Evaluation of D-limonene effects on intermediate metabolism of mice - relationship between limonene metabolism and health effects.

Abstract

The health effects of bioactive compounds found in fruits and vegetables have been studied for a long time and health-protective effects have been associated with different phytochemicals such as phytosterols, flavonoids, glucosinolates and many others. Despite their demonstrated bioactive effects in a variety of models, monoterpenoids are still overlooked when it comes to understanding their mechanism of action and efficacy in humans. The monoterpene d-limonene, found in high concentrations in the peel of citrus fruits is known for its anti-inflammatory, antinociceptive and hypolipemic effects. It is possible that due to its antimicrobial activity, this compound can modulate the composition of intestinal microbiota, which among other actions is responsible for converting primary bile acids into secondary ones with different physico-chemical properties and biological effects. Bile acids are agonists of receptors expressed in several tissues and are able to modulate fatty acid oxidation, gluconeogenesis, hormone secretion and other physiological processes related to intermediate metabolism. It has never been demonstrated, but we believe that changes in intestinal microbiota composition induced by d-limonene could affect bile acid metabolism and different physiological processes regulated by these molecules. The main goal of this study is to evaluate the effects of limonene on bile acid and intermediate metabolism. Male C57/Bl6-J mice will be distributed in 4 groups: 2 groups will receive a high fat-diet and the other 2 will be fed a control diet for 4 weeks. From the 5th week on, one group from each diet will be daily supplemented with 100 mg/Kg of d-limonene. The response to an oral glucose challenge will be evaluated at weeks 4 and 8. Hepatic lipid and glycogen content as well as other markers of lipid metabolism, including plasma triglyceride and cholesterol concentrations will be evaluated. Targeted metabolomics analysis (LC-QqQ) will be employed to analyse the concentration and composition of bile acids and markers of fatty acid/ amino acid oxidation (acylcarnitines) in plasma. Untargeted metabolomics (LC-qToF) will be used to determine the production of limonene metabolites and to assess whole-metabolome changes induced by limonene. The results obtained in this study will contribute to the elucidation of dietary monoterpenes mechanism of action and help assessing the potential use of d-limonene for the prevention/ treatment of metabolic diseases.