The effect of exercise in NAFLD and a GLP-1 receptor agonist in type 2 diabetes on lipid metabolism.

Background: Hypertriglyceridaemia increases the risk of developing an atherogenic lipoprotein phenotype (ALP) in patients with type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). An ALP is associated with an increased the risk of coronary heart disease (CHD) and cardiovascular disease (CVD). Objectives: To determine the effects of exercise and a glucagon like peptide-1 (GLP-1) receptor agonists on hypertriglyceridaemia and high-density lipoprotein (HDL) metabolism in patients with altered lipid metabolism, by conducting two clinical trials using stable isotope trace labelling technique: 1. To determine the effect of exercise on HDL apolipoprotein A-I (apoA-I) and very low-density lipoprotein (VLDL) apoB-100 subgroups (VLDL1-apoB-100 and VLDL2-apoB-100) kinetics in NAFLD. 2. To determine the effect of the GLP-1 receptor agonist lixisenatide on postprandial triacylglycerol-rich lipoprotein (TRL) apo-B-100 and B-48 and HDL-apoA-I kinetics in T2D. Study design: In the NAFLD study, participants were randomised into two groups for a period of 16 weeks. The first group received a supervised moderate-intensity exercise programme and the second group was the control group. Total HDL-apoA-I was measured using a primed constant intravenous infusion of 1-13C leucine for 9 hours in a total of 27 recruited participants; 15 participants in the exercise group and 12 in the control group. In the lixisenatide study participants were randomised in a double-blinded two-period cross-over design (lixisenatide versus placebo). Participants received treatment with lixisenatide or placebo for four weeks followed by a four-week washout period then another four weeks with the other treatment. TRL-apoB-100, TRL-apoB-48 and total HDL-apoA-I were measured using a primed constant intravenous infusion of 1-13C leucine for 8 hours during repeated meal feeding in a total of six participants. Laboratory protocol: For both studies, hourly blood samples were taken during the study period. TRL-apoB-100, TRL-apoB-48 and total HDL-apoA-I fractions were isolated using ultracentrifugation. Fractions were delipidated and separated by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands from SDS-PAGE were hydrolysed, purified, and then derivatised. The isotopic enrichment of 13C leucine in TRL-apoB-100, TRL-apoB-48 and total HDL-apoA-I were measured using gas chromatography – mass spectrometry (GC-MS). Fractional catabolic rate (FCR) and production rate (PR) were calculated for TRL-apoB-100, TRL-apoB-48 and total HDL-apoA-I. TRL-apoB-100 and TRL-apoB-48 concentrations were measured using competitive ELISA. Total HDL-apoA-I, lipid profile including triacylglycerol (TG), cholesterol, and free fatty acids (FFA), also called non-esterified fatty acids (NEFA), and glucose concentrations were measured using automatic analysers. Results: In the NAFLD study, sixteen weeks of exercise had no significant effect on HDL-apoA-I kinetics. However, the HDL-apoA-I pool size (PS) was significantly increased from [17.4±0.8 g/l to 18.9±0.75 g/l (P =0.05)] after exercise in the exercise group. VLDL1-apoB-100 FCR and PR were significantly increased between the exercise and control group; Exercise group FCR [(7.2±0.6) vs (10.9±1.5) pools/day P=0.02]; PR [(3.7±0.7) vs (5.5±0.5) mg/kg/day P= 0.003]. Fasting hypertriglyceridaemia was not significantly changed after exercise. In the lixisenatide study, TRL-apoB-100 FCR significantly increased after lixisenatide treatment versus placebo; (6.3±0.4 vs 4.1±0.6 pools/day P=0.01). TRL-apoB-100 PR was increased with borderline significance after lixisenatide treatment (P=0.06) versus placebo. TRL-apoB-48 and HDL-apoA-I kinetics were not significantly changed after lixisenatide treatment versus placebo. Fasting and postprandial plasma glucose concentrations were significantly lower after lixisenatide treatment (P=0.05 and P=0.001 respectively) versus placebo. Postprandial serum insulin concentration was significantly higher after lixisenatide treatment (P=0.001) versus placebo. Postprandial plasma TG, cholesterol and FFA concentrations were significantly lower after lixisenatide treatment (P=0.002, P=0.02 and P=0.05 respectively) versus placebo. Conclusion: Exercise and lixisenatide were both effective in increasing VLDL-apoB-100 FCR which has the potential to reduce plasma TG concentrations in patients with altered lipid metabolism. However, exercise did not correct fasting hypertriglyceridaemia in patients with NAFLD due to increased VLDL1-apoB-100 PR. Liver fat was reduced by over 50% in the exercise group although was not normalised suggesting hepatic IR was maintained. To correct fasting hypertriglyceridaemia, accumulated liver fat must be further cleared to restore hepatic insulin sensitivity which would decrease VLDL1-apoB-100 PR. A longer exercise period is therefore needed to remove more liver fat and to correct fasting hypertriglyceridaemia. Postprandial hypertriglyceridaemia was lowered after lixisenatide treatment despite the fact that VLDL-apoB-100 PR was increased with borderline significance. A decrease in plasma TG concentration would be expected to reduce the rate of transfer of TG and CE between TRL and HDL via cholesteryl ester transfer protein (CETP) and increase HDL-cholesterol (HDL-C) concentration. The lack of effect of exercise on HDL kinetics in the NAFLD study reflects the failure to lower hypertriglyceridaemia. The lack of effect of lixisenatide to increase HDL despite lower plasma TG may be due to the study being underpowered.