The intestinal absorption of n-3 and n-6 polyunsaturated fatty acids: proposed mechanisms from studies using human Caco-2 cells.

This thesis presents an innovative investigation of the process by which specific dietary fatty acids, the n-3 and n-6 polyunsaturated fatty acids (PUFAs), enter the intestinal absorptive cell. The intake of these particular fats and the ratio of n-6/n-3 PUFAs have been correlated with the development/regression of various diseases -like arteriosclerosis, non insulin-dependent diabetes, autoimmune disorders, breast and colon cancers. Their action and metabolism have been largely studied in various cell types. However, the mechanism by which they first enter absorptive cells - to be metabolised for release in blood circulation - is still not fully elucidated. Cellular and molecular aspects of fat-absorption have been neglected since it was long time believed that fatty acids simply diffuse through the epithelium. The discovery of a set of membrane and soluble proteins exhibiting a high affinity for long-chain fatty acids (LCFAs) in different tissues questioned the theory that lipid absorption was only the result of passive diffusion. However, since the gut exhibit unique environmental and cellular features it is not possible to extrapolate the mechanisms found in other organs, to the small intestine. For this mechanistic study, a human enterocyte cell model (Caco-2 cells) was used. A careful attention has been taken on the specific parameters that influence the transport characteristics of the Caco-2 cells and the accessibility of the fatty acids to the absorption site. The results presented in the first part of this study, show that only early passage Caco-2 cells cultured for 15 day post-confluence in 5 to 20% serum-supplemented medium present all the characteristics for long chain fatty acids uptake and metabolism. Besides, the study revealed that cellular uptake mainly is dependant on the degree of solubilisation of the fatty acids in bile salt micelles (which is affected by the concentration in bile salt, the pH and the degree of saturation of the FA). It was concluded that the LCFA solubilisation of in 10 mM sodium taurocholate solutions buffered between pH 6.8 and 7.0, allows a high, rapid and reproducible cellular uptake, over short incubation periods without affecting cell viability. After the optimisation of the study design, the process of the LCUFAs uptake in the intracellular space was kinetically analysed. It showed that all the fatty acids studied (oleic acid, linoleic acid, alpha-linolenic acids, arachidonic acids, eicosapentaenoic acid, docosahexaenoic acid) exhibited a time- and concentration-related saturable uptake, over the physiological FA concentration range. Further it revealed, for the first time, that each fatty acid enters the enterocyte simultaneously by different mechanisms over different phases. In the initial phase of the uptake, one or more facilitated transport process(es) top off to the passive diffusion. It was observed that the proportion of each process is dependant on the form (monomer or micellar aggregate) and concentration of the FA at the brush border membrane (BBM). As a complement, a delayed second phase of uptake was observed after 10 min of incubation for the PUFAs with 3 or more double bonds. Inhibition studies revealed that several FA transport systems coexist on the intestinal BBM, some including surface protein(s), and other composed of proteolysis resistant component(s). The third part of this study addresses the presence of specific membrane fatty acid transporter(s). CD36 or a close homolog, observed for the first time in Caco-2 membranes and cytosol, was revealed to be implicated in the process of PUFAs initial uptake by the use of newly synthesised specific inhibitors. Furthermore, it was observed that the cellular distribution of this protein was regulated by particular LCUFAs. This original observation opens interesting areas for further investigations. This unique comparative study allowed to propose a general mechanism for the uptake of the different LCUFAs. A lipid raft CD36-mediated vesicular transport was proposed for OA and n-6 PUFAs, in complement to a transport system more specific for the n-6 PUFAs. In addition to this, competition studies suggested the presence of a third system shared only by the n-3 PUFAs. In this new area, a better knowledge of the cellular and molecular mechanisms underlying the intestinal fat absorption might lead to new therapeutic strategies to correct lipid balance disorders.

Grantor dc:publisher.institution

Robert Gordon University

Year dc:date.issued

2006

Author dc:creator

• Girod-Roux, Emmanuelle

Advisor dc:contributor.advisor

• A. Dutta-Roy and N. Emisson

• N-3 and N-6 polyunsaturated fatty acids
• Caco-2 cells
• Human
• Long-chain fatty acids
• Metabolism
• Bile salt micelles
• Enterocyte
• Intestine

Language dc:language

en

Identifier

oai:rgu-repository.worktribe.com:2807395
https://doi.org/10.48526/rgu-wt-2807395

OAI identifier oai:identifier

oai:rgu-repository.worktribe.com:2807395