Palmitate-induced lipotoxicity alters acetylation of multiple proteins in clonal β cells and human pancreatic islets

Abstract

Type 2 diabetes is characterized by progressive β cell dysfunction, with lipotoxicity playing a possible pathogenetic role. Palmitate is often used to examine the direct effects of lipotoxicity and it may cause mitochondrial alterations by activating protein acetylation. However, it is unknown whether palmitate influences protein acetylation in β cells. We investigated lysine acetylation in mitochondrial proteins from INS-1E β cells (INS-1E) and in proteins from human pancreatic islets (HPI) after 24 h palmitate exposure. First, we confirmed that palmitate damages β cells and demonstrated that chemical inhibition of deacetylation also impairs INS-1E function and survival. Then, by 2-D gel electrophoresis, Western Blot and Liquid Chromatography-Mass Spectrometry we evaluated the effects of palmitate on protein acetylation. In mitochondrial preparations from palmitate-treated INS-1E, 32 acetylated spots were detected, with 13 proteins resulting over-acetylated. In HPI, 136 acetylated proteins were found, of which 11 were over-acetylated upon culture with palmitate. Interestingly, three proteins, glutamate dehydrogenase, mitochondrial superoxide dismutase, and SREBP-1, were over-acetylated in both INS-1E and HPI. Therefore, prolonged exposure to palmitate induces changes in β cell protein lysine acetylation and this modification could play a role in causing β cell damage. Dysregulated acetylation may be a target to counteract palmitate-induced β cell lipotoxicity.

Temporal Proteomic Analysis of Pancreatic β-Cells in Response to Lipotoxicity and Glucolipotoxicity

“…The PA stock solution was diluted in culture medium to which fatty-acid-free BSA had been added in a 1:19 molar ratio to prepare BSA-conjugated PA. Cells were incubated with the BSA-conjugated-PA at 0.5 mM without or with additional 16.7 mM glucose (PA or PAG, respectively) for different times (4,8,16,24, and 48 h).…”

“…Moreover, recent studies suggested that the metabolites of acyl-CoAs produced by FFAs and glucose can act as the substrates for post-translational modification (PTM) of proteins, such as palmitoylation and acetylation. These PTMs change protein function, which is highly linked to the dysfunction of ␤-cells (23,24). However, the interplay of the above molecular events is not well understood.…”

“…In this study, which aims to characterize in great detail the time course of lipotoxicity and glucolipotoxicity in pancreatic ␤-cells, we analyzed the protein changes of INS-1 rat insulinoma cell lines (INS-1 cells) treated with high palmitate without (PA) or with high glucose (PAG) at five consecutive time points (4,8,16,24, and 48 h) by multiplex TMT (Tandem Mass Tag) labeling-based quantitative proteomics approaches. Subsequent bioinformatics analysis of differentially expressed proteins, including time-course pattern clustering, proteinprotein interaction networks, gene set enrichment and KEGG pathway analysis, allowed us to recapitulate the lipotoxicityand glucolipotoxicity-related molecular events in more detail, including previously reported and newly identified features.…”

Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin‐secreting cells

“…We observed most marked hyperacetylation on ES1, IF1, TFEa, 3 lysine acetylation sites on the ADP-ATP translocase 1, isocitrate dehydrogenase [NADP], acyl-CoA thioesterase 8, mMDH2, and GDH1 in INS-1E SIRT3KO cells. Lysine acetylation of TFEa (38), ADP-ATP translocase 1 (39), isocitrate dehydrogenase [NADP] (36), MDH2 (1), and GDH1 (11,13) has been observed previously in other cell types or tissues. Importantly, for several of these enzymes (1,13,36,39), their activity is regulated by lysine acetylation (see also the earlier section of the Discussion).…”

“…However, we find that acute glucose stimulation (30 min) is not sufficient to alter the lysine acetylation status of the mitochondrial proteins identified. Lysine acetylation appears regulated mostly on a longer timescale responding to chronic changes in diet rather than rapid fluctuations of nutrients (1, 9, 11, 36).…”