of the enzyme telomerase (which is dependent on the expression of its catalytic protein TERT) which can help reverse this process and extend the lifespan of cells. When acting in this capacity, TERT is a nuclear protein, but recently it has been shown to possess the ability to shuttle between the nucleus and cytoplasm. The present study was performed with the aim of determining a possible role of TERT in insulin signalling in C2C12, primary mouse and human skeletal muscle cells. Confocal microscopy was used to study protein localisation; in parallel, changes in glucose uptake were determined by measuring 2-deoxyglucose. TERT protein in C2C12 cells was mostly non-nuclear (cytosolic) in localisation, and translocated to the plasma membrane in response to insulin. This translocation was inhibited by wortmannin and rapamycin indicating that this process was dependent on PI3K signalling (as is the insulin induced translocation of GLUT4) and also dependent on mTOR signalling. Altering TERT expression, through over-expression or RNAi-induced knock-down changed the intracellular distribution of GLUT1, GLUT4 and GLUT12, in a similar manner to the effect of insulin. GLUT1 and GLUT4 responded to insulin as expected in all the three cell systems, but there was a difference in the response of the recently identified GLUT12. GLUT12 in the C2C12 cells in the basal state was distributed throughout the cytoplasm and following insulin addition, became localised to peri-nuclear punctuate structures. By contrast, GLUT12 was localised to the plasma membrane (with some cytoplasmic presence) in primary mouse skeletal muscle tissue sections. Interestingly, under conditions of reduced plasma insulin levels in these mouse muscle sections GLUT12 remained abundantly present at the plasma membrane. Contrary to its actions in the C2C12 cells, insulin caused GLUT12 to mobilise from sub-plasma membrane pools and to translocate towards the plasma membrane in the human skeletal muscle cells. Furthermore, knocking-down GLUT12 expression in the C2C12 and primary mouse skeletal muscle cells decreased both basal and insulin dependent 2-deoxyglucose uptake. These observations suggest that this transporter may contribute towards overall glucose transport, but responds differently to an insulin stimulus in the C2C12 cells than in the primary mouse and human skeletal muscle cells. Importantly, GLUT12 distribution and expression were affected by a reduction in mTOR expression, but again there was a variation in the response between the C2C12 cells and both the other cell systems. However, over-expressing TERT increased 2-deoxyglucose uptake synergistically with the effect of insulin in all three cell systems. This increase in glucose transport was shown to be partly through the involvement of GLUT12, as knocking-down GLUT12 expression accounted for part of the increase in 2-deoxyglucose uptake seen in response to TERT over-expression. TERT protein was found to co-immunoprecipitate with all three glucose transporter proteins studied, suggesting that the effects of changes in TERT expression on GLUT intracellular localisation and glucose transport may be due to TERT-GLUT molecular interactions. Importantly, exposure to inhibitors of mTOR, PI3K and TERT did not diminish the ability of TERT to co-immunoprecipitate with GLUT1 and GLUT4 protein(s). However, geldanamycin, a Hsp90 inhibitor diminished the TERT-GLUT12 interaction, implying that the TERT-Hsp90-RAPTOR-mTOR complex formation may be important for this interaction. These findings suggest the existence of a novel extra-nuclear function of TERT and a possible mechanism that appears to regulate glucose transport, acting synergistically with insulin signalling. Although, TERT interacts with all the GLUT proteins studied, it appears to affect GLUT12 differently to GLUT1 and GLUT4 to regulate glucose transport and mTOR signalling appears to be important for GLUT12 function. Therefore, these results raise the prospect of the existence of a link between alterations in the extra-nuclear distribution of TERT and changes in insulin-dependent glucose transport.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology