Abstract
Cholesterol plays essential structural and signaling roles in mammalian cells, but too much cholesterol can cause cytotoxicity. Acyl-CoA:cholesterol acyltransferases 1 and 2 (ACAT1/2) convert cholesterol into its storage form, cholesteryl esters, regulating a key step in cellular cholesterol homeostasis. Adipose tissue can store >50% of whole-body cholesterol. Interestingly, however, almost no ACAT activity is present in adipose tissue, and most adipose cholesterol is stored in its free form. We therefore hypothesized that increased cholesterol esterification may have detrimental effects on adipose tissue function. Here, using several approaches, including protein overexpression, quantitative RT-PCR, immunofluorescence, and various biochemical assays, we found that ACAT1 expression is significantly increased in the adipose tissue of the ob/ob mice. We further demonstrated that ACAT1/2 overexpression partially inhibited the differentiation of 3T3-L1 preadipocytes. In mature adipocytes, increased ACAT activity reduced the size of lipid droplets (LDs) and inhibited lipolysis and insulin signaling. Paradoxically, the amount of free cholesterol increased on the surface of LDs in ACAT1/2-overexpressing adipocytes, accompanied by increased LD localization of caveolin-1. Moreover, cholesterol depletion in adipocytes by treating the cells with cholesterol-deficient media or β-cyclodextrins induced changes in cholesterol distribution that were similar to those caused by ACAT1/2 overexpression. Our results suggest that ACAT1/2 overexpression increases the level of free cholesterol on the LD surface, thereby impeding adipocyte function. These findings provide detailed insights into the role of free cholesterol in LD and adipocyte function and suggest that ACAT inhibitors have potential utility for managing disorders associated with extreme obesity.
Highlights
Cholesterol plays essential structural and signaling roles in mammalian cells, but too much cholesterol can cause cytotoxicity
We found that the protein level of ACAT1 was ϳ7-fold higher in the adipose tissue of the ob/ob mice than that of the WT mice (Fig. 1, A and B)
Adipose tissue can store more than 50% of whole-body cholesterol
Summary
Because normal adipose tissue maintains very low ACAT expression and activity, we suspect that increased ACAT expression/activity may exert adverse effects on adipose function and may be associated with dysfunctional adipose tissue. As revealed by filipin staining, there was appreciable free cholesterol accumulating on the LD surface instead of the plasma membrane when overexpressing ACAT1/2 in adipocytes on day 8 after differentiation, and ACAT2 overexpression showed stronger effects (Fig. 6, A and B). This phenotype was seen in mature adipocytes transiently overexpressing ACAT1 or ACAT2, respectively (Fig. 6, C and D). The free cholesterol level was significantly increased in mature adipocytes that were differentiated from 3T3-L1 preadipocytes stably overexpressing ACAT1/2 (Fig. 9A), as well as mature adipocytes transiently overexpressing ACAT1/2 (Fig. 9C) compared with control cells. These results indicate that ACAT overexpression perturbs cholesterol homeostasis in adipocytes
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