Acetylation of the Mitochondrial Chaperone GRP75 Governs ER‐Mitochondrial Calcium Homeostasis and Hepatocyte Insulin Resistance

Abstract Overnutrition exacerbates insulin resistance (IR) and is linked to excessive mitochondrial protein acetylation. However, the molecular mechanism by which mitochondrial protein acetylation influences hepatic IR remains incompletely elucidated. To investigate this biology, GCN5L1 liver knockout mice (LKO), which exhibit blunted mitochondrial protein acetylation are utilized. Interestingly, the hepatocytes of LKO mice exhibit impaired insulin signaling and exaggerated endoplasmic reticulum (ER) stress. To explore putative mechanisms, protein‐interaction and acetyl‐proteome analyses are conducted following hepatic induction of GCN5L1. The mitochondrial chaperone GRP75 interacts with GCN5L1 and is acetylated on lysine residues K567 and K612 by GCN5L1 overexpression. Furthermore, GRP75‐K567/612 acetylation reduces the assemble of IP3R1‐GRP75‐VDAC complex, which in turn leads to the maintenance of ER calcium homeostasis and insulin sensitivity. Interestingly, during high‐fat diet feeding, mitochondria‐localized GCN5L1 is significantly translocated to the cytosol. This translocation attenuates the acetylation of GRP75 at K567/612 and consequently enhances ER‐mitochondrial calcium flux and induces ER stress. In parallel, deacetylation‐mimicking mutated GRP75‐K567/612 promotes IR in vivo. Consequently, these findings demonstrate that the acetylation‐dependent modification of GRP75 plays a functional role in regulating overnutrition‐induced IR.