开题报告内容：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）
一、课题背景
The Na/K-ATPase, discovered in 1957 by Jens Skou, is a member of the P-type ATPase family and it is also an integral membrane protein maintaining cellular ion homeostasis by pumping Na and K across the cell membrane[1]. The protein consists of two noncovalently linked subunits, a catalytic subunit and an auxiliary subunit[2]. Four isoforms of the Na-K-ATPase alpha;-subunit are expressed in humans. While alpha;1 is expressed ubiquitously, alpha;2 is primarily found in myocytes and 3 in neurons, and alpha;4 is only detected in sperm [3] [4] [5][6] [7] [8] [9].
Na-K-ATPase is a fundamental component of ion transport. Recent studies show that the Na-K-ATPase may have some regulatory functions in addition to pumping ions across cell membranes. Also many studies have documented an important signaling function of the Na-K-ATPase.[10] [11] Specially, some scientists have shown that Na-K-ATPase alpha;1 is able to keep the Src kinase in an inactive state. [12] [13] Na, K ATPase and Src kinase form an inactivate complex, which is a receptor through which cardiotonic steroids such as ouabain stimulate protein and lipid kinase cascades and subsequently regulate many cellular activities like cell growth and metabolism.
The 1 isoform was found to mediate signal transduction through Src kinase, however, the Na-K-ATPase alpha;2, does not regulate Src and, therefore, may not serve the same role in signal transduction as alpha;1. This further demonstrated that the signaling mechanism of Na-K-ATPase is isoform specific, which supports a model where alpha;1 and alpha;2 isoforms play different roles in mediating contraction and signaling in myocytes.[14] Because of that, our project focused on the 1 signaling function.
Glycogen is a complex glucose polymer found in most species in animals. It serves as a storage form for glucose found in a variety of tissues.[15]
It has been understood for more than half a century that the ability of muscle to exercise is seriously compromised when the glycogen store is reduced to low levels, even when there is an abundance of other fuel sources.[16] Hultman and co-workers demonstrated a strong correlation between muscle glycogen content and endurance capacity during prolonged cycling exercise and an inability to continue such exercise when the glycogen stores were exhausted.[17] Also it was found that there is a close relationship between muscle glycogen content and fatigue resistance, both during prolonged (more than 1 h) and high-intensity intermittent exercises. [18] [19] [20] [21]
二、要解决的问题:
We came up with a hypothetical pathway through which the glycogen content in skeletal muscle will be affected by Na, K ATPase 1 iosform. And we are going to prove if this hypothesis is correct by determining the expression of important proteins in this pathway.
- 可行性分析
Na, K ATPase 1 is an isoform found to mediate signal transduction, and it is found that the 1 knock out mice have decreased endurance capacity. Based on the relationship between endurance capacity and glycogen content in skeletal muscle other scientists found, my project will talk about how the glycogen content in skeletal muscle is regulated by Na, K ATPase 1 isoform. And also there has been scientists in our lab proving that the glycogen content in skeletal muscle of fed mice is regulated by Na, K ATPase 1 isoform, what I plan to do is to prove similar pathway but in fasted mice.
- 研究方法和内容
Mice Exercise:
Treadmill testing: Fifteen weeks old female alpha;1 /- mice and litter mate controls were placed in the six lanes of an Exer 3/6 treadmill from Columbus Instruments. Animals were acclimated to the treadmill for 3 days at 5 m/min for 5 minutes at a 5° angle and were subjected to the testing protocol on the fourth day. Mice began the testing protocol running at 5 m/m for five minutes and increased by 2 m/min each minute up to 25 m/min, then continued running at 25 m/min until they reached fatigue. Fatigue was defined as 10 consecutive seconds spent on the shock grid, and the shock was discontinued to each mouse upon reaching fatigue. Distance at fatigue was recorded for each animal, and distance to fatigue was compared on Kaplan-Meier survival curves using the Gehan-Breslow-Wilcoxon test, where plt;0.05 was considered statistically significant.
Glycogen Levels Measurement:
We isolate the gastrocnemius muscle from fed mice, homogenized muscles and measured the glycogen levels according to the Glycogen Assay Kit II (Colorimetric) (ab169558) from Abcam.
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