Molecular characterization and functional analysis of apoptosis-inducing factor (AIF) in palmitic acid-induced apoptosis in Ctenopharyngodon idellus kidney (CIK) cells

Abstract

Palmitic acid (PA), the most common saturated free fatty acid, may cause apoptosis when overloaded in non-fat cells. Apoptosis-inducing factor (AIF) is known to translocate from the mitochondria into the nucleus to induce apoptosis. However, it remains to be investigated whether AIF involved in palmitic acid-induced lipoapoptosis in fish. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) AIF (CiAIF) gene, and determined its function in Ctenopharyngodon idellus kidney (CIK) cells. The open reading frame (ORF) of CiAIF gene is 1863 bp, encoding a precursor protein of 620 amino acids (aa). Sequence analysis indicated that CiAIF contains a mitochondrial localization sequence, a conserved Pyr_redox and a C-terminal domain. Phylogenetic analyses showed that the CiAIF gene tended to cluster with sequences from Danio rerio. CiAIF gene was ubiquitously expressed in all tested tissues, including heart, liver, spleen, muscle, brain, eye, kidney, intestine, and fat. Moreover, we demonstrated that PA treatment induced the expression level of CiAIF and increases in markers of endoplasmic reticulum (ER) stress and apoptosis. Meanwhile, ER stress-inducing agent thapsigargin (TG) induced CiAIF translocated into the nucleus in CIK cells, whereas the suppression of ER stress inhibited PA-induced CiAIF expression and apoptosis. In addition, overexpression of CiAIF caused apoptosis by upregulating capase9, capase8, and capase3b, and affects protein translation via directly interacting with CieIF3g. Taken together, our data indicate that in Ctenopharyngodon idellus, PA is key elements that affect not only ER stress and mitochondrial apoptosis but also different physiological functions, such as protein translation, and CiAIF might play a key role in this progress.