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Inhibitors of hepatic microsomal triacylglycerol hydrolase decrease very low density lipoprotein secretion¹

DEAN GILHAM^2,, SAMUEL HO^*,2, MEHDI RASOULI^*,3, PAUL MARTRES, DENNIS E. VANCE and RICHARD LEHNER^*,,4

Departments of
^* Pediatrics,
Cell Biology,
Biochemistry, CIHR Group on the Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta, Canada T6G 2S2; and
GlaxoSmithKline, 91951 Les Ulis cedex, France

⁴Correspondence: Departments of Pediatrics and Cell Biology, University of Alberta, 328 Heritage Medical Research Centre, Edmonton, Alberta, Canada T6G 2S2. E-mail: richard.lehner{at}ualberta.ca

SPECIFIC AIMS

Triacylglycerol hydrolase (TGH) is an intracellular lipase that has been proposed to catalyze the mobilization of hepatic triacylglycerol (TG) stores for very low density lipoprotein (VLDL) assembly. The aim of the current study was to examine the effects of inhibition of this lipase activity on lipid and apolipoprotein (apo) B synthesis and secretion, revealing a potential pharmacological target for reducing risk of coronary artery disease and stoke from atherosclerosis.

PRINCIPAL FINDINGS

1. Diethyl-p-nitrophenylphosphate (E600) inhibits TGH activity
We have previously reported the purification of a hepatic TGH and hypothesized that this lipase is involved in mobilizing stored TG for VLDL assembly via a lipolysis and reesterification pathway. The present research explores the relationship between TGH and the provision of lipid for VLDL and apoB secretion. TGH has been localized to the endoplasmic reticulum (ER) and can associate with lipid droplets. The enzyme is absent from the liver-derived cell lines HepG2 and McArdle RH7777. These cell lines are inefficient at mobilizing stored TG for lipoprotein assembly and secretion. Transfection of McArdle RH7777 cells with rat TGH cDNA resulted in increased mobilization of intracellular TG and more robust lipidation of apoB100. Incubation of TGH transfected McArdle RH7777 cells with the lipase inhibitor diethyl-p-nitrophenylphosphate (E600), followed by analysis of remaining microsomal lipolytic activity, indicated that E600 entered the cell and inhibited TGH activity. Mobilization and secretion of stored TG were dramatically reduced.

2. Mobilization of lipids in primary rat hepatocytes
Inhibition studies were carried out in primary rat hepatocytes where the lipolysis/reesterification pathway in VLDL formation is well established. Rat hepatocytes produce both forms of apoB: apoB48 and apoB100. Cellular lipid synthesis, turnover, and secretion were assessed by tracing the acyl acceptor (glycerol backbone of glycerolipids) and acyl donor (fatty acid) by labeling hepatocytes with [¹⁴C]glycerol and [³H]oleate. After prelabeling of cellular lipids, cells were incubated 4 h in the presence or absence of E600. During this period, 57% of preformed cellular glycerol-labeled TG was turned over in the absence of the inhibitor (P<0.009) compared with only 19% turnover (not significant) in the presence of the inhibitor. Inhibition of intracellular TG turnover resulted in the inhibition of lipid secretion (Fig. 1 ). Secretion of stored glycerol-labeled TG was decreased by 41% (P<0.015) in the presence of E600. This is further evidence that lipolysis is an essential step for secretion of stored TG. Only 18–20% of TG labeled in the glycerol backbone was secreted, indicating a dilution of the [¹⁴C]glycerol released by lipolysis with endogenous unlabeled glycerol before reesterification. Inhibiting the mobilization of preformed TG by E600 dramatically decreased oleate-labeled TG secretion. Decreased TG secretion from hepatocytes treated with the lipase inhibitor was confirmed by measurements of TG mass in the media. Reduction of TG secretion was accompanied by increased cellular TG levels.

View larger version (29K): [in this window] [in a new window]   Figure 1. Secretion of lipids from primary rat hepatocytes incubated with (fill) or without (no fill) E600. Hepatocytes were incubated 4 h ± E600 (100 µM) in the presence of 0.4 mM [3H]oleic acid and [14C]glycerol (pulse). Cells not treated with E600 were washed for 1 h in DMEM, followed by a second wash ± E600 (100 µM) and subsequent incubation for 4 h ± E600 (100 µM) (chase). Media were collected and radioactivities associated with the various lipids were determined. Data represent the mean ± SD from 3 independent experiments. *P < 0.015; **P < 0.0001; ***P < 0.001; ****P < 0.005. TG, triacylglycerol; PC, phosphatidylcholine; CE, cholesteryl ester.

Although E600 had no effect on the incorporation of fatty acid into PC or TG, there was a significant decrease in the incorporation of the fatty acid into cholesterol ester (CE) during labeling (34%, P<0.04) (Fig. 1) . It is unlikely that E600 inhibited acyl-CoA:cholesterol acyltransferase (ACAT) activity since another inhibitor from the same series [bis(4-nitrophenyl)phosphate)] at concentrations used in our experiments had no effect on ACAT activity in vitro. The lower incorporation of fatty acid into CE in the presence of the inhibitor may be related to the inhibition of TG turnover as the released fatty acids might also serve as a substrate for ACAT.

3. Lipase inhibitors decrease apoB secretion
The effect of E600 treatment on the secretion of apoB was examined by labeling newly synthesized proteins with [³H]leucine. Preincubation of hepatocytes with E600 resulted in decreased secretion of [³H]leucine-labeled apoB48 (40%, P<0.02) and apoB100 (90%, P<0.0001). Inhibitor treatment decreased cellular apoB48 levels by 30% (P<0.05) and apoB100 levels by 80% (P<0.0001). Intracellular degradation of apoB in hepatoma cell lines has been postulated to be mediated by the proteasome; however, inclusion of the proteasome inhibitor lactacystin during incubations with primary hepatocytes did not significantly increase cellular or secreted apoB levels. Our results are in support of recent data showing that a degradation process within a post-ER compartment is involved in the loss of apoB100 in hepatocytes. We confirmed our metabolic labeling studies by immunoblot analysis of total apoB secretion. ApoB100 secretion from inhibitor-treated cells was substantially decreased (70%) whereas apoB48 secretion was more resistant to the inhibitor (30% decrease) (Fig. 2 ).

View larger version (31K): [in this window] [in a new window]   Figure 2. Secretion of apoB from primary rat hepatocytes incubated with or without E600. Hepatocytes were incubated with 0.4 mM oleate to generate triacylglycerol stores, washed, and incubated 1 h ± E600 (100 µM). Cells were subsequently incubated for 4 h ± E600 (100 µM). Media apoB were collected by cab-o-sil precipitation and analyzed by immunoblotting. A) Immunoblot, B) densitometries of the immunoblot.

To determine the specificity of the effects obtained with E600, we performed studies with a specific TGH inhibitor, 4,4,4-trifluoro-2-[2-(3-methylphenyl)hydrazono]-1-(2-thienyl)butane-1,3-dione (GR148672X). Under the same conditions as with E600 in primary hepatocytes, except using 10 µM GR148672X, there was a dramatic decrease in mobilization of stored TG and of apoB100 secretion. ApoB100 secretion was decreased by 42% (P<0.03) and apoB48 by 20% (P<0.06). GR148672X inhibited TG mass secretion by 54% (P<0.02). Neither E600 nor GR148672X showed any effect on rates of apoB or albumin syntheses, albumin secretion, or hepatocyte integrity.

CONCLUSIONS AND SIGNIFICANCE

High levels of plasma VLDL and its main protein component, apoB, are recognized risk factors for the development of atherosclerosis. Progression of this arterial disease is an underlying cause of heart attack and stroke, the major cause of death in North America and Europe. Reducing VLDL production and levels in plasma would decrease circulating atherogenic lipids and slow the progression of the disease. Assembly of VLDL is a complex process requiring coordinated protein synthesis and provision of neutral lipid and phospholipid. The bulk (60–70%) of the TG secreted with VLDL from liver and primary cultured hepatocytes arises from stored, intracellular TG rather than de novo synthesis. This mobilization involves lipolysis, followed by reesterification.

We propose a model for the role of TGH in the liver in which the TGH substrate pool exists in lipid droplets found in the ER lumen (Fig. 3 ). The TGH substrate may be derived from cytosolic stores via a process that involves microsomal triglyceride transfer protein (MTP). MTP has been shown to be required for secretion of TG-rich apoB-containing lipoproteins. From recent studies in mice in which the MTP gene was disrupted and through MTP inhibition studies, it has become apparent that MTP function may be required for the formation of a luminal TG storage droplet. The luminal droplet may be the primary source of VLDL-TG. It has been hypothesized that the luminal droplet may "fuse" with the nascent, TG-poor primordial apoB particle to form fully lipidated VLDL, but no data directly support the occurrence of such a fusion. In our model, lipolysis of the luminal droplet by TGH to partial acylglycerols and fatty acids would provide substrates for TG resynthesis by luminally localized acyltransferases, followed by TG loading onto nascent apoB-containing particles. TG that is not assembled into apoB may be returned to cytosolic or luminal storage pools (futile cycle). There is experimental evidence for the existence of a latent (luminal) diacylglycerol acyltransferase activity and luminal TG synthesis. Alternately, TGH may access TG at the inner ER membrane as TGH has been shown to adsorb to lipid monolayers and contains a "lipid binding domain." Upon contact with the ER, TGs could egress from the cytosolic lipid droplet into the ER bilayer. Bilayers can incorporate up to 5 mol% of TG without affecting the membrane integrity. Another putative function of TGH would be the delipidation of secretion-incompetent or misfolded apoB particles, facilitating their degradation. The idea behind this hypothesis is that misfolded apoB particles could potentially interfere with the production of correctly assembled lipoproteins by sequestering chaperones, including MTP. However, TGH inhibitor treatment did not result in increased cellular apoB levels. Our recent studies suggest that apoB associated TG is not a substrate for TGH. Furthermore, inhibition of TGH did not affect the secretion of albumin, indicating our results were not due to a general effect of the compounds used on protein translation or the secretory pathway.

View larger version (36K): [in this window] [in a new window]   Figure 3. Model of triacylglycerol (TG) mobilization for very low density lipoprotein (VLDL) assembly. The substrate for triacylglycerol hydrolase (TGH) may be derived from cytosolic stores via a process that involves microsomal TG transfer protein (MTP). MTP function may be required for the formation of a luminal TG storage droplet. The luminal droplet may be the primary source of VLDL-TG. Lipolysis of the luminal droplet by TGH to partial acylglycerols such as diacylglycerol (DG) and fatty acids would provide substrates for TG resynthesis by luminally localized acyltransferases (diacylglycerol acyltransferase, DGAT), followed by TG loading onto nascent apoB-containing particles. TG that is not assembled into apoB may be returned to cytosolic or luminal storage pools (futile cycle). Alternately, TGH may access TG at the inner endoplasmic reticulum (ER) membrane. Upon contact with the ER, TGs could egress from the cytosolic lipid droplet into the ER bilayer.

Our data show that inhibition of intracellular TG lipolysis reduces TG and apoB100 secretion. The effects of inhibition of lipolysis were most profound on secretion of apoB100-containing particles, likely because apoB48-containing lipoproteins can be secreted as a relatively lipid-poor, high density particle, while lipid-poor apoB100-containing lipoproteins more readily undergo intracellular degradation. The data support our previous observations in McArdle RH7777 cells transfected with rat TGH cDNA, where preferential lipidation of apoB100 was observed (i.e., increased apoB100 VLDL formation).

The observation of marked decreases of apoB and TG secretion by the inhibition of microsomal lipolytic activity provides further support to the concept that a lipolysis/reesterification pathway is a regulated step in VLDL assembly. TGH may therefore be an attractive target for pharmacological intervention that would decrease the levels of circulating atherogenic lipids.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0728fje; doi: 10.1096/fj.02-0728fje

² Present address: Promega Corporation, Madison, WI 53711-5399, USA.

³ Present address: Department of Biochemistry and Genetics, University of Mazandarah, Sari, Iran.

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