Evidence that microsomal triglyceride transfer protein is limiting in the production of apolipoprotein B-containing lipoproteins in hepatic cells

Abstract

The microsomal triglyceride transfer protein (MTP) is a heterodimeric lipid transfer protein that is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins. A key unresolved question is whether the MTP-mediated step is rate limiting. To address this, a unique experimental strategy was used that allowed the in situ modulation and measurement of MTP triglyceride transfer activity. In order to accomplish this, an irreversible photoaffinity inhibitor, BMS-192951, was designed and synthesized. When incubated with purified MTP and irradiated with UV light at 360 nm, BMS-192951 inhibits triglyceride transfer by covalently binding to the protein. HepG2 cells were treated with either increasing concentrations of BMS-192951 (0-15 m ) with 5 min of ultraviolet irradiation, or 3.0 m BMS-192951 with various lengths (0-15 min) of ultraviolet irradiation. Microsomal extracts were prepared exhaustively dialyzed to remove unbound inhibitor, and assayed for MTP-mediated triglyceride transfer activity. BMS-192951 was shown to reduce MTP activity in both a dose-and UV exposure time-dependent fashion. Measurement of apoB concentration in the media showed that apoB secretion was reduced in proportion to the in situ inhibition of MTP activity, while no change was observed in apoA-I secretion. Experiments performed in McArdle RH-7777 rat hepatoma cells and primary rat hepatocytes gave nearly identical results; the decrease in apoB secretion was proportional to the decrease in MTP activity. These results indicate that MTP-mediated lipid transfer is limiting in the assembly and secretion of apoB-containing lipoproteins in hepatic cells under the conditions tested.

Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B

“…This finding is particularly interesting as our experimental model was the wild-type C57BL/6 mouse on normal chow without addition of lipid to the diet. Our results suggest that MTP is rate-limiting for hepatic VLDL secretion, as recently suggested by Jamil et al (29) and Liao, Kobayashi, and Chan (30) based on in vitro studies. In vitro studies using pharmacological MTP inhibitors have established that decreased MTP activity results in a decrease of TG and apoB secretion (23)(24)(25)(26).…”

“…However, the question of whether increased MTP activity over basal levels of expression results in increased VLDL and apoB secretion in vivo has not yet been answered. Interestingly, a recent in vitro study suggested that MTP might be limiting for the secretion of apoB-containing lipoproteins (29). The same study reported that attempts to increase MTP activity by transfecting hepatoma cell lines with an expression plasmid encoding MTP were unsuccessful (29).…”

“…Interestingly, a recent in vitro study suggested that MTP might be limiting for the secretion of apoB-containing lipoproteins (29). The same study reported that attempts to increase MTP activity by transfecting hepatoma cell lines with an expression plasmid encoding MTP were unsuccessful (29). Recently, adenovirus-mediated overexpression of MTP was shown to increase apoB secretion in HepG2 cells (30).…”

Effects of overexpression of the amino-terminal fragment of apolipoprotein B on apolipoprotein B and lipoprotein production

“…There are several types of data on cells and animals suggesting that MTP is rate limiting for production of lipoproteins. Using a photo-affinity inhibitor in rat hepatoma cells, Jamil et al (7) indicated that the MTP-mediated lipid transfer step was the rate-limiting step in the production of apoB-containing lipoproteins. By using an MTP inhibitor, Wetterau et al (33) showed that apoB-containing lipoproteins within the plasma of experimental animals were decreased proportionally with druginduced MTP inhibition.…”

Lipoprotein assembly capacity of the mammary tumor-derived cell line C127 is due to the expression of functional microsomal triglyceride transfer protein

“…5A , lanes 2 and 4). This quantitative difference in secretion of apoB-41F from C127 cells in the presence or absence of cotransfected MTP suggests that, although MTP may be present in these cells, it is limiting (31).…”