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

Abstract

The presence of elevated circulating triacylglycerol (TG)-rich very low density lipoprotein (VLDL) and apolipoprotein B-100 (apoB-100) levels represents an independent risk factor for coronary artery disease. Triacylglycerol hydrolase catalyzes the mobilization of cytoplasmic TG stores. To test the hypothesis that the enzyme plays a role in the provision of core lipids for the assembly of VLDL, we inhibited the lipase activity in primary rat hepatocytes and analyzed lipid and apoB synthesis and secretion. Inhibition of lipolysis resulted in a dramatic decrease in secretion of TGs. In addition, secretion of cholesteryl ester and phosphatidylcholine was substantially decreased. Analysis of secreted apolipoproteins indicated that apoB-100 secretion was much more sensitive to lipase inhibition than was apoB-48 secretion, perhaps because of the ability of apoB-48 to be secreted as a relatively lipid-poor particle. The results agreed with those obtained with hepatoma cells transfected with triacylglycerol hydrolase cDNA, in which preferential lipidation of apoB-100 was observed. Together, our findings provide evidence that inhibition of intracellular TG hydrolysis significantly decreases apoB-100 secretion and suggest that triacylglycerol hydrolase may be a suitable pharmacological target in efforts to lower plasma lipid levels.

Attenuation of Adipocyte Triacylglycerol Hydrolase Activity Decreases Basal Fatty Acid Efflux

“…First-strand cDNA synthesis from 2 g of total RNA was performed using SuperScript TM II reverse transcriptase (Invitrogen) primed by oligo(dT) [12][13][14][15][16][17][18] primers. The following primers were used: aP2 (adipocyte lipid-binding protein), 5Ј-GAA CCT GGA AGC TTG TCT TCG-3Ј (forward (F)) and 5Ј-ACC AGC TTG TCA CCA TCT CG-3Ј (reverse (R)); cyclophilin, 5Ј-TCC AAA GAC AGC AGA AAA CTT TCG-3Ј (F) and 5Ј-TCT TCT TGC TGG TCT TGC CAT TCC-3Ј (R); ATGL, 5Ј-TGG AAC ATC TCA TTC GCT GG-3Ј (F) and 5Ј-AAT GCC GCC ATC CAC ATA G-3Ј (R); adiponutrin, 5Ј-GTT TGC AGG CTG CGG CTT CC-3Ј (F) and 5Ј-GGC AGA TGT CAT GCT CAC CG-3Ј (R); peroxisome proliferator-activated receptor ␥, 5Ј-ATG TCT CAT AAT GCC ATC-3Ј (F) and 5Ј-CTA GTA CAA GTC CTT GTA-3Ј (R); diacylglycerol acyltransferase 1, 5Ј-ATT CAC GGA TCA TTG AGC G-3Ј (F) and 5Ј-CTG CCA TGT CTG AGC ATA GG-3Ј (R); diacylglycerol acyltransferase 2, 5Ј-CTA CGT TGG CTG GTA ACT TCC-3Ј (F) and 5Ј-AAC CAG ATC AGC TCC ATG G-3Ј (R).…”

“…Lipase Assay-Differentiated 3T3-L1 adipocytes were incubated in the presence or absence of lipase inhibitors for 4 h. The concentration of the inhibitors used in the experiments was previously determined to be effective in inhibition of TGH activity both in vitro and in intact cells (13). Cells were then washed with Tris-buffered saline and harvested in an ice-cold homogenization buffer, and cytosolic and membrane fractions were prepared as described above.…”

“…Media were centrifuged at 1500 rpm for 5 min in a microcentrifuge to remove cell debris. Lipids were extracted from cell homogenates and media with chloroform/methanol (2:1, v/v) in the presence of nonradioactive lipid carriers (phosphatidylcholine, oleic acid, dioleoylglycerol, trioleoylglycerol, and cholesteryl oleate) as described previously (12,13). The chloroform phase containing lipids was dried under nitrogen, re-dissolved in a small volume of chloroform, and applied to silica gel H thinlayer chromatography plates.…”

“…The chloroform phase containing lipids was dried under nitrogen, re-dissolved in a small volume of chloroform, and applied to silica gel H thinlayer chromatography plates. The plates were developed in heptane/isopropyl ether/acetic acid 60:40:4 (by volume) to separate neutral lipids (FA, TG, and cholesteryl esters) (12,13). Lipid classes were visualized by exposure to iodine vapor, bands were scraped, and the associated radioactivity was determined by scintillation counting.…”

“…We have characterized another neutral intracellular TG hydrolase (TGH) in hepatic microsomes (10). TGH (also termed Ces3) associates with hepatic lipid droplets (11) and hydrolyzes stored hepatic TG (12)(13)(14)(15). TGH is also expressed in 3T3-L1 adipocytes (16,17) and adipose tissue (18 -20) where it has been also shown to associate with adipocyte lipid droplets (19).…”