| 研究実績の概要 |
In the FY2019, we further explored adipose tissue cell populations in the diet-induced obesity mouse model. In the FY2018, we have established this model and collected several cell popoulations in the visceral white adipose tissue from these mice. This year, we have optimized the workflow and increased the number of replicates to get the data with better statistical significance. Due to the limited amount of protein from FACS-sorted cells, we have applied the tandem mass tag (TMT) labeling strategy in protein quantification.
Importantly, we have collected cell samples from three time points during dietary interference (i.e. high-fat diet (HFD) vs. control diet (CD)), including 1 week, 8 weeks and 16 weeks. Adipose tissue inflammation during obesity involves time-dependent changes of the adipose tissue cells and their interactions, but the proteome changes in these cells remain largely unknown. Thus, our data can provide unprecedented insights in the molecular basis of adipose tissue inflammation.
In addition, we have found a few candidate proteins from the 8-week results for functional validation. This includes chloride intracellular channel protein 4 (Clic4), Sn1-specific diacylglycerol lipase beta (Daglb) and a cluster of V-type proton ATPase subunits. The abundances of these proteins significantly increased after 8 weeks of HFD feeding. We have established ex vivo cell culture system using bone marrow-derived macrophages (BMDM) and we will use macrophage inflammatory markers and lipid metabolism as readout for the validation.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Some cell populations, e.g. adipose tissue T cells and B cells, have very limited cell numbers, especially in young and/or mice under control diet. Thus, we have spent quite some time to optimized the workflow, both for samples collection (FACS) and protein quantification. As the diet interference takes time, e.g. 8 or 16 weeks, it has been time-consuming to repeat the experiment and apply the optimized workflow. By the end of FY2019, we have made some progress and generated some data with reasonably good quality.
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| 今後の研究の推進方策 |
We will finish collecting the samples from different time points of dietary interference. We will then perform protein quantification in each cell population independently using tandem mass tag proteomics strategy, and compared the protein abundances between high-fat and control diet, as well as during the development of diet-induced obesity. In addition, we will validate the function of Clic4, Daglb and v-ATPases in macrophage activation and lipid metabolism using bone marrow-derived macrophages.
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