Kirkpatrick, Euan
(2024)
The roles of muscle contraction and pharmaceuticals on
restoring glucose uptake in a mouse skeletal muscle
model of immobilization.
PhD thesis, University of Nottingham.
Abstract
Current understanding of muscle atrophy during immobilization and weightlessness is
incomplete, partially due to the barriers to entry in the study of whole muscle. For this reason,
a readily available model based on the C2C12 cell line is highly desirable as it would
significantly increase the number of researchers able to engage in study of changes to skeletal
muscle as a result of inactivity. The work presented here was in part funded as part of a newly
established Astropharmacy cohort, which aims to tackle problems facing future manned space
exploration. As such, the experiments performed here were planned and analysed with this
perspective in mind. Inactivity is known to result in both muscle atrophy and insulin resistance.
Terrestrially this is thought to be a key step in the development of diabetes as skeletal muscle is
critical for the disposal of glucose during physical activity. For this reason, we are interested in
establishing a pharmacological model of immobilisation with associated features including
reduced glucose uptake (insulin resistance) and then restoring activity either by EPS (electric
pulse stimulation) or further pharmaceutical intervention.
Chapter 2
Firstly, we confirmed the suitability of C2C12 cells in our hands as a platform for measurement
of glucose uptake using 2-Deoxyglucose (2DG). We determined that at a final concentration of
25µM per well 2-deoxyglucose could be used to trace glucose uptake over a 24-hour period in
both the basal and EPS treated state. We confirmed normal cell responses to EPS in terms of
anabolic signalling changes, glucose uptake and lactate production. The ability of cells to
respond to insulin was also tested and confirmed.
Chapter 3
After establishing the suitability of the platform, we were interested in determining the effect
of repeated bouts of contraction throughout a 24-hour period effected the endpoints
measured previously- glucose uptake, lactate output and anabolic signalling- as well as the
addition of cell glycogen content. We found that there were no significant differences in
glucose uptake, cell glycogen, lactate output or p-P70 (T389), p-4EBP1 (T37/46), p-mTOR
(S2448) at the 24-hour timepoint with any of the frequency/ duration combinations that we
attempted. This was attributed to a combination of the lack of a maintenance pulse during the
rest periods and possible proximity effect.
Chapter 4
Returning to 24-hour continuous EPS we established the ability of combined CPA and
blebbistatin (CB) treatment to inhibit contraction in c2c12 cells at concentrations of 100 µM
and 10µM respectively. This was paired with increases in biomarkers (ATP2A1 and CALM1) that
indicate elevated cytosolic calcium. When treated with EPS the CPA and blebbistatin model
showed reduced glucose uptake, reduced P70, ERK1/2 phosphorylation and elevated 4EBP1
phosphorylation compared to cells treated with EPS alone, indicating prevention of contraction
and consequent downstream effects in line with immobilisation.
Chapter 5
Lastly, we attempted to restore glucose uptake, glycogen, lactate output, anabolic signalling, p
EEF2 (T56), p-PKB (T3080), P-ERK1/2 (S217/221) to baseline values by application of
combinations of EPS, AICAR and dantrolene. We found that application of these drugs reduced
glucose uptake beyond what treatment with CB alone had. However, lactate output and
markers of cytosolic calcium improved with dantrolene treatment. In cells that did not receive
CB treatment, we found that EPS had a neutral or negative effect in the case of AICAR on the
ability of drug treatment to stimulate additional glucose uptake, which may suggest
incompatibility between pharmacological and exercise-based uptake methods, possibly due to
conflicting signalling between EPS which favours transient changes and drug treatments which
favour sustained changes.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Brook, Matthew
Wilkinson, Daniel
Atherton, Philip
Williams, Philip
Greenhaff, Paul |
| Keywords: |
Glucose uptake; skeletal muscle |
| Subjects: |
Q Science > QP Physiology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
77412 |
| Depositing User: |
Kirkpatrick, Euan
|
| Date Deposited: |
17 Jul 2024 04:40 |
| Last Modified: |
17 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/77412 |
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