Cell culture models of fatty acid overload: Problems and solutions

Abstract

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.

Keywords: Cells and tissues; Fatty acid metabolism; Fatty acids; Lipids; Lipotoxicity.

Figure 1. BSA decreases cellular lipid content

INS1 cells were grown to 70% confluence in 10% FBS RPMI culturing media, after which they were treated with 100 µM BSA plus 1% FBS or 400 µM palmitate conjugated to BSA/FBS in a 4:1 ratio for 16 h. Oleate (100 µM) was conjugated directly to 10% FBS. Cells were then fixed with 4% PFA for 15 min and stained with Hoechst and BODIPY493 for 15 min. Cells were washed twice with PBS and imaged using the Operetta imager, after which the lipid droplets per cell were quantified. Typical images shown left, quantifications of the number of lipid dropets and lipid droplet area, right. Note the decrease in lipid content with BSA. * p < 0.05 versus BSA plus 1% FBS.

Figure 2. The unbound FFA fraction in solution increases with FA:BSA molar ratios

The unbound fraction was calculated for BSA and palmitate or oleate by multiple stepwise equilibrium analysis as described in [123].