BIOFEEDBACK


X-box binding protein 1 (XBP1s) and C/EBP homologous protein (CHOP), as well as the proapoptotic BCL-2 family member death protein 5 (DP5; also known as harakiri). Exposure to palmitate using the different BSA preparations induced similar levels of ATF3, XBP1s, CHOP, and DP5 mRNA expression (Figure 2, A–D), with somewhat greater induction for precomplexed palmitate. The activation of the PERK and IRE1 branches of the ER stress response and the induction of DP5 mRNA by oleate is milder compared with palmitate (28,29). Overall, gene expression was modified by oleate in a comparable manner across the different oleate/ BSA preparations (Figure 2). Human islets exposed to palmitate with 1% charcoal-absorbed or 0.67% FFA-free BSA showed a similar induction of ATF3 and CHOP mRNA expression (Figure 2, E and F). Taken together, the gene expression data are in keeping with the apoptosis findings. The results obtained with different


FFA and BSA preparations might be explained by changes in the unbound FFA concentrations. These concentra- tions can be theoretically calculated using the multiple stepwise equilibrium model (10,11), but more recently it has become possible to directly measure them using the f luorescent probe ADIFAB2, which is an acrylodan-deriva- tized intestinal fatty acid binding protein (33). Increasing the concentration of FFA-free BSA in the medium resulted in decreased unbound concentrations of FFAs (Figure 3A) in a linear manner across the range of concentrations examined (Figure 3B). In keeping with the comparable cellular responses to FFAs (Figures 1 and 2), FFAs used in the presence of 1% charcoal-absorbed BSA, 0.75% FFA-free BSA, or precom- plexed FFAs used in the presence of 0.67% FFA-free BSA resulted in similar unbound FFA concentrations, ~26 nM for palmitate and ~35 nM for oleate (Figure 3A). These data confirm that the FFA-BSA precomplexing process probably results in some loss of FFAs due to aggregation. The measurement of unbound FFA


concentrations allowed us to compare apoptosis induced by similar unbound concentrations of oleate (24.8 ± 1.4 nM) and palmitate (24.4 ± 1.0 nM) (n = 3–6,


Vol. 58 | No. 5 | 2015


P = 0.9). As expected, the saturated FFA palmitate induced significantly more cell apoptosis (17 ± 3%) than unsaturated oleate (7 ± 1%) (n = 4, P < 0.001). Because of the importance of the


unbound FFA concentrations, the use of fetal bovine serum (FBS) in cell culture also deserves consideration. FBS contains albumin and other FFA binding proteins, which will lower unbound FFA concentrations. The albumin concen- tration of FBS is ~2.5 g/dl. The use of 10% FBS in medium will increase the albumin concentration by 0.25%, which may significantly alter unbound FFA concentrations and cellular effects (Figure 1A and Figure 3A). The mixture of bovine FFAs present in FBS may also affect experimental results. The addition of unsaturated fatty acids is protective against palmitate, at least in part through modulation of the ER stress response (28). An


equimolar combinat ion of


palmitate and oleate (total concen- tration 0.5 mM) in medium containing 0.75% FFA-free BSA resulted in an unbound FFA concentration of 34 nM, a value close to that observed for oleate. In conclusion, when using FFAs for


in vitro experiments, the concentra- tions and preparation of FFAs and BSA should be carefully considered. Our results demonstrate that the source of albumin and the method of FFA conju- gation affect the unbound FFA concen- trations and, consequently, cellular outcomes. In publications using FFAs, the FFA and BSA concentrations and the FFA/albumin molar ratio need to be provided. In addition, we recommend measuring unbound FFA concentra- tions when using FFAs for in vitro exper- iments.


Author contributions


D.A.C. and M.C. contributed to the experimental design of the study. A.F.O., D.A.C., L.L., and M.I.E. carried out experiments and data analysis. M.B. and P.M. contributed materials. A.F.O., D.A.C., and M.C. wrote the manuscript.


Acknowledgments


This work was suppor ted by the European Union (project BetaBat


in 232


Framework Program 7), the Fonds National de la Recherche Scientifique (FNRS), and Actions de Recherche Concer tées


de la Communauté


Française (ARC), Belgium. D.A.C. is an FNRS post-doctoral fellow. We thank Michael Pangerl, Anyishaï Musuaya, and Isabelle Millard, ULB Center for Diabetes Research, for expert technical assistance, and Décio L Eizirik, ULB Center for Diabetes Research, and Alan M Kleinfeld, Fluoresprobe Sciences LLC, San Diego, CA, for helpful discussions and comments on the manuscript.


Competing interests The authors declare no competing interests.


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