Michael Stallings and Professor Chad Hancock, Department of Nutrition, Dietetics, and Food Science
Type 2 Diabetes Mellitus is a public health concern for the United States and is becoming a global health crisis. The hallmark of type 2 diabetes is high blood sugar, which leads to a host of other health issues. In healthy subjects, insulin acts as a signal to increase uptake of glucose from the blood stream into skeletal muscle. However, with the onset of diabetes skeletal muscle becomes resistant to insulin signaling. One of the many factors involved in skeletal muscle insulin resistance is Forkhead Box Protein 1a
(FOXO1a) [1].
FOXO1a is an important protein that helps regulate blood glucose levels. It is a transcription factor, which means it can increase the levels of other proteins in the cell. It has been shown to interact with the transcription site of Thioredoxin Interacting Protein (TXNIP) [2]. TXNIP has been shown in some models to regulate peripheral glucose uptake [3]. I investigated the link between FOXO1a and TXNIP levels in mouse skeletal muscle to determine if they affected each other.
Western Blotting is one way to determine protein levels in skeletal muscle. I homogenized the hind limb muscle of our test mice and treated the tissues in several solutions to prepare for Western blotting. I then prepared for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) by performing a protein assay and making samples for the blot.
SDS-PAGE was performed on precast gels. Protein was transferred from the gels to nitrocellulose membranes after equilibrating in transfer buffer. The membranes were then blocked in a five percent milk solution for one hour and incubated with primary antibody overnight at 5 degrees Celsius. The membranes were then washed and incubated for an hour in secondary antibody at room temperature. After a final wash, electrochemical luminescent reagents were applied to the membrane and signal strength was determined via autoradiography film.
After scanning the developed film, pixel saturation was quantified using National Institutes of Health software as a measure of protein level. Groups were compared using 2-way Analysis of Variance with Sigma Stat software.
There were statistically significant changes in FOXO1a levels in several skeletal muscles. Mice have a variety of muscle types. Some are better suited for endurance activity and others for fast twitch movement. We saw a spectrum of increased FOXO1a levels in response to the treatment. FOXO1a was not increased in red quadriceps, a small but significant increase occurred in white quadriceps and the largest increase was seen in the tibialis anterior. Importantly, this response was not reflected in levels of TXNIP.
These data show that in male mice, increases in the transcription factor FOXO1a do not change levels of TXNIP in a variety of skeletal muscle fiber types. Although aberrant glucose metabolism in skeletal muscle is associated with increased FOXO1a, these aberrations are not mediated by changes in TXNIP.
References
- Müssig K , Staiger H , et al. (2009). “Association of common genetic variation in the FOXO1 gene with beta-cell dysfunction, impaired glucose tolerance, and type 2 diabetes.” Journal of Clinical Endocrinology and Metabolism, Volume 94 Issue 4:1353-
60. - de Candia, P. et al. (2008). “A Combination of Genomic Approaches Reveals the Role of FOXO1a in Regulating an Oxidative Stress Response Pathway.” PLoS ONE Volume 3, Issue 2: e1670.
- Muoio, M. (2007). “TXNIP Links Redox Circuitry to Glucose Control.” Cell Metabolism, Volume 5, Issue 6, Pages 412-414.