• Cholesterol esterification is another way to prevent the accumulation of free cholesterol within cells. The human acyl-coenzyme A:cholesterol acyltransferase (ACAT) can regulate the storage or secretion of cholesterol through esterification, maintaining the balance between free cholesterol and cholesterol esters. ACAT1 is widely expressed in cells throughout the body, but its expression is significantly higher in macrophages, epithelial cells, and steroidogenic cells compared to other cell types. Mammals have two isoenzymes, ACAT1 and ACAT2. Both are transmembrane proteins, with ACAT1 predicted to have nine transmembrane domains and ACAT2 having two to five domains. The first 140 amino acids of ACAT1 are located in the cytoplasm and mediate the formation of a tetramer composed of two homodimers. Whether ACAT2 exists in a polymeric form is unknown.
• Cholesterol is an allosteric activator of ACAT1. Cholesterol can activate ACAT1 by directly binding to it, and the activated ACAT1 can esterify intracellular free cholesterol, storing it in lipid droplets. This positive feedback mechanism allows for rapid regulation of intracellular free cholesterol levels. ACAT2 is primarily expressed in intestinal epithelial cells, with trace amounts in the liver. 
• In mice, blocking ACAT1-mediated cholesterol esterification has been shown to improve Alzheimer's disease and inhibit the growth of pancreatic and prostate cancer tumors. Inhibiting ACAT1 in CD8+ T cells increases plasma membrane cholesterol, promotes T cell receptor clustering and immune synapse formation, ultimately enhancing the anti-tumor activity of these cells. This suggests that ACAT1 could be a novel therapeutic target for cancer treatment. ACAT1 inhibitors have previously been shown to alleviate amyloid pathology, reduce the size of hepatocellular carcinoma tumors, inhibit the growth and metastasis of pancreatic cancer tumors, prevent prostate cancer, and enhance the anti-tumor response of CD8+ T cells, as well as immunotherapy. Additionally, knocking out ACAT1 can alleviate atherosclerotic phenotypes.
• The exons 2-12 of mouse Acat1 gene were knocked out in B-Acat1 KO mice. 
• Protein expression analysis: Mouse ACAT1 was only detected in heart, liver, spleen, lung, kidney and brain of wild-type mice, but not in homozygous B-Acat1 KO mice.
• mRNA expression analysis: Mouse Acat1 mRNA was only detectable in heart and brain of wild-type mice, but not in homozygous B-Acat1 KO mice.

Gene targeting strategy for B-Acat1 KO mice. The exons 2-12 of mouse Acat1 gene were knocked out in B-Acat1 KO mice. 

Western blot analysis of ACAT1 protein expression in homozygous B-Acat1 KO mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-Acat1 KO mice (-/-), and then analyzed by western blot with anti-ACAT1 antibody (Proteintech, 16215-1-AP). 40 μg total proteins were loaded for western blotting analysis. Mouse ACAT1 was only detected in heart, liver, spleen, lung, kidney and brain of wild-type mice, but not in homozygous B-Acat1 KO mice.

Strain specific analysis of Acat1 mRNA expression in wild-type C57BL/6JNifdc and B-Acat1 KO mice by RT-PCR. Heart and brain RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-Acat1 KO mice (-/-), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Acat1 primers. Mouse Acat1 mRNA was only detectable in wild-type mice, not in homozygous B-Acat1 KO mice.