In white adipocytes, the lipid ester core consists almost exclusively of triglycerides, whereas in many non-adipocytes LDs contain both TG and cholesterol esters in various ratios. TG synthesis is facilitated in the presence of excess fatty acids. In many non-adipocytes in culture, only a small number of LDs exist under normal conditions, but the addition of unsaturated fatty acids such as oleic acid to the medium induces abundant TG-rich LDs. CE metabolism has been studied most actively using macrophage foam cells, which take up significant quantities of plasma lipoproteins; in contrast, the general conditions that induce CE accumulation in other cell types are not well known. Degradation mechanisms have also been more thoroughly analyzed for TG than for CE. The regulatory mechanism of cytosolic lipases, including adipocyte triglyceride lipase and hormone-sensitive lipase, has been rapidly unveiled. In contrast, the enzymes engaged in CE hydrolysis have not been firmly established, even in macrophage foam cells. A recent study revealed that autophagy is involved in the degradation of LDs in hepatocytes, but it is not yet known in detail whether and to what extent this process is active in other cell types. In the present study, we found that treatment with protein translation inhibitors causes a significant increase in BMS-354825 CE-rich LDs. Translation inhibitors are frequently used in cell biological experiments, but the effect observed in the present study has not been given attention in the past. Earlier studies showed that treatment with cycloheximide suppresses autophagy. More recently, inhibition of protein synthesis was shown to activate mTORC1. We aimed to investigate whether the increase in CE-rich LDs that results from treatment with translation inhibitors was caused by mTORC1 activation and/ or suppression of autophagy. In the present study, we found that protein translation inhibitors cause a significant increase in CE-rich 
LDs. Because translation inhibitors are known to cause mTORC1 activation and autophagy suppression, we initially supposed that those processes were responsible for the increase in CE-rich LDs. Yet this increase in CE and LDs was observed even in the presence of mTORC1 inhibitors and in autophagy-deficient cells, indicating the engagement of other mechanisms. As a possible cause of the observed phenomena, we speculate that translation inhibitors may cause a down-regulation of CE hydrolysis: that is, CE hydrolytic enzymes may have a relatively short half-life and may decrease quickly when protein synthesis is suppressed. Hormone-sensitive lipase may be engaged in CE hydrolysis, but if its decrease were the main cause of the CE increase in CHX-treated cells, TG would be expected to increase simultaneously, and this was not observed in the present experiment. Other than HSL, several neutral CE hydrolases have been reported to be critical for CE digestion in macrophage foam cells, but their role in other cell types is not clear. Thus we are yet to examine the aforementioned possibility. We observed that the CHX-induced increase in CE and LDs also occurs in autophagy-deficient Atg5-null MEF, but this does not preclude the possibility that autophagy is involved in CE metabolism and LD turnover. In fact we found that significantly larger amounts of CE were observed in Atg5-deficient MEF than in wild-type MEF both before and after CHX treatment. Moreover, the PR-171 seemingly complete suppression of the autophagic flow in cells treated with CHX and Torin1 caused a significantly higher increase of CE than in cells treated with CHX alone, in which a low level of autophagy was occurring.