Figure 5. CHX induced increases in LDs and CE even in the presence of mTORC1 inhibitors. (A) 3Y1 cells were treated without or with 10 mg/ml CHX, 10 mg/ml CHX and 0.4 mM rapamycin, or 10 mg/ml CHX and 0.25 mM Torin1 for 8 hr. Addition of rapamycin or Torin1 decreased phospho-S6K significantly. GAPDH is shown as a loading control. Each lane was loaded with 20 mg protein. (B) 3Y1 cells were treated with 10 mg/ml CHX alone or together with 0.4 mM rapamycin or 0.25 mM Torin1 for 18 hr. LDs increased to a similar degree irrespective of the presence of rapamycin or Torin1. Bar, 10 mm. (C) 3Y1 cells were treated without or with 10 mg/ml CHX, 10 mg/ml CHX and 0.4 mM rapamycin, or 10 mg/ml CHX and 0.25 mM Torin1 for 18 hr. CE increased in response to CHX treatment even when rapamycin or Torin1 was given simultaneously. Mean 6 SD is shown. The difference from the control sample was examined by Student’s t test (*p,0.01). (D) 3Y1 cells were treated in the same manner as in Fig. 5A. The autophagic flux was examined by adding 20 mM chloroquine for 1 hr immediately before sample preparation. A low level of LC3-II increase was caused by chloroquine in samples treated with CHX alone or CHX and rapamycin, but not in samples treated with CHX and Torin1. GAPDH is shown as a loading control. Each lane was loaded with 50 mg protein.
activation is inhibition of autophagy (for a recent review, see Jung et al., 2010), and the autophagic flux in 3Y1 cells was in fact suppressed significantly by CHX (Fig. 4B). To examine the possibility that down-regulation of autophagy by CHX caused the increase in CE-rich LDs, we turned to autophagy-deficient MEF taken from Atg5 knockout mice [16]. It was confirmed that mTORC1 was activated similarly by CHX in wild-type and Atg5-null MEF (Fig. 4C). However, CE and LDs were observed to increase as a result of CHX treatment in both cell types (Fig. 4D; data not shown). These results demonstrated that the CHX-induced increase in CE-rich LDs does not depend on suppression of autophagy. It is notable, however, that significantly larger amounts of CE were found in Atg5-null cells than in wild-type cells, both without and with CHX treatment (Fig. 4D), indicating that autophagy may also be engaged in degrading CE-rich LDs.CE-rich LDs Increased Even When mTORC1 was Inhibited
Since mTORC1 is a hub of many intracellular signaling pathways, its activation could cause various other effects in addition to autophagic suppression [27]. To test whether the CHX-induced LD increase occurred as a result of mTORC1 activation, cells were treated with either rapamycin or Torin1along with CHX. It was confirmed that phosphorylation of S6K was suppressed effectively by either 0.4 mM rapamycin or 0.25 mM Torin1 (Fig. 5A). Even in the presence of these mTORC1 inhibitors, CHX induced a significant increase in LDs (Fig. 5B) and CE (Fig. 5C), indicating that mTORC1 activation is not involved in the mechanism. Interestingly, in cells treated with rapamycin or Torin1 along with CHX, even though mTORC1 was inhibited, autophagy was not activated (Fig. 5D): the autophagic flux was low (rapamycin) or appeared nonexistent (Torin1). The seemingly complete suppression of autophagy with CHX and Torin1 may explain the significantly higher CE increase in those cells than that in cells treated with CHX alone or with CHX and rapamycin (Fig. 5C). On the other hand, in the absence of CHX, Torin1 alone induced a slight but significant increase of CE (Fig. S1). The result suggested that Torin1 enhanced the CHX-induced CE increase by suppressing mTORC2, which is likely to be a negative regulator of lipid accumulation [28,29].
Discussion
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 [13,14,15] and autophagy suppression [11,12], 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 (HSL) may be engaged in CE hydrolysis [30], 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 [9]. 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 [10]. 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 (Fig. 4D). Moreover, the 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 (Fig. 5C, 5D). These results showed that autophagic suppression is not the main cause of the CE increase induced by CHX, but nonetheless they also indicated that autophagy is an important mechanism of CE degradation. LDs that consist predominantly of TG in white adipocytes are degraded effectively by the sequential action of ATGL, HSL, and monoacylglycerol lipase [8]. To degrade LDs that are enriched with CE, however, lysosomal acid lipase, which has CE hydrolytic activity, may be involved, as it is for degradation of cholesterolloaded macrophages [31]. For LDs containing CE and TG in comparable amounts, CE hydrolysis may be a prerequisite for effective TG degradation because CE may surround the TG core, forming concentric layers on the surface [24]. In this context, it is notable that the deficiency of lysosomal acid lipase thatcharacterizes Wolman disease manifests as an accumulation of CE as well as TG [32]. It was surprising that, upon treatment with translation inhibitors, TIP47 was recruited to the CE-rich LDs even though the total amount of TIP47 decreased drastically. TIP47 was previously shown to be recruited to TG-rich LDs induced by unsaturated fatty acids, but in such cases the overall expression of TIP47 also increased [26]. The present result indicates that TIP47 recruitment to LDs does not depend on the increased expression of TIP47 or on the composition of the lipid esters in LDs; rather, it is directly related to the increment of lipid esters. On the other hand, the increased recruitment of TIP47 to LDs should reduce TIP47 in the soluble cytoplasmic fraction, especially when the total amount is downregulated. Although the non-LD function of TIP47 remains controversial [33,34], it must be determined whether any result seen in the presence of translation inhibitors can be explained by a decrease in TIP47 in the cytoplasm. The phenomena observed in the present study need to be taken into account in interpreting experimental results obtained using translation inhibitors. Yet the implications of this study are not limited to such artificial conditions, given that, in cells exposed to various stresses, protein synthesis is suppressed [35] and LDs increase [36,37]. LDs that increase in cultured cells under ER stress are enriched with CE (Ohsaki et al., unpublished observation). The detailed mechanism underlying CE-rich LD formation as well as the impact of this process are worthy of further studies in this context.
