BIOFEEDBACK
Figure 2. Induction of markers of endoplasmic reticulum (ER) stress and the mitochondrial pathway of apoptosis. (A) ATF3 mRNA expression in INS-1E cells cultured 16 h in the absence (CTL) or presence of 0.5 mM palmitate (PAL) or oleate (OLE) in medium containing 1% charcoal-absorbed BSA, 0.75% FFA-free BSA, or 0.67% FFA-free BSA precomplexed to palmitate or oleate (n = 5–8). (B) Spliced XBP1 (XBP1s), (C) CHOP and (D) DP5 mRNA expression in INS-1E cells treated as noted in (A) (n = 5–8). (E) ATF3 and (F) CHOP mRNA expression in human islets cultured 48 h in the absence (CTL) or presence of 0.5 mM palmitate (PAL) in medium containing 1% charcoal-absorbed BSA or 0.67% FFA-free BSA precomplexed to palmitate (n = 6). Gene expression was normalized for the geometric mean of the expression levels of reference genes GAPDH, b-actin, and OAZ1. For INS-1E cells, the normalized data were expressed as fold induction of CTL. Results represent mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001 for the comparison against CTL. ##
P < 0.01 for the comparison between FFA preparations.
ratio of 3.3, the theoretical unbound palmitate and oleate concentrations are 27 nM and 47 nM, respectively (11). Under normal physiological condi-
tions, an average of 2 FFA molecules are bound to each albumin molecule in the circulation, corresponding to concentrations of unbound FFAs less than 20 nM. The FFA/albumin molar
Vol. 58 | No. 5 | 2015
ratio can rise to as high as 6 in patho- logical conditions (12,13). In experi- mental systems designed to evaluate mechanisms of lipotoxicity relevant to pathophysiological states, variable and sometimes high molar ratios are used (14–23). Ratios greater than 5 should be avoided as they may exceed FFA solubility, potentially inducing artifacts.
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In spite of the crucial role of albumin in determining biologically available FFA levels, the BSA concentration or FFA/ albumin molar ratio are frequently not mentioned in scientific reports (24,25). Albumin preparations may contain
variable amounts of contaminants (26), which can affect FFA binding. Charcoal treatment of BSA allows the removal of
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