BIOFEEDBACK
some contaminants, including endog- enous bovine FFAs (26), and has been used by our group in previous publi- cations (11,27–30). FFA-free albumins are commercially available and have a high degree of purity. FFAs can be added directly to albumin-containing culture medium or be precomplexed with albumin before being added to the medium. Here we present a systematic
comparison of the effects of different preparations of palmitate or oleate and BSA on lipotoxicity in pancreatic b-cells. Charcoal-absorbed BSA and FFA-free BSA were used to prepare FFA solutions, as described in the Materials and methods section in the Supplementary Material. Compared with charcoal-absorbed BSA, FFA-free BSA has the advantage of having less residual FFAs bound to it. Exposure of INS-1E cells for 16 h to 0.5 mM palmitate in 1% FFA-free BSA-containing medium (FFA/albumin ratio of 3.3) induced significantly less apoptosis compared with medium containing 1% charcoal- absorbed BSA (Figure 1A). We therefore asked whether the serial filtration steps that follow the charcoal incubation of BSA affect the final albumin concen- tration of the medium. The BSA concentration in the 1% charcoal- absorbed BSA preparation was 25%
lower compared with 1% FFA-free BSA (n = 3–4, P = 0.03). In keeping with this result, a reduction from 1% to 0.75% FFA-free BSA (FFA/albumin ratio of 4.4) resulted in apoptosis levels compa- rable to the 1% charcoal-absorbed BSA preparation (Figure 1A). We next prepared FFA solutions by precom- plexing palmitate with FFA-free BSA before diluting it into the medium (see Materials and methods section in the Supplementary Material). Compared with FFA stock solutions prepared in ethanol that are typically not kept for more than a month, the FFA-albumin complexes can be prepared in large amounts and stored for several years at -20°C. Precomplexed palmitate (0.5 mM, 0.75% FFA-free BSA) induced less apoptosis compared with the equivalent non-precomplexed preparation (Figure 1A). Similar apoptosis levels were only reached when the BSA concentration was lowered to 0.67% (FFA/albumin ratio of 5.0) (Figure 1A). We hypothe- sized that during the precomplexing of FFAs to BSA, some of the FFAs form aggregates resulting in lower FFA concentrations. Oleate (0.5 mM) used in the presence
of 1% charcoal-absorbed BSA or 0.75% FFA-free BSA, or precomplexed oleate used in the presence of 0.67% FFA-free BSA, induced comparable levels of
apoptosis (Figure 1B), in keeping with the palmitate data. We also compared cell death induced in human islets by precomplexed FFA preparations against 1% charcoal-absorbed BSA, as used in previous reports (28–30). Because primary human islets are less suscep- tible to lipotoxicity than clonal rat INS-1E cells (29,31,32), islet cell death was assessed after 72 h of exposure. As in INS-1E cells, a reduction in the amount of BSA to 0.67% resulted in similar levels of palmitate- or oleate- induced cell death (Figure 1, C and D). These data illustrate that dose- response experiments are of value for determining suitable experimental conditions. Based on the cell death data, we chose 1% charcoal-absorbed BSA, 0.75% FFA-free BSA, or precom- plexed FFAs in the presence of 0.67% FFA-free BSA for further signal trans- duction studies. FFAs cause b-cell apoptosis via the
induction of endoplasmic reticulum (ER) stress and activation of the mitochon- drial pathway of cell death through up-regulation of pro-apoptotic BCL-2 proteins (29). We compared the effect of di fferent palmi tate and oleate preparations on the induction of transcription factors that mediate the ER stress response, namely activating transcription factor 3 (ATF3), spliced
Figure 3. Unbound free fatty acid (FFA) concentrations in different FFA/albumin preparations. (A) Unbound FFA concentrations were measured using the ADIFAB2 probe in 0.5 mM (total concentration) palmitate (PAL)- or oleate (OLE)-containing phenol red–free RPMI medium containing 1% charcoal-absorbed BSA, the indicated concentration of FFA-free BSA, or 0.67% FFA-free BSA precomplexed to palmitate or oleate. (B) Relationship between unbound palmitate or oleate concentrations and the percentage of FFA-free BSA in the solution. Linear regression is shown for palmitate (R2
= 0.99) and oleate (R2 Vol. 58 | No. 5 | 2015 = 0.91). Results represent mean ± SEM of 3–9 independent experiments. 230
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