α-Tocopherol reduces VLDL secretion through modulation of intracellular ER-to-Golgi transport of VLDL.

Avoiding hepatic steatosis is crucial for preventing liver dysfunction, and one mechanism by which this is accomplished is through synchronization of the rate of very low density lipoprotein (VLDL) synthesis with its secretion. Endoplasmic reticulum (ER)-to-Golgi transport of nascent VLDL is the rate-limiting step in its secretion and is mediated by the VLDL transport vesicle (VTV). Recent in vivo studies have indicated that α-tocopherol (α-T) supplementation can reverse steatosis in nonalcoholic fatty liver disease, but its effects on hepatic lipoprotein metabolism are poorly understood. Here, we investigated the impact of α-T on hepatic VLDL synthesis, secretion, and intracellular ER-to-Golgi VLDL trafficking using an in vitro model. Pulse-chase assays using [3H]-oleic acid and 100 µmol/L α-T demonstrated a disruption of early VLDL synthesis, resulting in enhanced apolipoprotein B-100 expression, decreased expression in markers for VTV budding, ER-to-Golgi VLDL transport, and reduced VLDL secretion. Additionally, an in vitro VTV budding assay indicated a significant decrease in VTV production and VTV-Golgi fusion. Confocal imaging of lipid droplet (LD) localization revealed a decrease in overall LD retention, diminished presence of ER-associated LDs, and an increase in Golgi-level LD retention. We conclude that α-T disrupts ER-to-Golgi VLDL transport by modulating the expression of specific proteins and thus reduces VLDL secretion.

Reticulon 3 regulates very low density lipoprotein secretion by controlling very low density lipoprotein transport vesicle biogenesis.

Secretion of very low density lipoprotein (VLDL) by the liver is an important physiological process; however, the rate of VLDL secretion is determined by its transport from the endoplasmic reticulum (ER) to the Golgi. This transport event is facilitated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). We have reported earlier a detailed VTV proteome, which revealed that reticulon 3 (RTN3) is uniquely present in the VTV. Our immunoblotting and electron microscopic data demonstrate that RTN3 is enriched in the VTV; however, other ER-derived vesicles do not contain RTN3. Co-immunoprecipitation data coupled with confocal microscopic analyses strongly suggest that RTN3 interacts with VLDL core protein, apoB100, at the ER level. Our data show that either blocking of RTN3 using specific antibodies or RTN3 knockdown resulted in significant reduction in VTV biogenesis from hepatic ER membranes. Additionally, VLDL secretion from hepatocytes was significantly decreased when RTN3 was silenced by RTN3 siRNA. We conclude that RTN3 regulates VLDL secretion by controlling VTV-mediated ER-to-Golgi transport of nascent VLDL.

Cathepsin B regulates hepatic lipid metabolism by cleaving liver fatty acid-binding protein.

Synthesis and secretion of hepatic triglycerides (TAG) associated with very-low-density lipoprotein (VLDL) play a major role in maintaining overall lipid homeostasis. This study aims to identify factors affecting synthesis and secretion of VLDL-TAG using the growth hormone-deficient Ames dwarf mouse model, which has reduced serum TAG. Proteomic analysis coupled with a bioinformatics-driven approach revealed that these mice express greater amounts of hepatic cathepsin B and lower amounts of liver fatty acid-binding protein (LFABP) than their wildtype littermates. siRNA-mediated knockdown of cathepsin B in McA-RH7777 cells resulted in a 39% increase in [3H]TAG associated with VLDL secretion. Cathepsin B knockdown was accompanied by a 74% increase in cellular LFABP protein levels, but only when cells were exposed to 0.4 mm oleic acid (OA) complexed to BSA. The cathepsin B knockdown and 24-h treatment with OA resulted in increased CD36 expression alone and additively. Co-localization of LFABP and cathepsin B was observed in a distinct Golgi apparatus-like pattern, which required a 1-h OA treatment. Moreover, we observed co-localization of LFABP and apoB, independent of the OA treatment. Overexpression of cathepsin B resulted in decreased OA uptake and VLDL secretion. Co-expression of cathepsin B and cathepsin B-resistant mutant LFABP in McA-RH7777 cells resulted in an increased TAG secretion as compared with cells co-expressing cathepsin B and wildtype LFABP. Together, these data indicate that cathepsin B regulates VLDL secretion and free fatty acid uptake via cleavage of LFABP, which occurs in response to oleic acid exposure.

Silencing of Small Valosin-containing Protein-interacting Protein (SVIP) Reduces Very Low Density Lipoprotein (VLDL) Secretion from Rat Hepatocytes by Disrupting Its Endoplasmic Reticulum (ER)-to-Golgi Trafficking.

The transport of nascent very low density lipoprotein (VLDL) particles from the endoplasmic reticulum (ER) to the Golgi determines their secretion by the liver and is mediated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Our previous studies have shown that the formation of ER-derived VTV requires proteins in addition to coat complex II proteins. The VTV proteome revealed that a 9-kDa protein, small valosin-containing protein-interacting protein (SVIP), is uniquely present in these specialized vesicles. Our biochemical and morphological data indicate that the VTV contains SVIP. Using confocal microscopy and co-immunoprecipitation assays, we show that SVIP co-localizes with apolipoprotein B-100 (apoB100) and specifically interacts with VLDL apoB100 and coat complex II proteins. Treatment of ER membranes with myristic acid in the presence of cytosol increases SVIP recruitment to the ER in a concentration-dependent manner. Furthermore, we show that myristic acid treatment of hepatocytes increases both VTV budding and VLDL secretion. To determine the role of SVIP in VTV formation, we either blocked the SVIP protein using specific antibodies or silenced SVIP by siRNA in hepatocytes. Our results show that both blocking and silencing of SVIP lead to significant reduction in VTV formation. Additionally, we show that silencing of SVIP reduces VLDL secretion, suggesting a physiological role of SVIP in intracellular VLDL trafficking and secretion. We conclude that SVIP acts as a novel regulator of VTV formation by interacting with its cargo and coat proteins and has significant implications in VLDL secretion by hepatocytes.

Mature VLDL triggers the biogenesis of a distinct vesicle from the trans-Golgi network for its export to the plasma membrane.

Post-Golgi trafficking of mature VLDL (very-low-density lipoprotein) is crucial in maintaining normal TAG (triacylglycerol) homoeostasis of hepatocytes; however, the mechanism that regulates the exit of mature VLDL from the TGN (trans-Golgi network) is not known. We developed an in vitro TGN-budding assay that allowed us to examine the formation of secretory vesicles from the TGN in primary rat hepatocytes. We isolated TAG-rich PG-VTVs (post-TGN VLDL transport vesicles) using a continuous sucrose density gradient. PG-VTVs were distributed in low-density fractions, whereas protein transport vesicles were present in relatively higher-density fractions of the same sucrose gradient. EM revealed large intact PG-VTVs ranging 300-350 nm in size. The biogenesis of PG-VTVs from the TGN required cytosol, ATP, GTP hydrolysis and incubation at 37°C. PG-VTVs concentrated the VLDL proteins: apolipoproteins apoB100, apoAIV, apoAI and apoE, but did not contain either albumin or transferrin. Proteinase K treatment did not degrade VLDL core proteins, suggesting that PG-VTVs were sealed. PG-VTVs were able to fuse with and deliver VLDL to the PM (plasma membrane) in a vectorial manner. We conclude that we have identified a new TGN-derived vesicle, the PG-VTV, which specifically transports mature VLDL from the TGN to the PM.

CideB protein is required for the biogenesis of very low density lipoprotein (VLDL) transport vesicle.

Nascent very low density lipoprotein (VLDL) exits the endoplasmic reticulum (ER) in a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Similar to protein transport vesicles (PTVs), VTVs require coat complex II (COPII) proteins for their biogenesis from the ER membranes. Because the size of the VTV is large, we hypothesized that protein(s) in addition to COPII components might be required for VTV biogenesis. Our proteomic analysis, supported by Western blotting data, shows that a 26-kDa protein, CideB, is present in the VTV but not in other ER-derived vesicles such as PTV and pre-chylomicron transport vesicle. Western blotting and immunoelectron microscopy analyses suggest that CideB is concentrated in the VTV. Our co-immunoprecipitation data revealed that CideB specifically interacts with VLDL structural protein, apolipoprotein B100 (apoB100), but not with albumin, a PTV cargo protein. Confocal microscopic data indicate that CideB co-localizes with apoB100 in the ER. Additionally, CideB interacts with COPII components, Sar1 and Sec24. To investigate the role of CideB in VTV biogenesis, we performed an in vitro ER budding assay. We show that the blocking of CideB inhibits VTV budding, indicating a direct requirement of CideB in VTV formation. To confirm our findings, we knocked down CideB in primary hepatocytes and isolated ER and cytosol to examine whether they support VTV budding. Our data suggest that CideB knockdown significantly reduces VTV biogenesis. These findings suggest that CideB forms an intricate COPII coat and regulates the VTV biogenesis.

Tolfenamic acid suppresses cytochrome P450 2E1 expression in mouse liver.

Non-steroidal anti-inflammatory drugs (NSAIDs) play a significant role in the chemoprevention of cancer. We recently showed the chemopreventive response of a NSAID, 2-[(3-chloro-2-methylphenyl)amino]benzoic acid) known as tolfenamic acid (TA) in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in rats. Pre-clinical studies showed that TA inhibits Specificity protein (Sp) transcription factors and acts as an anti-cancer agent in several cancer models; however the pertinent mechanisms associated with its chemopreventive response in esophageal cancer are not known. Since the bioactivation of carcinogens through cytochrome P450 (CYP) is critical for the induction of cancer, we have studied the effect of TA on critical CYP isozymes in mouse liver samples. Athymic nude mice were treated with vehicle (corn oil) or TA (50 mg kg(-1), 3 times per week) for 4 weeks. Protein extracts (whole cell lysates and microsomal fractions) were prepared from liver tissue and the expression of various CYP isozymes was determined by Western blot analysis. Rat (Sprague-Dawley) livers were harvested and primary hepatocyte cultures were treated with vehicle (DMSO) or TA (50 µM) and cell viability was assessed at 2 and 5 days post-treatment. TA caused remarkable decrease in the expression of CYP2E1 in both liver lysates and sub-cellular fraction, while its response on other tested isozymes was marginal. TA did not affect the body weight of animals (mice) and viability of rat hepatocytes. These results demonstrate that TA modulates the expression of CYP2E1 which is associated with the bioactivation of carcinogens without causing apparent toxicity. These data suggest that TA-induced inhibition of CYP2E1 attenuates the bioactivation of carcinogens potentially leading to the chemoprevention of NMBA-induced esophageal tumorigenesis in rats.

Proteomic analysis of the very low density lipoprotein (VLDL) transport vesicles.

The VLDL transport vesicle (VTV) mediates the transport of nascent VLDL particles from the ER to the Golgi and plays a key role in VLDL-secretion from the liver. The functionality of VTV is controlled by specific proteins; however, full characterization and proteomic profiling of VTV remain to be carried out. Here, we report the first proteomic profile of VTVs. VTVs were purified to their homogeneity and characterized biochemically and morphologically. Thin section transmission electron microscopy suggests that the size of VTV ranges between 100 nm to 120 nm and each vesicle contains only one VLDL particle. Immunoblotting data indicate VTV concentrate apoB100, apoB48 and apoAIV but exclude apoAI. Proteomic analysis based on 2D-gel coupled with MALDI-TOF identified a number of vesicle-related proteins, however, many important VTV proteins could only be identified using LC-MS/MS methodology. Our data strongly indicate that VTVs greatly differ in their proteome with their counterparts of intestinal origin, the PCTVs. For example, VTV contains Sec22b, SVIP, ApoC-I, reticulon 3, cideB, LPCAT3 etc. which are not present in PCTV. The VTV proteome reported here will provide a basic tool to study the mechanisms underlying VLDL biogenesis, maturation, intracellular trafficking and secretion from the liver.

The identification of the SNARE complex required for the fusion of VLDL-transport vesicle with hepatic cis-Golgi.

VLDLs (very-low-density lipoproteins) are synthesized in the liver and play an important role in the pathogenesis of atherosclerosis. Following their biogenesis in hepatic ER (endoplasmic reticulum), nascent VLDLs are exported to the Golgi which is a physiologically regulatable event. We have previously shown that a unique ER-derived vesicle, the VTV (VLDL-transport vesicle), mediates the targeted delivery of VLDL to the Golgi lumen. Because VTVs are different from other ER-derived transport vesicles in their morphology and biochemical composition, we speculated that a distinct set of SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins would form a SNARE complex which would eventually facilitate the docking/fusion of VTVs with Golgi. Our results show that Sec22b is concentrated in VTVs as compared with the ER. Electron microscopic results show that Sec22b co-localizes with p58 and Sar1 on the VTV surface. Pre-treatment of VTV with antibodies against Sec22b inhibited VTV-Golgi fusion, indicating its role as a v-SNARE (vesicle SNARE). To isolate the SNARE complex, we developed an in vitro docking assay in which VTVs were allowed to dock with the Golgi, but fusion was prevented to stabilize the SNARE complex. After the docking reaction, VTV-Golgi complexes were collected, solubilized in 2% Triton X-100 and the SNARE complex was co-immunoprecipitated using anti-Sec22b or GOS28 antibodies. A approximately 110 kDa complex was identified in non-boiled samples that was dissociated upon boiling. The components of the complex were identified as Sec22b, syntaxin 5, rBet1 and GOS28. Antibodies against each SNARE component significantly inhibited VTV-Golgi fusion. We conclude that the SNARE complex required for VTV-Golgi fusion is composed of Sec22b, syntaxin 5, rBet1 and GOS28.