Development of a liquid chromatography-mass spectrometry method to investigate branched chain amino acid and acylcarnitine metabolism in type 2 diabetes

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

Development of a liquid chromatography-mass spectrometry method to investigate branched chain amino acid and acylcarnitine metabolism in type 2 diabetes

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

EMPIRICAL MODELING OF SURFACE ROUGHNESS AND METAL REMOVAL RATE IN CNC MILLING OPERATION

Surface finish and material removal rate are two important factors in the manufacturing organization which affect acceptability of the product which in turn reflects on the profitability of the organization. Ability of the production setup to produce the components with high material removal rate without sacrificing the surface requirements can play vital role in sustainability and profitability of the organization. In this paper, the effect of process parameters on metal removal rate and surface roughness has been investigated in milling of SAE52100 tool steel. Cutting speed, feed and depth of cut have been taken as input factors in three level full factorial orthogonal arrays used for experimentation. Mathematical models have been developed using response surface methodology to predict surface finish, and metal removal rate in term of machining parameters.  Depth of cut and feed rate are found to be a dominant parameter for surface roughness; whereas feed rate mainly effects the metal removal rate. The results of mathematical models have been compared with the experimental and found to be in good agreement. The results of predicted model can be used in selection of process parameters to insure desired quality and improved productivity

Two weeks of early time-restricted feeding (eTRF) improves skeletal muscle insulin and anabolic sensitivity in healthy men

Copyright © The Author(s) on behalf of the American Society for Nutrition 2020. BACKGROUND: Altering the temporal distribution of energy intake (EI) and introducing periods of intermittent fasting (IF) exert important metabolic effects. Restricting EI to earlier in the day [early time-restricted feeding (eTRF)] is a novel type of IF. OBJECTIVES: We assessed the chronic effects of eTRF compared with an energy-matched control on whole-body and skeletal muscle insulin and anabolic sensitivity. METHODS: Sixteen healthy males (aged 23±1 y; BMI 24.0±0.6 kg·m-2) were assigned to 2 groups that underwent either 2 wk of eTRF (n=8) or control/caloric restriction (CON:CR; n=8) diet. The eTRF diet was consumed ad libitum and the intervention was conducted before the CON:CR, in which the diet was provided to match the reduction in EI and body weight observed in eTRF. During eTRF, daily EI was restricted to between 08:00 and 16:00, which prolonged the overnight fast by ∼5 h. The metabolic responses to a carbohydrate/protein drink were assessed pre- and post-interventions following a 12-h overnight fast. RESULTS: When compared with CON:CR, eTRF improved whole-body insulin sensitivity [between-group difference (95% CI): 1.89 (0.18, 3.60); P=0.03; η2p=0.29] and skeletal muscle uptake of glucose [between-group difference (95% CI): 4266 (261, 8270) μmol·min-1·kg-1·180 min; P=0.04; η2p=0.31] and branched-chain amino acids (BCAAs) [between-group difference (95% CI): 266 (77, 455) nmol·min-1·kg-1·180 min; P=0.01; η2p=0.44]. eTRF caused a reduction in EI (∼400 kcal·d-1) and weight loss (-1.04±0.25 kg; P=0.01) that was matched in CON:CR (-1.24±0.35 kg; P=0.01). CONCLUSIONS: Under free-living conditions, eTRF improves whole-body insulin sensitivity and increases skeletal muscle glucose and BCAA uptake. The metabolic benefits of eTRF are independent of its effects on weight loss and represent chronic adaptations rather than the effect of the last bout of overnight fast. This trial was registered at clinicaltrials.gov as NCT03969745

Characterisation of Aberrant Metabolic Pathways in Hepatoblastoma Using Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS)

Hepatoblastoma (HB) is a rare childhood tumour with an evolving molecular landscape. We present the first comprehensive metabolomic analysis using untargeted and targeted liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS) of paired tumour and non-tumour surgical samples in HB patients (n = 8 pairs). This study demonstrates that the metabolomic landscape of HB is distinct from that of non-tumour (NT) liver tissue, with 35 differentially abundant metabolites mapping onto pathways such as fatty acid transport, glycolysis, the tricarboxylic acid (TCA) cycle, branched-chain amino acid degradation and glutathione synthesis. Targeted metabolomics demonstrated reduced short-chain acylcarnitines and a relative accumulation of branched-chain amino acids. Medium- and long-chain acylcarnitines in HB were similar to those in NT. The metabolomic changes reported are consistent with previously reported transcriptomic data from tumour and non-tumour samples (49 out of 54 targets) as well as metabolomic data obtained using other techniques. Gene set enrichment analysis (GSEA) from RNAseq data (n = 32 paired HB and NT samples) demonstrated a downregulation of the carnitine metabolome and immunohistochemistry showed a reduction in CPT1a (n = 15 pairs), which transports fatty acids into the mitochondria, suggesting a lack of utilisation of long-chain fatty acids in HB. Thus, our findings suggest a reduced metabolic flux in HB which is corroborated at the gene expression and protein levels. Further work could yield novel insights and new therapeutic targets

Acute effects of prior dietary fat ingestion on postprandial metabolic responses to protein and carbohydrate co-ingestion in overweight and obese men: A randomised crossover trial

Background: Obesity and insulin resistance are associated with an impaired sensitivity to anabolic stimuli such as dietary protein (anabolic resistance). Omega-3 polyunsaturated fatty acids (n-3 PUFA) may be protective against the deleterious effects of saturated fatty acids (SFA) on insulin resistance. However, the contribution of excess fat consumption to anabolic and insulin resistance and the interaction between SFA and n-3 PUFA is not well studied. Aim: The primary aim of this study was to investigate the effects of an oral fat pre-load, with or without the partial substitution of SFA with fish oil (FO)-derived n-3 PUFA, on indices of insulin and anabolic sensitivity in response to subsequent dietary protein and carbohydrate (dextrose) co-ingestion. Methods: Eight middle-aged males with overweight or obesity (52.8 ± 2.0 yr, BMI 31.8 ± 1.4 kg·m−2) ingested either an SFA, or isoenergetic SFA and FO emulsion (FO), or water/control (Con), 4 h prior to a bolus of milk protein and dextrose. Results: Lipid ingestion (in particular FO) impaired the early postprandial uptake of branched chain amino acids (BCAA) into the skeletal muscle in response to protein and dextrose, and attenuated the peak glycaemic response, but was not accompanied by differences in whole body (Matsuda Index: Con: 4.66 ± 0.89, SFA: 5.10 ± 0.94 and FO: 4.07 ± 0.59) or peripheral (forearm glucose netAUC: Con: 521.7 ± 101.7; SFA: 470.2 ± 125.5 and FO: 495.3 ± 101.6 μmol·min−1·100 g lean mass·min [t = 240–420 min]) insulin sensitivity between visits. Postprandial whole body fat oxidation was affected by visit (P = 0.024) with elevated rates in SFA and FO, relative to Con (1.85 ± 0.55; 2.19 ± 0.21 and 0.65 ± 0.35 kJ·h−1·kg−1 lean body mass, respectively), however muscle uptake of free fatty acids (FFA) was unaffected. Conclusion: Oral lipid preloads, consisting of SFA and FO, impair the early postprandial BCAA uptake into skeletal muscle, which occurs independent of changes in insulin sensitivity. Clinical trial registry number: ClinicalTrials.gov Identifier NCT03146286

Effect of acute and short-term dietary fat ingestion on postprandial skeletal muscle protein synthesis rates in middle-aged, overweight and obese men

Muscle anabolic resistance to dietary protein is associated with obesity and insulin resistance. However, the contribution of excess consumption of fat to anabolic resistance is not well studied. The aim of these studies was to test the hypothesis that acute and short-term dietary fat overload will impair the skeletal muscle protein synthetic response to dietary protein ingestion. Eight overweight/obese males [46.4±1.4 years, BMI 32.3±5.4 kg/m2] participated in the acute feeding study, which consisted of 2 randomised crossover trials. On each occasion, subjects ingested an oral meal (with and without fat emulsion) 4h before the coingestion of milk protein, intrinsically labelled with [1-13C]phenylalanine, and dextrose. Nine overweight/obese males [44.0±1.7 years, BMI 30.1±1.1 kg/m2] participated in the chronic study, which consisted of a baseline 1-week isocaloric diet followed by a 2-week high fat diet (+25% energy excess). Acutely, incorporation of dietary amino acids into the skeletal muscle was 2-fold higher (P<0.05) in the lipid trial compared to control. There was no effect of prior lipid ingestion on indices of insulin sensitivity (muscle glucose uptake, PDC activity and Akt phosphorylation) in response to the protein/dextrose drink. Fat overfeeding had no effect on muscle protein synthesis or glucose disposal in response to whey protein ingestion, despite increased muscle DAG C16:0 (P=0.06) and ceramide C16:0 (P<0.01) levels. Neither acute nor short-term dietary fat overload has a detrimental effect on skeletal muscle protein synthetic response to dietary protein ingestion in overweight/obese men, suggesting dietary-induced accumulation of intramuscular lipids per se is not associated with anabolic resistance

Two weeks of early time-restricted feeding (eTRF) improves skeletal muscle insulin and anabolic sensitivity in healthy men

Copyright © The Author(s) on behalf of the American Society for Nutrition 2020. BACKGROUND: Altering the temporal distribution of energy intake (EI) and introducing periods of intermittent fasting (IF) exert important metabolic effects. Restricting EI to earlier in the day [early time-restricted feeding (eTRF)] is a novel type of IF. OBJECTIVES: We assessed the chronic effects of eTRF compared with an energy-matched control on whole-body and skeletal muscle insulin and anabolic sensitivity. METHODS: Sixteen healthy males (aged 23±1 y; BMI 24.0±0.6 kg·m-2) were assigned to 2 groups that underwent either 2 wk of eTRF (n=8) or control/caloric restriction (CON:CR; n=8) diet. The eTRF diet was consumed ad libitum and the intervention was conducted before the CON:CR, in which the diet was provided to match the reduction in EI and body weight observed in eTRF. During eTRF, daily EI was restricted to between 08:00 and 16:00, which prolonged the overnight fast by ∼5 h. The metabolic responses to a carbohydrate/protein drink were assessed pre- and post-interventions following a 12-h overnight fast. RESULTS: When compared with CON:CR, eTRF improved whole-body insulin sensitivity [between-group difference (95% CI): 1.89 (0.18, 3.60); P=0.03; η2p=0.29] and skeletal muscle uptake of glucose [between-group difference (95% CI): 4266 (261, 8270) μmol·min-1·kg-1·180 min; P=0.04; η2p=0.31] and branched-chain amino acids (BCAAs) [between-group difference (95% CI): 266 (77, 455) nmol·min-1·kg-1·180 min; P=0.01; η2p=0.44]. eTRF caused a reduction in EI (∼400 kcal·d-1) and weight loss (-1.04±0.25 kg; P=0.01) that was matched in CON:CR (-1.24±0.35 kg; P=0.01). CONCLUSIONS: Under free-living conditions, eTRF improves whole-body insulin sensitivity and increases skeletal muscle glucose and BCAA uptake. The metabolic benefits of eTRF are independent of its effects on weight loss and represent chronic adaptations rather than the effect of the last bout of overnight fast. This trial was registered at clinicaltrials.gov as NCT03969745

Demonstration of therapeutic window of Cerebrolysin in embolic stroke: A prospective, randomized, blinded, and placebo-controlled study

Background and aims In an effort to characterize the effects of Cerebrolysin for treatment of stroke that are essential for successful clinical translation, we have demonstrated that Cerebrolysin dose dependently enhanced neurological functional recovery in experimental stroke. Here, we conduct a prospective, randomized, placebo-controlled, blinded study to examine the therapeutic window of Cerebrolysin treatment of rats subjected to embolic stroke. Methods Male Wistar rats age 3-4 months (n = 100) were subjected to embolic middle cerebral artery occlusion. Animals were randomized to receive saline or Cerebrolysin daily for 10 consecutive days starting 4, 24, 48, and 72 h after middle cerebral artery occlusion. Neurological outcome was measured weekly with a battery of behavioral tests (adhesive removal test, modified neurological severity score (mNSS), and foot-fault test). Global test was employed to assess Cerebrolysin effect on neurological recovery with estimation of mean difference between Cerebrolysin and control-treated groups and its 95% confidence interval in the intent-to-treat population, where a negative value of the mean difference and 95% confidence interval \u3c 0 indicated a significant treatment effect. All rats were sacrificed 28 days after middle cerebral artery occlusion and infarct volume was measured. Results Cerebrolysin treatment initiated within 48 h after middle cerebral artery occlusion onset significantly improved functional outcome; mean differences and 95% confidence interval were -11.6 (-17.7, -5.4) at 4 h, -7.1 (-13.5, -0.8) at 24 h, -8.4 (-14.2, -8.6) at 48 h, and -4.9 (-11.4, 1.5) at 72 h. There were no differences on infarct volume and mortality rate among groups. Conclusions With a clinically relevant rigorous experimental design, our data demonstrate that Cerebrolysin treatment effectively improves stroke recovery when administered up to 48 h after middle cerebral artery occlusion

Demonstration of therapeutic window of Cerebrolysin in embolic stroke: A prospective, randomized, blinded, and placebo-controlled study

Background and aims In an effort to characterize the effects of Cerebrolysin for treatment of stroke that are essential for successful clinical translation, we have demonstrated that Cerebrolysin dose dependently enhanced neurological functional recovery in experimental stroke. Here, we conduct a prospective, randomized, placebo-controlled, blinded study to examine the therapeutic window of Cerebrolysin treatment of rats subjected to embolic stroke. Methods Male Wistar rats age 3-4 months (n = 100) were subjected to embolic middle cerebral artery occlusion. Animals were randomized to receive saline or Cerebrolysin daily for 10 consecutive days starting 4, 24, 48, and 72 h after middle cerebral artery occlusion. Neurological outcome was measured weekly with a battery of behavioral tests (adhesive removal test, modified neurological severity score (mNSS), and foot-fault test). Global test was employed to assess Cerebrolysin effect on neurological recovery with estimation of mean difference between Cerebrolysin and control-treated groups and its 95% confidence interval in the intent-to-treat population, where a negative value of the mean difference and 95% confidence interval \u3c 0 indicated a significant treatment effect. All rats were sacrificed 28 days after middle cerebral artery occlusion and infarct volume was measured. Results Cerebrolysin treatment initiated within 48 h after middle cerebral artery occlusion onset significantly improved functional outcome; mean differences and 95% confidence interval were -11.6 (-17.7, -5.4) at 4 h, -7.1 (-13.5, -0.8) at 24 h, -8.4 (-14.2, -8.6) at 48 h, and -4.9 (-11.4, 1.5) at 72 h. There were no differences on infarct volume and mortality rate among groups. Conclusions With a clinically relevant rigorous experimental design, our data demonstrate that Cerebrolysin treatment effectively improves stroke recovery when administered up to 48 h after middle cerebral artery occlusion