Type 2 diabetes mellitus (T2DM) is mainly caused by genetic modifications and inappropriate life styles. The complexity of T2DM has brought us challenges for a comprehensive understanding of altered metabolic pathways that contributing to the development of T2DM. Therefore, a comprehensive metabolic analysis is needed. To date, taking regular exercise is a common and effective therapeutic way known to antagonize the metabolic disorders of T2DM. However, the regulatory effects of exercise on T2DM or T2DM induced complications have not been clearly characterized. Here, we present the effect of physical activity on biochemical changes in diabetic db/db mice in plasma, urine, skeletal muscle and kidney samples. Based on liquid chromatography coupled with high resolution Orbitrap mass spectrometry (LC-MS) and gas chromatography coupled with mass spectrometry (GC-MS), two major approaches, untargeted and targeted metabolomics studies, have been developed to delineate metabolic signatures in various kinds of biofluid and tissue samples. Targeted quantification methods on acylcarnitines and acyl-CoA have been developed. Untargeted metabolomics analysis by GC-MS and LC-MS have also been developed to draw a more comprehensive view of the metabolic changes in response to T2DM and exercise on db/db diabetic mice. The transcript expressions of mRNA in pathways of interest have also been measured to confirm the hypothesis. Firstly, a targeted quantification method of acylcarnitines by using high resolution parallel reaction monitoring (PRM) on LC-MS platform has been developed. A total of 117 acylcarnitines were detected from plasma and urine samples. The application of targeted profiling of acylcarnitines in db/m+ control and db/db diabetic mice indicated incomplete amino acid and fatty acid oxidation in diabetic mice. Interestingly, the reduction of medium odd-numbered chain acylcarnitines in urine samples was firstly observed between db/m+ and db/db mice. The high resolution PRM method makes it possible to monitor the widespread metabolic changes of the acylcarnitines in response to stimuli. Besides, the accurate MS and MS/MS spectra data of the 117 acylcarnitines could be used as mass spectrometric resources for the identification of acylcarnitines. In addition to targeted metabolomics analysis, untargeted metabolomics profiling analysis in plasma samples indicated that db/db diabetic mice may be more susceptible to exercise for energy expenditure. Interestingly, all the results from plasma, skeletal muscle and kidney samples may demonstrate that physical activity could mitigate insulin resistance in T2DM mice through improving fatty acid β-oxidation (FAO) and eliminating overloaded intermediate which contribute to insulin resistance. Specifically, the results from kidney samples demonstrated that exercise exhibit beneficial effect in reducing hyperlipidemia, expression levels of inflammatory markers (TNFα, IL-6 and COX2) and fibrosis markers (Collagen 1), and alleviating diabetic nephropathy (DN) induced mesangial expansion in kidneys of diabetic mice. The results of metabolic changes in kidney of db/db mice revealed that the accumulation of acyl-CoA, phospholipids and hydroxylated acylcarnitines were substantially ameliorated by exercise, and the reduction of important enzymes CTP1α and Acadl in FAO were partially reversed. In addition, branched-chain amino acids (BCAA) metabolism which positively related to inflammation (TNFα) was down-regulated in DN mice by exercise. What’s more, the accumulation of uric acid, which contributes to inflammation and tubulointestitial fibrosis in kidney disease, together with its six precursors have also been substantially reduced. The results in kidney samples demonstrated that in addition to beneficial effect in alleviating lipotoxicity through improving FAO efficiency, exercise also ameliorated diabetic induced inflammation and fibrosis via promoting BCAA catabolism and accelerating the elimination of uric acid. Together, the mass spectrometry-based metabolomics study is a powerful tool to investigate the regulatory effect of exercise on complex metabolic diseases. The results may provide informative insights into the underlying the mechanism of exercise on T2DM and T2DM induced complications.
|Date of Award||19 Mar 2018|
|Supervisor||Zongwei CAI (Supervisor)|
- Animal models
- Exercise therapy
- Mice as laboratory animals.