Translation Inhibitors Induce Formation of Cholesterol Ester-Rich Lipid Droplets

<div><p>Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy.</p> </div

Translation inhibitors induced an increase in cytoplasmic lipid droplets.

<p>(A) Time course of the LD increase in 3Y1 cells treated with 10 µg/ml cycloheximide (CHX). LDs and nuclei were stained with BODIPY493/503 and DAPI, respectively. Small LDs were detectable as early as 4 hr after the addition of CHX, but the increase in LDs became more prominent with longer incubation. Bar, 10 µm. (B) Effect of different translation inhibitors. 3Y1 cells under normal culture conditions were compared with those treated with 10 µg/ml CHX, 2 µM puromycin, or 20 µM emetine for 18 hr. LDs increased in both size and number upon treatment with any of the reagents. Bar, 10 µm. (C) Electron microscopy. LDs in 3Y1 cells that were treated with either 10 µg/ml CHX or 0.4 mM oleic acid (OA) for 18 hr were observed as round electron-lucent structures. High-magnification figures of the rectangular areas are shown in the inset. Notably, the perimeters of LDs induced by CHX were lined with an electron-dense rim, which was not seen in those induced by OA (arrowheads in the inset). Bar, 1 µm.</p

CHX treatment caused an increase in cellular cholesterol ester content.

<p>(A) Thin layer chromatography (TLC) of the total lipid extract from 3Y1 cells. Cholesterol esters (CE) increased significantly after treatment with 10 µg/ml CHX for 18 hr, whereas triglycerides (TG) did not show a significant change. In contrast, in cells treated with 0.4 mM OA, TG rather than CE increased. Lipids extracted from cells with an equal protein content were compared. (B) Quantification of TG, CE, and free cholesterol (FC) in 3Y1 cells. The experimental conditions were the same as in (A). CHX increased CE significantly, whereas OA induced a prominent increase in TG. The FC content was equivalent in the three samples. Mean ± standard deviation (SD) is shown. The difference from the control sample was examined by Student’s <i>t</i> test (*<i>p</i><0.01, **<i>p</i><0.05). (C) Time course of the CE increase after CHX treatment. The increase in CE in 3Y1 cells was detectable by means of TLC as early as 4 hr after treatment with 10 µg/ml CHX. (D) TLC of the total lipid extract from Huh7 and 293A cells treated without or with 10 µg/ml CHX for 18 hr. The increase in CE was observed in both cell types. (E) Quantification of CE and FC in Huh7 cells that were treated without or with 0.25 mM methyl-β-cyclodextrin-cholesterol complex (MβCD-FC) or with 10 µg/ml CHX for 18 hr. Both treatments increased CE without affecting the FC content. Mean ± SD is shown. The difference from the control sample was examined by Student’s <i>t</i> test (*<i>p</i><0.01).</p

CHX caused the CE increase even in autophagy-deficient cells.

<p>(A) 3Y1 cells were treated without or with either 10 µg/ml CHX or 0.4 mM OA for 18 hr. CHX caused a significant increase in phospho-S6K. (B) The autophagic flux was examined by incubating 3Y1 cells with 20 µM chloroquine for 1 hr immediately before sample preparation. Chloroquine caused a significant increase in LC3-II in the control, but not in cells pretreated with 10 µg/ml CHX for 18 hr. (C) Wild-type and Atg5-null MEF were treated without or with 10 µg/ml CHX for 18 hr. The increase in phospho-S6K was observed in a comparable degree in both cell lines. GAPDH was probed as a loading control. (D) Wild-type and Atg5-null MEF were treated without or with 10 µg/ml CHX for 18 hr. CE increased significantly in response to CHX treatment in both cell lines. Mean ± SD is shown. The difference from the respective control was examined by Student’s <i>t</i> test (*<i>p</i><0.01).</p

CHX induced increases in LDs and CE even in the presence of mTORC1 inhibitors.

<p>(A) 3Y1 cells were treated without or with 10 µg/ml CHX, 10 µg/ml CHX and 0.4 µM rapamycin, or 10 µg/ml CHX and 0.25 µM 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 µg protein. (B) 3Y1 cells were treated with 10 µg/ml CHX alone or together with 0.4 µM rapamycin or 0.25 µM Torin1 for 18 hr. LDs increased to a similar degree irrespective of the presence of rapamycin or Torin1. Bar, 10 µm. (C) 3Y1 cells were treated without or with 10 µg/ml CHX, 10 µg/ml CHX and 0.4 µM rapamycin, or 10 µg/ml CHX and 0.25 µM Torin1 for 18 hr. CE increased in response to CHX treatment even when rapamycin or Torin1 was given simultaneously. Mean ± SD is shown. The difference from the control sample was examined by Student’s <i>t</i> test (*<i>p</i><0.01). (D) 3Y1 cells were treated in the same manner as in Fig. 5A. The autophagic flux was examined by adding 20 µM 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 µg protein.</p

Asymmetrical Distribution of Choline Phospholipids Revealed by Click Chemistry and Freeze-Fracture Electron Microscopy

Choline-containing phospholipids (Cho-PLs) are major components of all cellular membranes. We developed an electron microscopic technique to investigate the poorly understood problem of how Cho-PLs are distributed between membrane leaflets. Our method relies on generating freeze-fracture replicas of cells metabolically labeled with the choline analog, propargylcholine, followed by “click” reaction to conjugate biotin to propargylcholine head groups, and immunodetection of biotin with colloidal gold. Using this method in budding yeast, we found that, surprisingly, the Golgi and plasma membrane display a cytoplasmic leaflet-dominant asymmetry in Cho-PL distribution; in contrast, Cho-PLs are evenly distributed between the exoplasmic and cytoplasmic leaflets of other organelle membranes. In mammalian culture cells, the plasma membrane shows symmetrical Cho-PL distribution between leaflets, suggesting a fundamental difference between yeast and mammals. Our method should be expandable to other classes of lipids and will be useful for deciphering the mechanism responsible for generating lipid asymmetry in biological membranes

Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet

<div><p>We showed previously that UBXD8 plays a key role in proteasomal degradation of lipidated ApoB in hepatocarcinoma cell lines. In the present study, we aimed to investigate the functions of UBXD8 in liver <i>in vivo</i>. For this purpose, hepatocyte-specific UBXD8 knockout (UBXD8-LKO) mice were generated. They were fed with a normal or high-fat diet, and the phenotypes were compared with those of littermate control mice. Hepatocytes obtained from UBXD8-LKO and control mice were analyzed in culture. After 26 wk of a high-fat diet, UBXD8-LKO mice exhibited macrovesicular steatosis in the periportal area and microvesicular steatosis in the perivenular area, whereas control mice exhibited steatosis only in the perivenular area. Furthermore, UBXD8-LKO mice on a high-fat diet had significantly lower concentrations of serum triglyceride and VLDL than control mice. A Triton WR-1339 injection study revealed that VLDL secretion from hepatocytes was reduced in UBXD8-LKO mice. The decrease of ApoB secretion upon UBXD8 depletion was recapitulated in cultured primary hepatocytes. Accumulation of lipidated ApoB in lipid droplets was observed only in UBXD8-null hepatocytes. The results showed that depletion of UBXD8 in hepatocytes suppresses VLDL secretion, and could lead to periportal steatosis when mice are fed a high-fat diet. This is the first demonstration that an abnormality in the intracellular ApoB degradation mechanism can cause steatosis, and provides a useful model for periportal steatosis, which occurs in several human diseases.</p></div

Histological evaluation of liver at 30 wk of age.

<p>For both control and UBXD8-LKO mice, 15 females and 15 males were examined by microscopy of paraffin sections stained with H&E. Statistical significance of the data was analyzed by Fisher’s exact test.</p><p>*P<0.001 by Fisher's exact test.</p><p>Histological evaluation of liver at 30 wk of age.</p

Analysis of serum lipoproteins.

<p>(A) Lipoprotein profile obtained by gel filtration–HPLC of mouse serum at 30 wk old. A representative result on TG (blue) and total cholesterol (red) is shown for sera of normal and UBXD8-LKO male mice fed a normal (A) or a high-fat diet (B). Sera from female mice gave similar results. Sera for the UBXD8-LKO group fed a high-fat diet were taken from mice showing periportal steatosis. Lower panels show a magnified view of the VLDL portion. FG: free glycerol. (B) VLDL-TG was lower in UBXD8-LKO mice than in normal mice, even when they were fed a normal diet. The difference between the control and UBXD8-LKO mouse became significantly larger when mice were fed a high-fat diet. P values were obtained by non-paired Student's t test (n = 3; means ± SEM). (C) The TG-to-cholesterol ratio in VLDL tended to be lower in the UBXD8-LKO than in the control mouse. P values were obtained by non-paired Student's t test (n = 3; means ± SEM).</p

TG secretion was lower in UBXD8-LKO mice than in controls.

<p>TG secretion estimated by the Triton WR-1339 injection method. Control and UBXD8-LKO mice, both male and female, were fed a normal diet until 30–33 wk of age. Serum TG levels were measured before and 1 and 2 h after the Triton WR-1339 injection. P values were obtained by non-paired Student's t test (n = 4; means ± SEM).</p