Development of Biomarkers for Inhibition of SLC6A19 - A Potential Target to treat Metabolic Disorders

Abstract

Dietary protein restriction has beneficial impacts on metabolic health. SLC6A19 (B0AT1) is the major transporter of neutral amino acids at the intestinal epithelium and absorbs the bulk of the diet-derived neutral amino acids from the intestinal lumen. It also reabsorbs neutral amino acids in the renal proximal tubules. Mice lacking SLC6A19 show signs of protein restriction, such as high FGF21 levels and low mTORC1 activity. Moreover, they have improved glucose homeostasis and resist diet-induced obesity. Pharmacological blockage of this transporter could be used to induce protein restriction and to treat metabolic diseases such as type 2 diabetes, fatty liver and phenylketonuria. A few novel inhibitors of SLC6A19 have recently been identified using in vitro compound screening, but it remains unclear whether these compounds block the transporter in vivo. To evaluate the efficacy of SLC6A19 inhibitors, biomarkers are required that can reliably detect successful inhibition of the transporter in mice. A gas chromatography mass spectrometry (GC-MS)-based untargeted metabolomics approach was used to discriminate global metabolite profiles in plasma, urine and faecal samples from SLC6A19ko and wt mice. Due to inefficient absorption in the intestine and lack of reabsorption in the kidney, significantly elevated amino acids levels were observed in urine and faecal samples. By contrast, a few neutral amino acids were reduced in the plasma of male SLC6A19ko mice as compared to other biological samples. Metabolites of bacterial protein fermentation such as p-cresol-glucuronide and 3-indole-propionic acid were more abundant in SLC6A19ko mice, indicating protein malabsorption of dietary amino acids. Consistently, plasma appearance rates of [14C]-labelled neutral amino acids were delayed in SLC6A19ko mice as compared to wt after intra-gastric administration of a mixture of amino acids. Receiver operating characteristic (ROC) curve analysis was used to validate the potential use of these metabolites as biomarkers. Feasibility of volatile biomarkers in breath was also explored in this thesis. While I did not identify any breath-based volatiles due to limitation in method development, I identified a few volatile organic compounds (VOCs) in the headspace of mouse urine that illustrate the potential of breath analysis in the analysis of SLC6A19 activity. Moreover, I also investigated the relationship between protein restriction and dietary protein intake in wt and SLC6A19ko mice. When SLC6A19ko mice were fed diets containing 5%, 25%, or 52% of their total calories derived from protein, no differences in food intake or weight gain were observed. All essential amino acids positively correlated with increasing dietary casein content in the wt mice. The SLC6A19ko mice showed reduced postprandial levels of essential amino acids in plasma, particularly following high-protein diets. Upon fasting, essential amino acids were the same in wt and SLC6A19ko mice due to reduced amino acid catabolism. Bacterial metabolites originating from amino acid fermentation correlated with the dietary protein content, but showed a complex profile in the blood of the SLC6A19ko mice. The findings in this study provide putative metabolite biomarkers that can be used to detect protein malabsorption and the inhibition of this transporter in intestine and kidney. It also highlights the potential of SLC6A19 as a knock-out or inhibition target to induce protein restriction for the treatment of metabolic disorders. Show more