Study of potential mechanisms involved in fat mass-lowering effects of conjugated linoleic acid and calcium in mice by focusing on gut microbiota, bone remodelling and epigenetic marks

Abstract

Obesity is currently increasing at an epidemic rate and is associated with a wide array of co-morbidities, such as metabolic syndrome, cancer and heart disease, among others. One of the approaches in order to tackle this issue is the identification of dietary factors which could be able to counteract the development of obesity. Within this context, our laboratory has previously carried out studies to determine the potentially beneficial effects of conjugated linoleic acid (CLA) and calcium on body weight loss and fat mass accretion, and has shown that supplementation with either of these compounds in mice under diet-induced obesity causes decreased weight gain and lower adiposity levels. The aim of this Thesis has been to study potential mechanisms by which these two compounds could act, by focusing on ones that to our knowledge have not been previously analysed: 1) the effect on gut microbiota; 2) the relationship between bone and energy metabolism; 3) their potential effect on key tissues and organs involved in energy metabolism; and 4) DNA methylation of genes of interest within this context. In line with the previous work done in our lab, the study was carried out in an animal model of adult mice receiving a high-fat (HF) enriched with CLA or calcium. Results show that, on one hand, CLA stimulates the expression of gastric leptin and ghrelin and exerts a prebiotic action on specific bacteria in the caecum, particularly Bacteroidetes/ Prevotella and Akkermansia muciniphila. However, supplementation with this compound does not counteract the negative effect of a HF diet on Bifidobacterium spp., which was decreased in both groups. On the other hand, calcium has a similar effect, by also conferring a prebiotic effect, in particular on Bifidobacteirum spp. and Bacteroidetes/Prevotella, and by decreasing Clostridium spp. This modulation by calcium correlated with healthy metabolic parameters, suggesting that the effect observed on gut microbiota could partially explain the overall healthier metabolic profile of these animals. A faecal microbiota transplant, using faeces from calcium animals to animals receiving HF diet, revealed that a transplant alone is not able to confer the beneficial effects seen in the microbiota of animals from the calcium group. This suggests that calcium itself provides an adequate environment for the growth of the studied bacteria and without its presence such bacteria are not able to colonise the colon. Moreover, calcium supplementation contributes to the maintenance of bone mass, although effects are mainly seen when both CLA and calcium are co-supplemented, suggesting a beneficial effect of these two compounds on bone health in obesity treatment. A similar effect was observed when analysing the methylation profile of specific genes, which is significantly altered in a gene- and tissue-specific manner, in particular by calcium and cosupplemented with CLA. The changes observed in DNA methylation profile, induced by dietary supplementation, were mainly seen in those animals which presented an overall healthier metabolic profile, introducing the need to consider the potential of epigenetic modulation strategies for obesity management. Overall, this Thesis shows that a diet supplemented with calcium has beneficial effects on body weight and adiposity in mice, which could be mediated by the modulation of gut microbiota and the methylation profile of genes associated to lipid metabolism. In addition, combined treatment with CLA shows beneficial effects on bone mass and parameters associated with their formation.