Pabla, Pardeep
(2021)
Development of a liquid chromatography-mass spectrometry method to investigate branched chain amino acid and acylcarnitine metabolism in type 2 diabetes.
PhD thesis, University of Nottingham.
Abstract
The global prevalence of obesity and type 2 diabetes (T2D) continues to rise at an alarming rate. Despite the well-established association between obesity and an increased risk of developing T2D, the mechanisms underlying the pathogenesis of T2D with obesity remain unclear. Skeletal muscle is a major site for the disposal of ingested carbohydrate in healthy individuals. It is generally accepted that chronic overnutrition leads to accumulation of fat and fatty acid metabolites within the skeletal muscle which are believed to play a pivotal role in the progression of insulin resistance to carbohydrate metabolism which is one of the pathological hallmarks of T2D. Whilst several metabolites have been implicated, there appears to be no consensus over which metabolite is mediating skeletal muscle insulin resistance.
In recent years, circulating concentrations of acylcarnitines, which are intermediates in glucose, fatty acid and branched chain amino acids (BCAAs) metabolism have been identified as potential novel biomarkers of insulin resistance and T2D. Furthermore, fatty acid derived acylcarnitines have since been shown to impair insulin signalling in vitro. Interestingly, BCAAs and their associated short-chain acylcarnitines appear to be more closely associated with insulin resistance than any marker of fatty acid metabolism, giving rise to the hypothesis that BCAAs and their catabolites may also play a causative role in the development of skeletal muscle insulin resistance. However, the simultaneous and quantitative assessment of acylcarnitines, BCAAs and related metabolites in human skeletal muscle is lacking or limited to one metabolite group. Therefore, the main aim of this this thesis was to develop a quantitative analytical method for the assessment of BCAAs and acylcarnitines in human muscle to extend upon much of published literature which has been limited to investigations in fasting plasma samples and to explore their role in the development of insulin resistance and T2D.
In Chapter 3 a novel liquid chromatography coupled to high resolution mass spectrometry method was developed and optimised to enable the quantitative assessment of a full range of BCAA and fatty acid derived acylcarnitines, BCAAs and related catabolites in both human plasma and muscle samples. The commonly cited challenges of metabolite quantification from biological tissues were systematically addressed using stable isotope internal standards allowing metabolite concentrations to be determined with a high degree of confidence.
In Chapter 4, the method was validated by quantitatively assessing BCAAs and acylcarnitine concentrations in fasting plasma and skeletal muscle samples of patients with T2D and an age matched obese control subjects. The results revealed striking elevations of BCAAs and BCAA derived acylcarnitines in both plasma and muscle of patients with T2D compared to control subjects. Furthermore, these metabolites were significantly correlated with fasting blood glucose. Surprisingly, no significant differences in fatty acid derived acylcarnitines were observed between groups in either plasma or muscle. These data show that plasma profiles may not always reflect muscle profiles as suggested by previous reports.
In Chapter 5, skeletal muscle BCAA and acylcarnitine metabolism was investigated in the fasted and insulin stimulated state. In order to determine if the elevations observed in fasting state in the previous persist in the face of insulin thereby allowing some indication of whether they could be causative of insulin resistance. A group of young and old lean, and old overweight/obese individuals was investigated in a cross sectional design. Ageing was associated with a (30%) decline in muscle BCAA content and decreased insulin sensitivity. And increased adiposity was associated with a (20%) increase in BCAA content. In response to insulin infusion, there was an attenuated decline in muscle BCAA and BCAA catabolite content in the old lean group only. In addition, fatty acid derived acylcarnitines were suppressed in all groups despite differing glucose disposal during insulin infusion. These findings appear to dissociate muscle BCAA content, ageing and insulin resistance and suggest fatty acid derived acylcarnitnes may not be associated with insulin resistance.
In Chapter 6, the potential interactions of BCAA and fatty acid metabolism were explored in a group of middle aged and older aged patients with T2D. Middle aged T2D patients had elevated meal derived fatty acid oxidation and endogenous fatty acid delivery to muscle during an oral glucose tolerance test (OGTT) when compared to age and BMI matched control subjects. This was associated with elevated fasting muscle BCAA content and an attenuated suppression of plasma BCAA and BCAA catabolites during the OGTT. Remarkably, these metabolic perturbations were absent in the older aged T2D patients, despite a similar duration of diabetes and insulinaemic responses to OGTT.
Collectively, the work in this thesis provides quantitative assessment of muscle BCAA catabolism and fatty acid metabolism in humans. T2D and obesity-induced insulin resistance are characterised by elevated BCAAs and BCAA derived acylcarnitines but ageing per se exerts the opposite effects. In addition, the results of this thesis suggest that fatty acid derived acylcarnitines may not be associated with muscle insulin resistance in vivo. The potential role of BCAA derived acylcarnitines in insulin resistance and their role as biomarkers of progression to T2D requires further investigation.
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