The Effect of Oleic Acid on Metformin Transport and Toxicity in a Caco-2 Cell Model

Abstract

Metformin is the first-line drug for the management of Type 2 Diabetes (T2DM) in Aotearoa/New Zealand. Approximately ~20% of patients are intolerant to metformin. This intolerance presents as gastrointestinal distress, nausea and diarrhoea. Metformin is almost exclusively transported into cells, its site of action, by transport proteins of the Solute Carrier family (SLC). Two transcription factor families PPAR and HNF may dynamically regulate the expression of these SLC transporters. Their endogenous ligands, free fatty acids (FFA), are often substantially elevated in T2DM patients. This research hypothesises that high levels of circulating free fatty acids, specifically palmitic and oleic acid, activate PPAR and HNF, altering the expression of transporters relevant to the distribution of metformin within the enterocyte, leading to intolerance. Using non-differentiated Caco-2 cells as an in vitro model of the gut, the aims of this thesis were to 1) determine the ability of FFA to induce steatosis in Caco2 cells, 2) determine whether steatosis alters metformin-induced changes in Caco2 cell viability, and 3) determine whether steatosis alters the facilitated intracellular transport of metformin in Caco2 cells. At a soluble, nontoxic, concentration (100 μM), palmitic acid did not induce steatosis. At 1 mM, oleic acid for 24 h was found to induce steatosis, without toxicity. Concentration-dependent effect experiments determined oleic acid did not alter the cytotoxicity of metformin compared with controls, as determined visually and by two assays of cell viability after 24 - 72 h of exposure. The kinetics of radiolabelled metformin were established in steatotic and non-steatotic cells. Oleic acid exposure (72 h) slightly decreased the active transport of metformin, relative to vehicle control. These preliminary findings suggest oleic acid may alter the active transport of metformin but that this alteration is unlikely to be relevant for cytotoxicity. Phenotypic differences between the enterocyte and non-differentiated Caco-2 cell, however, indicate further studies are required to fully understand the impact of steatosis on metformin kinetics.