Basic Sciences

AUP1 (Ancient Ubiquitous Protein 1) Is a Key Determinant of Hepatic Very-Low–Density Lipoprotein Assembly and SecretionHighlights

Jing Zhang, Mostafa Zamani, Christoph Thiele, Jennifer Taher, Mohsen Amir Alipour, Zemin Yao, Khosrow Adeli
https://doi.org/10.1161/ATVBAHA.117.309000
Arteriosclerosis, Thrombosis, and Vascular Biology. 2017;37:633-642
Originally published February 9, 2017

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Abstract

Objective—AUP1 (ancient ubiquitous protein 1) is an endoplasmic reticulum–associated protein that also localizes to the surface of lipid droplets (LDs), with dual role in protein quality control and LD regulation. Here, we investigated the role of AUP1 in hepatic lipid mobilization and demonstrate critical roles in intracellular biogenesis of apoB100 (apolipoprotein B-100), LD mobilization, and very-low–density lipoprotein (VLDL) assembly and secretion.

Approach and Results—siRNA (short/small interfering RNA) knockdown of AUP1 significantly increased secretion of VLDL-sized apoB100-containing particles from HepG2 cells, correcting a key metabolic defect in these cells that normally do not secrete much VLDL. Secreted particles contained higher levels of metabolically labeled triglyceride, and AUP1-deficient cells displayed a larger average size of LDs, suggesting a role for AUP1 in lipid mobilization. Importantly, AUP1 was also found to directly interact with apoB100, and this interaction was enhanced with proteasomal inhibition. Knockdown of AUP1 reduced apoB100 ubiquitination, decreased intracellular degradation of newly synthesized apoB100, and enhanced extracellular apoB100 secretion. Interestingly, the stimulatory effect of AUP1 knockdown on VLDL assembly was reminiscent of the effect previously observed after MEK–ERK (mitogen-activated protein kinase kinase–extracellular signal-regulated kinase) inhibition; however, further studies indicated that the AUP1 effect was independent of MEK–ERK signaling.

Conclusions—In summary, our findings reveal an important role for AUP1 as a regulator of apoB100 stability, hepatic LD metabolism, and intracellular lipidation of VLDL particles. AUP1 may be a crucial factor in apoB100 quality control, determining the rate at which apoB100 is degraded or lipidated to enable VLDL particle assembly and secretion.

Introduction

Lipid droplets (LDs) are dynamic organelles that serve as the main intracellular storage sites for neutral lipids.1 They are composed of a highly hydrophobic core surrounded by a phospholipid monolayer and have been shown to be associated with various proteins.2 Proteomic studies have revealed 229 mammalian LD-associated proteins, including AUP1 (ancient ubiquitous protein 1), which was listed in endoplasmic reticulum (ER)–associated protein category,3 and more recently, AUP1 was found to be abundantly expressed on the surface of LDs.4 Hepatic LDs play a particularly important role in maintaining lipid homeostasis through the regulation of very-low–density lipoprotein (VLDL) secretion.5–8 Hepatic VLDL overproduction, a common feature of insulin-resistant states, results in increased circulating levels of triglyceride and cholesterol, which are major risk factors for cardiovascular disease and coronary atherosclerosis.9,10 Therefore, understanding the link between LD biology and VLDL assembly and secretion could have important clinical implications.

See accompanying editorial on page 609

AUP1 is known as an ER-associated protein that is involved in ER-associated degradation (ERAD) of misfolded proteins.11 Its ability to localize to both LDs and the ER membrane is mainly because of the presence of a hydrophobic domain close to the N terminus, called the LD-targeting domain.4,12,13 AUP1 is inserted into the ER membrane via a monotopic hairpin fashion and transported to the LD membrane.13 Additional domains of AUP1 include a predicted acyltransferase domain, a known G2-binding region, and a coupling of ubiquitin conjugated to ERAD domain at the C terminus.13 In line with the presence of a coupling of ubiquitin conjugated to ERAD domain, AUP1 has been copurified with gp78, an E3 ubiquitin ligase, and has been shown to recruit ubiquitin-conjugating enzyme Ubc7 to the surface of LDs.4,11 It was recently shown to be involved in ERAD of both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and Insig-1 at the LD surface, which suggests a key role for AUP1 in the maintenance of cholesterol homeostasis.14 The ability for AUP1 to additionally regulate hepatic lipoprotein metabolism through its actions at the surface of LDs has not yet been investigated.

apoB100 (apolipoprotein B-100) is the main structural protein component of hepatic VLDL particles. The apoB100 gene is constitutively transcribed and translated in hepatocytes. Therefore, the primary way in which this protein is regulated is through its intracellular degradation.10 Studies have suggested the involvement of both ERAD and the post-ER degradative pathways in apoB100 degradation,15 with ERAD acting as the primary pathway in cultured hepatocytes such as HepG2 cells.16,17 Ubiquitination can regulate the assembly of VLDL particles in HepG2 cells and is considered the essential step in targeting apoB100 to the ERAD pathway.18 Recent studies by our laboratory and others have shown that apoB100 associates with many proteins involved in the ERAD pathway, mostly molecular chaperones known as heat-shock proteins.19–21 We have previously shown the importance of BiP, an ER luminal chaperone, in proteasomal targeting and ER quality control of misfolded apoB100.22 These studies suggested that the interaction of apoB100 chains with lipids reduced BiP-mediated ER retention and facilitated transport of apoB100 out of the ER. Alternatively, nascent apoB100 chains retained by BiP may subsequently be targeted to the proteasome via interaction with p97, a cytosolic ATPase anchored to the ER membrane.22 Suzuki et al23 showed that lipidated apoB100 could be translocated from the ER lumen to the surface of LDs. This observation suggested that LDs could act as a site of proteasomal degradation for lipidated apoB100 molecules.

Our previous work has shown that HepG2 cells, normally defective in apoB100 lipidation and VLDL assembly, can synthesize and secrete fully sized VLDL particles after MEK–ERK (mitogen-activated protein kinase kinase–extracellular signal-regulated kinase) inhibition by U0126 (a highly selective inhibitor of both MEK1 and MEK2).24 The MEK/ERK pathway involves cross talk with ERAD, and its inhibition in gastric cancer cells can block ER stress–mediated upregulation of GRP7825,26 and also specifically reduces apoB100 ubiquitination in HepG2 cells when inhibited by U0126.18 In the present study, we investigated the role of AUP1 in the regulation of apoB100 stability, LD mobilization, and VLDL particle assembly and secretion in HepG2 cells under basal (U0124, an inactive analog of U0126) and MEK–ERK–inhibited (U0126) conditions. Our data suggest that AUP1 plays a key role in intracellular apoB100 biogenesis, degradation, and lipidation. Furthermore, it may act as a critical regulator of hepatic VLDL assembly before secretion.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

Knockdown of AUP1 in HepG2 Cells Markedly Enhanced the Secretion of VLDL Particles

To examine the effects of AUP1 deficiency on apoB100 synthesis and lipoprotein secretion, metabolic labeling and fractionation of lipoproteins in ancient ubiquitous protein 1 siRNA ([short/small interfering RNA] AUP1siRNA)–transfected HepG2 cells was performed under various conditions. AUP1 was knocked down using siRNA, leading to a 70% reduction in mRNA (Figure 1A) and a 92% decline in protein expression (Figure 1B). This caused a major shift in the apoB100-containing lipoprotein profile, leading to a marked increase in secretion of large VLDL-sized lipoproteins. Under basal conditions (U0124 treated), knockdown of AUP1 significantly increased the secretion of VLDL1- and VLDL2-sized apoB100-containing lipoprotein particles from HepG2 cells by 5-fold and 2-fold, respectively, and also significantly increased larger sized intermediate-density lipoprotein (IDL) apoB100-containing particles by 2-fold. However, it did not change the size of apoE (apolipoprotein E)–containing control lipoprotein particles, although it changed amount of the apoE in some IDL, LDL, and high-density lipoprotein (HDL) fractions (Figure 1C). A similar effect on VLDL size was observed after MEK–ERK inhibition using U0126, as previously reported.24 Knockdown of AUP1 significantly increased VLDL1- and VLDL2-sized apoB100-containing lipoprotein particles by 3.5-fold and 2-fold, respectively, with minimal effect on the density profile of apoE-containing particles (Figure 2A). The data from Figure 1C and Figure 2A also show that knockdown of AUP1 markedly increased the ratio of apoB in fractions 1 to 3 compared with fractions 4 to 8 by 2.7-fold (basal condition, U0124) and 3.2-fold (under MEK–ERK inhibition with U0126 treatment), with no significant effect on apoE. Interestingly, when the above experiments were performed side by side, the effects of MEK–ERK inhibition and AUP1 knockdown on VLDL secretion were found to be additive (Figure 2B). HepG2 cells transfected with AUP1siRNA and treated with U0126 exhibited the highest level of VLDL secretion (Figure 2B). However, there was no change on the size of apoE-containing particles (Figure 2B). Additionally, we observed that AUP1 was abundantly expressed in HepG2 cells, and U0126 treatment caused a small but significant decrease by 12% in AUP1 mRNA level (Figure I in the online-only Data Supplement). These data suggest the involvement of 2 distinct mechanisms underlying the effects of AUP1 knockdown and MEK–ERK inhibition but does not rule out potential interactions between these pathways.

Figure 1.
Figure 1.

Knockdown of AUP1 (ancient ubiquitous protein 1) in HepG2 cells markedly enhances secretion of very-low–density lipoprotein (VLDL)–sized apoB100 (apolipoprotein B-100)–containing lipoprotein particles. A, AUP1 mRNA expression level from HepG2 cells transfected with negative control siRNA ([short/small interfering RNA] NCsiRNA) or ancient ubiquitous protein 1 siRNA (AUP1siRNA). B, AUP1 expression level from HepG2 cells transfected with NCsiRNA or AUP1siRNA. C, HepG2 cells were transfected with NCsiRNA or AUP1siRNA, treated with U0124 and oleic acid (OA), and pulsed with [35S]-Met/Cys. The media was collected, and apoB100-containing lipoprotein profile was determined by NaBr gradient ultracentrifugation (apoE [apolipoprotein E]–containing lipoprotein profile as a control). Results are expressed as mean±SD. Analysis by 2-tailed unpaired Student t test and 2-way ANOVA with Bonferroni post hoc test; one sample represents one well of a 6-well plate for (A) and (B), but one sample represents a pool of all wells from a 6-well plate for (C); n=3; **P<0.01; ***P<0.001.

Figure 2.
Figure 2.

Knockdown of AUP1 (ancient ubiquitous protein 1) in HepG2 cells markedly enhances secretion of very-low-density lipoprotein (VLDL)–sized apoB100 (apolipoprotein B-100)–containing lipoprotein particles after MEK–ERK (mitogen-activated protein kinase kinase–extracellular signal-regulated kinase) inhibition. A, HepG2 cells were transfected with negative control siRNA ([short/small interfering RNA] NCsiRNA) or ancient ubiquitous protein 1 siRNA (AUP1siRNA), treated with U0126 and oleic acid (OA), and pulsed with [35S]-Met/Cys. The media was collected and apoB100-containing lipoprotein profile was determined by NaBr gradient ultracentrifugation (apoE [apolipoprotein E]–containing lipoprotein profile as a control). B, The pooled lipoprotein profiles of HepG2 (data from Figure 1C and Figure 2A). Results are expressed as mean±SD. Analysis by 2-way ANOVA with Bonferroni post hoc test; one sample represents a pool of all wells from one 6-well plate; n=3, *P<0.05, **P<0.01, ***P<0.001.

Knockdown of AUP1 Significantly Increased the Average Size of LDs and Protein Level of Cell Death–Inducing DFFA (DNA Fragmentation Factor Alpha Subunit)-Like Effector B in HepG2 Cells

To investigate whether LDs contributed to the effect of AUP1 knockdown, we used BODIPY 493/503 to visualize total LDs in HepG2 cells (Figure 3A). The fluorescence imaging showed that knockdown of AUP1 significantly increased the average size of LDs under both basal (U0124 treated; by 89%) and MEK–ERK–inhibited (U0126 treated; by 76%) conditions (Figure 3B). Additionally, treatment with U0126 alone also led to increases in the average size of LDs in AUP1siRNA-transfected HepG2 cells by 40%, and a nonsignificant similar trend was observed in the control (negative control siRNA [NCsiRNA] transfected) cells by 49% (Figure 3B). A nonsignificant trend toward a higher number of LDs in cells after either AUP1 knockdown or U0126 treatment was also observed (data not shown).

Figure 3.
Figure 3.

Knockdown of AUP1 (ancient ubiquitous protein 1) significantly increased the average size of lipid droplets (LDs) and protein level of CIDEB (cell death–inducing DFFA [DNA fragmentation factor alpha subunit]-like effector B). A, The representative confocal images showed total LDs and nuclei in negative control siRNA ([short/small interfering RNA] NCsiRNA)– or ancient ubiquitous protein 1 siRNA (AUP1siRNA)–transfected HepG2 cells (U0124 or U0126, and oleic acid [OA] treated). Bar=22 μm. B, The average size of LDs. C, HepG2 cells were transfected with negative control siRNA (NCsiRNA) or AUP1siRNA, treated with U0124 or U0126, and OA. Cell lysates were projected to Western blots. D, The analyzed data of Western blots. Results are expressed as mean±SD. Analysis by 1-way ANOVA with Bonferroni multiple comparison test; (A) and (B): n=6; (C) and (D): n=3, *P<0.05, **P<0.01, ***P<0.001.

To further investigate changes in LD formation, we used BODIPY 558/568 C12 to image newly formed LDs in HepG2 cells under the conditions described above (Figure IIA in the online-only Data Supplement). Treatment with both oleic acid and BODIPY 558/568 C12 led to rapid label incorporation into intracellular LDs under basal conditions. AUP1 knockdown significantly increased the average size of newly formed LDs by 36% after U0124 treatment, but only a small, nonsignificant increase was observed under U0126-treated (MEK–ERK–inhibited) conditions (Figure IIB in the online-only Data Supplement). Similarly, treatment with U0126 significantly increased the average size of LDs in control (NCsiRNA transfected) HepG2 cells by 34%, but only a small nonsignificant similar trend was observed after U0126 treatment of AUP1siRNA-transfected HepG2 cells (Figure IIB in the online-only Data Supplement). Finally, there was a trend toward a higher number of newly formed LDs in cells after either AUP1 knockdown or U0126 treatment, but these changes did not reach statistical significance (data not shown).

We further assessed changes in LD-associated proteins by Western blot (Figure 3C; Figure IIIA in the online-only Data Supplement). Knockdown of AUP1 in HepG2 cells significantly increased CIDEB protein (cell death–inducing DFFA-like effector B) level in the absence of U0126, and a similar trend was found in the presence of U0126 (Figure 3D). Treatment of U0126 showed the same trend, especially in NCsiRNA-transfected cells (Figure 3D). Furthermore, U0126 treatment led to a small nonsignificant increase in TIP47 protein level; however, AUP1 knockdown had no effect in modulating the expression of TIP47 (Figure 3D).

Knockdown of AUP1 Significantly Increased Metabolically Labeled Triglyceride in Cells and in Secreted VLDL-Sized Lipoprotein Particles

Knockdown of AUP1 significantly increased the average size of LDs in HepG2 cells, suggesting enhanced lipidation and increased triglyceride content of VLDL particles. To examine triglyceride mobilization, HepG2 cells were used for metabolically labeling triglyceride using [3H]-glycerol. The radioactive triglyceride levels were measured in cellular and secreted lipoprotein fractions. Under U0124 and lipid-rich conditions, knockdown of AUP1 significantly increased radioactive triglycerides in cells (by 9%) and in secreted VLDL-sized lipoprotein particles (by 59%). This did not affect the triglyceride content of small, HDL-sized particles (Figure 4A). Similarly, when the MEK–ERK pathway was inhibited by U0126 under lipid-rich conditions, knockdown of AUP1 also significantly increased radioactive triglyceride levels in VLDL-sized lipoproteins (by 2-fold) but had no effect on HDL-sized particles (Figure 4B). Again, MEK–ERK inhibition by U0126 seemed to augment the stimulatory effects of AUP1 knockdown on VLDL triglyceride (Figure 4B).

Figure 4.
Figure 4.

Knockdown of AUP1 (ancient ubiquitous protein 1) significantly increased metabolically labeled triglyceride (TG) content in very-low–density lipoprotein (VLDL)–sized lipoprotein particles with or without MEK–ERK (mitogen-activated protein kinase kinase–extracellular signal-regulated kinase) inhibition. HepG2 cells were transfected with NCsiRNA or AUP1siRNA, treated with U0124 (A) or U0126 (B), and oleic acid (OA), followed by a incubation with [3H]-glycerol. Media was subjected to lipoprotein particle fractionation. VLDL-sized fractions and high-density lipoprotein (HDL)–sized fractions were pooled separately. Lipids were extracted from each pool and separated by thin layer chromatography. C, HepG2 cells were transfected with NCsiRNA or AUP1siRNA, treated with U0124 or U0126, and OA. mRNA levels of some lipogenic genes were measured by real-time polymerase chain reaction. Results are expressed as mean±SD. Analysis by 2-tailed unpaired Student t test for (A) and (B), analysis by 1-way ANOVA with Bonferroni multiple comparison test for (C); one sample represents a pool of all wells from a 6-well plate for (A) and (B), but one sample represents one well of a 6-well plate for (C); n=3, *P<0.05, **P<0.01, ***P<0.001.

We further performed real-time quantitative polymerase chain reaction to investigate whether knockdown of AUP1 affects mRNA levels of apoB, MTP (microsomal triglyceride transfer protein), and lipogenic genes in HepG2 cells (Figure 4C). U0126 treatment alone was sufficient to decrease apoB and SREBP1c mRNA levels and increase MTP and ACC (acetyl-CoA carboxylase alpha) mRNA levels (Figure 4C). mRNA levels of LXR-α (liver X receptor alpha), FAS (fatty acid synthase), and SCD1 (stearoyl-CoA desaturase) were significantly increased by AUP1 knockdown only under U0126-treated conditions and increased by U0126 treatment only in AUP1siRNA-transfected cells. Additionally, AUP1 knockdown decreased SREBP1c mRNA only in the absence of U0126. We also found that U0126 significantly decreased LPCAT1 at protein level (Figure IIIB in the online-only Data Supplement). Overall, de novo lipogenesis may be increased by AUP1 knockdown only in the presence of U0126.

To investigate whether knockdown of AUP1 affects fatty acid uptake, we used alkyne-oleate to trace fatty acid metabolism (Figure IVA in the online-only Data Supplement). AUP1 knockdown or MEK–ERK inhibition showed a trend of increased fatty acid uptake mostly because of an increasing trend in labeled PE (Figure IVB and IVC in the online-only Data Supplement).

We next assessed fatty acid oxidation in HepG2 cells by measuring respiration. It is well known that palmitate stimulates fatty acid oxidation, and etomoxir (CPT1a inhibitor) decreases fatty acid oxidation. Our data consistently confirmed this and also showed that knockdown of AUP1 significantly increased the maximum respiration of HepG2 in the presence of palmitate under basal condition (U0124) but did not affect the basal respiration (Figure VA and VB in the online-only Data Supplement). Because of interference of U0126 with the assay, oxygen consumption rate could not be determined under these conditions.

Furthermore, fatty acid oxidation was assessed by real-time quantitative polymerase chain reaction of key factors involved in fatty acid oxidation. CPT1a mRNA was significantly increased after AUP1 knockdown only in the presence of U0126 and increased with U0126 treatment only in AUPsiRNA-transfected cells (Figure VC in the online-only Data Supplement). U0126 also increased peroxisome proliferator–activated receptor-α mRNA and decreased AMPK (5′ adenosine monophosphate-activated protein kinase) mRNA, but AUP1 knockdown had no significant effect on expression of peroxisome proliferator–activated receptor-α and only had a nonsignificant decreasing effect on AMPK mRNA (Figure VC in the online-only Data Supplement).

Evidence for Direct AUP1–apoB Interaction

Because AUP1 has been implicated in quality control of misfolded proteins, we investigated whether AUP1 directly interacts with apoB. We first performed confocal imaging of AUP1 and apoB in HepG2 cells under different conditions. As shown in Figure 5A and Supplemental Figure VI in the online-only Data Supplement, AUP1 was found to colocalize with apoB, and this colocalization seemed to occur partly on the surface of LDs. AUP1–apoB colocalization seemed to be enhanced after treatment with MG132, an inhibitor of proteasomal degradation, likely because of stabilization of the apoB pool available to interact with AUP1 (Figure 5A; Figure VII in the online-only Data Supplement). We then repeated this imaging experiment in both control (NCsiRNA transfected) and AUP1siRNA-transfected HepG2 cells after oleic acid and MG132 treatment. NCsiRNA-transfected cells showed strong AUP1–apoB colocalization, similar to that seen in nontransfected cells, but this colocalization was lost with AUP1siRNA transfection (Figure 5A; Figure VIII in the online-only Data Supplement). This further confirms AUP1 colocalization with apoB100 in HepG2 cells.

Figure 5.
Figure 5.

Evidence for direct interaction of AUP1 (ancient ubiquitous protein 1) with apoB (apolipoprotein B) in HepG2 cells. A, The confocal images showed AUP1, apoB, and nuclei in nontransfected or siRNA (short/small interfering RNA)-transfected HepG2 cells treated with dimethyl sulfoxide (DMSO) or MG132, in the presence or absence of oleic acid (OA). n=3 with representative images shown. Bar=16 μm. B, The representative images from in situ proximity ligation assay showed AUP1–apoB interaction (red fluorescent signals) and nuclei in negative control siRNA (NCsiRNA)– or ancient ubiquitous protein 1 siRNA (AUP1siRNA)–transfected HepG2 cells treated with DMSO or MG132 under lipid-rich condition. n=3 with representative images shown. Bar=25 μm. C, HepG2 cells were treated with DMSO or MG132, followed by cross-linking with dithiobis succinimidyl propionate (DSP). Cell lysates were subjected to immunoprecipitation and immunoblotting. n=3 with representative images shown.

To further confirm the AUP1–apoB interactions, an in situ proximity ligation assay (Duolink) was performed in HepG2 cells (Figure 5B; Figure IX in the online-only Data Supplement), which confirmed a direct interaction, as visualized by the red spots. When cells were treated with MG132, there was a marked increase in the interaction, similar to the observations made with the confocal studies described above.

Finally, direct cross-linking and coimmunoprecipitation experiments in the presence of MG132 (Figure 5C; Figure XA and XB in the online-only Data Supplement) showed coimmunoprecipitation of apoB100 and AUP1. Immunoprecipitated apoB also contained p97, and the apoB100–p97 interactions were enhanced with MG132 treatment, as expected.22 These interactions were specific to apoB100 as neither AUP1 nor p97 could be detected in samples immunoprecipitated with transferrin (Figure 5C), apoA I (Figure XA in the online-only Data Supplement), apoE (Figure XB in the online-only Data Supplement), or normal goat serum.

Effect of AUP1 Knockdown on apoB100 Ubiquitination and Intracellular Stability

We further measured ubiquitination level of apoB100 in AUP1siRNA-transfected HepG2 cells (and NCsiRNA-transfected cells as negative control) in the presence and absence of MG132 (Figure XIA and XIB in the online-only Data Supplement). Knockdown of AUP1 showed a nonsignificant trend toward a lower ubiquitination level of apoB100 in the presence or absence of MG132 (Figure XIC in the online-only Data Supplement). As expected, MG132 treatment led to higher ubiqutination level of apoB100 in either control or AUP1siRNA-transfected HepG2 cells, but these changes were not statistically significant (Figure XIC in the online-only Data Supplement). Concomitantly, apoB100 mass was significantly increased by 71% in AUP1siRNA-transfected HepG2 cells under MG132 treatment (Figure XID in the online-only Data Supplement). Knockdown of AUP1 seemed to cause a slight but nonsignificant increase in apoB100 mass, suggesting protection from proteasomal degradation (Figure XID in the online-only Data Supplement).

To investigate whether AUP1 knockdown affects apoB100 stability, pulse-chase experiments were performed using HepG2 cells transfected with AUP1siRNA (or NCsiRNA as negative control) and treated with oleic acid under basal condition (U0124). The counts of [35S] recovered from apoB100 bands showed that AUP1 knockdown significantly increased cellular apoB100 at 0, 10, and 30 minutes and further significantly increased secretion of apoB100 at 60 minutes and 120 minutes (Figure 6A and 6B). Total apoB100 levels were significantly increased at 0, 10, and 30 minutes by AUP1 knockdown (Figure 6B). However, when we set cellular [35S] counts at end of the pulse period (0 minutes chase) to 100%, we found that cellular, medium, and total apoB100 profiles were identical between control NCsiRNA-transfected cells and AUP1siRNA-transfected cells (Figure XIIA in the online-only Data Supplement). Taken together, these data seem to indicate that AUP1 knockdown may stabilize apoB100 cotranslationally (as there was a marked increase in labeled apoB recovered at the end of pulse), but there was no change in post-translational stability of apoB. AUP1 knockdown did not show any significant effects on the stability of the control protein albumin (Figure 6A and 6C; Figure XIIB in the online-only Data Supplement).

Figure 6.
Figure 6.

Effect of AUP1 (ancient ubiquitous protein 1) knockdown on intracellular stability of apoB100 (apolipoprotein B-100). A, HepG2 cells were transfected with negative control siRNA ([short/small interfering RNA] NCsiRNA) or ancient ubiquitous protein 1 siRNA (AUP1siRNA) and treated with U0124 and oleic acid (OA). Cells were pulsed with [35S]-Met/Cys and chased at 0, 10, 30, 60, and 120 min. For each time point, the media and cell lysate were collected for immunoprecipitation of apoB and albumin. B, Counts of [35S] recovered from apoB100 and albumin bands. Results are expressed as mean±SD. Analysis by 2-way ANOVA with Bonferroni post hoc test; n=3, *P<0.05, ***P<0.001.

Discussion

The assembly and secretion of VLDL particles is a complex process that occurs uniquely in hepatocytes and is critically dependent on the availability of freshly translated apoB100 protein and a sufficient supply of core lipids for lipoprotein formation.9 Defective apoB100 synthesis or an inadequate lipid supply can curtail VLDL production and lead to rapid apoB100 degradation. Identifying cellular factors that determine the rate of VLDL secretion are physiologically and clinically important because this process is critical to lipid homeostasis in the liver and is a key determinant of the circulating levels of atherogenic lipids and lipoproteins.27 Evidence presented here supports a potentially important role for the LD-associated protein AUP1 in apoB regulation. In recent years, AUP1 has been implicated in quality control of misfolded proteins in the ER and in LD particle clustering.12 Our data suggest that it may also play a decisive role in determining whether apoB100 is degraded or lipidated for VLDL assembly and secretion.

The link between AUP1 function and VLDL assembly may arise from the close association of AUP1 with LDs and its involvement in LD biogenesis. Using BODIPY 493/503, we found that the average size of LDs was noticeably increased after AUP1 knockdown in HepG2 cells. Staining of newly formed LDs using BODIPY 558/568 C12 for 4 hours further supported the link between AUP1 expression and LD size. In contrast to our observations presented here, an earlier study12 found that AUP1 knockdown in A431 cells caused LD declustering and its overexpression in COS7 cells led to LD clustering. These contrasting observations may be because of differences in lipid metabolism between the cell lines and the unique role of hepatocytes in packaging and secreting VLDL particles, a function absent in COS7 and A431 cell lines. Importantly, LD-associated AUP1 seemed to interact directly with apoB100 based on both the confocal microscopy and proximity ligation assay results. Under lipid-rich conditions, and after treatment with the proteasome inhibitor MG132, AUP1 and apoB100 were found to abundantly colocalize on the surface of LDs. Previous studies have reported the association of AUP1,4 and to a lesser extent apoB, with the surface of LDs,28 and our data suggest that AUP1 and apoB can directly interact on the LD surface. Additionally, an alternate experimental approach, classical coimmunoprecipitation experiment, also supported a direct intracellular interaction between AUP1 and apoB100. This interaction was more evident in the presence of a proteasome inhibitor, indicating that that the apoB100 pool associated with AUP1 is likely destined for rapid proteasomal degradation. A single N-terminal domain of AUP1 is important for its insertion into the ER membrane in a monotopic hairpin fashion and further allows for LD localization.13 Thus, it is possible that some LDs are extrusions of the ER and that AUP1 may simultaneously interact with and link the ER and adjoining LDs.

The observation that knockdown of AUP1 leads to a marked increase in hepatic assembly and secretion of large triglyceride-rich VLDL particles is particularly intriguing. This suggests that removal of AUP1 function effectively corrects the well-known defect of VLDL production in HepG2 cells. In HepG2 cells under basal conditions, the majority of apoB is secreted in particles of low-density lipoprotein density.29,30 Although the addition of exogenous fatty acid enhances apoB secretion from HepG2 cells, most of the secreted particles remain in the low-density lipoprotein density range.29,30 The defect in apoB-VLDL secretion has been attributed to inefficient mobilization of cytosolic triglyceride storage pools,30,31 and inefficient transfer of triglyceride to the secretion-coupled microsomal pool is believed to be critical for VLDL formation.31 Our data presented here suggest that AUP1 function may be a key determinant of the lipid pool accessible to apoB to form secretion-competent VLDL particles. Interaction of AUP1 with apoB on the surface of LDs may hamper apoB lipidation and VLDL assembly. In support of this hypothesis, knockdown of AUP1 led to a correction in lipid mobilization in HepG2 cells and a marked increase in formation of fully-lipidated triglyceride-rich VLDL particles.

This novel observation for AUP1 is reminiscent of the effects seen on inhibition of the ERK-signaling pathway in HepG2 cells.24 We have previously reported that the defective VLDL assembly in HepG2 cells may be linked to hyperactivity of the MEK–ERK pathway. Inhibition of the overactive ras–MEK–ERK pathway in HepG2 cells corrected the defect in VLDL assembly, leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK–ERK cascade in VLDL assembly in the HepG2 model.24 Studies by Fisher et al18 have further extended these findings, showing that the inhibition of the MEK–ERK pathway and stimulation of VLDL secretion may be linked to reduced ubiquitination of apoB100. In the present study, we found that the stimulatory effects of AUP1 knockdown and MEK–ERK inhibition on VLDL secretion, although additive, are likely independent. However, we observed a small decrease in AUP1 mRNA on MEK–ERK inhibition by U0126, implying that modulation of AUP1 expression may account for part of the effects of MEK–ERK inhibition on VLDL production. Interestingly, MEK–ERK inhibition by U0126 seemed to augment the effect of AUP1 knockdown on VLDL assembly and triglyceride content. Given the potential involvement of AUP1 in cellular signaling pathways,32,33 we postulated that AUP1 knockdown may alter expression levels of key protein factors involved in hepatic de novo lipogenesis and LD lipolysis. mRNA analysis of factors involved in de novo lipogenesis and fatty acid oxidation supported this finding. In addition, Alkyne-oleate tracing experiments showed a trend toward increasing alkyne-oleate uptake after AUP1 knockdown, which may provide more lipid substrate for lipogenesis and VLDL assembly. This is consistent with the results of AUP1 knockdown increasing the average size of newly formed LDs and also increasing the maximum respiration of HepG2 under basal condition in the presence of palmitate.

Recent proteomic studies suggest that LD-associated proteins have extensive functional properties in the areas of cellular signaling, protein degradation, and membrane trafficking.34 Knockout of ADRP (adipose differentiation–related protein), a LD-associated protein, in Lep (ob/ob) mice was shown to increase VLDL secretion and decrease hepatic triglyceride. ADRP knockout also improved glucose tolerance and insulin sensitivity in liver and muscle, whereas no change in lipogenic gene expression was observed.35 Furthermore, Bell et al36 reported that double knockdown of TIP47 and ADRP increased LD size, decreased LD number, and induced insulin resistance in AML12 mouse liver cells. Interestingly, the absence of ADRP has been shown to protect mice from hepatic ER stress.37 Another LD-associated protein, CIDEB, localized to both the ER and LDs, has 2 domains that bind apoB and localize to LDs, respectively. These 2 domains are both required for CIDEB to promote triglyceride-enriched VLDL assembly and secretion.38 Our data showed that AUP1 knockdown increased CIDEB in HepG2 cells and also increased the size and possibly the number of LDs. These results are consistent with the previous report that CIDEB increases LD size by facilitating LD clustering and fusion.39 Therefore, additional lipids may be provided for VLDL assembly when AUP1 is knocked down in HepG2 cells. These findings suggest a close connection between ER and cytoplasmic LDs and that many LD-associated proteins including CIDEB, ADRP, and AUP1 may play critical roles in lipid mobilization and hepatic VLDL assembly and secretion.

Interestingly, knockdown of AUP1 showed a trend toward increased protection of apoB100 from degradation even when the proteasome pathway was inhibited using MG132. This might be because of the involvement of post–ER-degradative pathways such as autophagy. Our previous work showed autophagy involved in ER stress–induced degradation of misfolded apoB.40 Because autophagy can also use ubiquitin tags for substrate protein recognition and degradation, this pathway might become more active in HepG2 cells when the proteasome pathway is inhibited using MG132, although proteasome degradation is the primary pathway in HepG2 cells under basal condition.

Taken together, the experimental findings reported here implicate AUP1 in apoB100 quality control and in determining the rate at which apoB100 is lipidated to enable VLDL particle assembly and secretion (Figure XIII in the online-only Data Supplement). AUP1 seems to directly interact with apoB100 on the surface of LDs, negatively controlling both apoB100 protein and triglyceride availability. Our data also showed that AUP1 can modulate the size of LDs as AUP1 knockdown significantly enhanced LD size. It would be interesting to determine whether modulation of AUP1 can regulate hepatic insulin sensitivity via alterations in hepatic lipid homeostasis. These findings may have clinical implications, as alterations in the size of LDs and dysregulation of VLDL secretion have been reported in metabolic disorders such as hepatic steatosis and insulin resistance. Pharmacological manipulation of AUP1 might be an attractive approach to controlling hepatic lipid metabolism and ameliorating diabetic dyslipidemia.

Sources of Funding

This work was supported in part by an operating grant from the Heart and Stroke Foundation of Ontario to K. Adeli and The Canadian Institutes of Health Research (CIHR) grant no MOP 123279 to Z. Yao. M. Zamani was supported by the CIHR Training Program in Protein Folding and a Mitacs accelerate scholarship. J. Taher is supported by an NSERC (Natural Sciences and Engineering Research Council of Canada) PGS (Postgraduate Scholarships)-doctoral scholarship.

Disclosures

None.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.117.309000/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    apoB100
    apolipoprotein B-100
    apoE
    apolipoprotein E
    AUP1
    ancient ubiquitous protein 1
    AUP1siRNA
    ancient ubiquitous protein 1 siRNA
    CIDEB
    cell death–inducing DFFA (DNA fragmentation factor alpha subunit)-like effector B
    ER
    endoplasmic reticulum
    ERAD
    endoplasmic reticulum–associated degradation
    LD
    lipid droplet
    NCsiRNA
    negative control siRNA
    VLDL
    very-low–density lipoprotein

  • Received June 13, 2016.
  • Accepted January 23, 2017.

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Highlights

  • AUP1 (ancient ubiquitous protein 1) is an endoplasmic reticulum–associated protein that also localizes to the surface of lipid droplets, with dual role in protein quality control and lipid droplet regulation.

  • siRNA (short/small interfering RNA) knockdown of AUP1 significantly increased secretion of very-low–density lipoprotein–sized apoB100 (apolipoprotein B-100)–containing particles from HepG2 cells, correcting a key metabolic defect in these cells that normally do not secrete much very-low–density lipoprotein.

  • AUP1 was found to directly interact with apoB100. Knockdown of AUP1 reduced apoB100 ubiquitination, decreased intracellular degradation of newly synthesized apoB100, and enhanced extracellular apoB100 secretion.

  • Our experimental findings implicate AUP1 in apoB100 quality control and in determining the rate at which apoB100 is lipidated to enable very-low-density lipoprotein particle assembly and secretion.

  • AUP1 plays critical roles in intracellular biogenesis of lipid droplet mobilization, apoB stability, and very-low–density lipoprotein assembly and secretion.

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  • AUP1 (Ancient Ubiquitous Protein 1) Is a Key Determinant of Hepatic Very-Low–Density Lipoprotein Assembly and SecretionHighlights
    Jing Zhang, Mostafa Zamani, Christoph Thiele, Jennifer Taher, Mohsen Amir Alipour, Zemin Yao and Khosrow Adeli
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2017;37:633-642, originally published February 9, 2017
    https://doi.org/10.1161/ATVBAHA.117.309000
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