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Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.

ABSTRACT: The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed.

SUBMITTER: Owen MR

PROVIDER: S-EPMC1218291 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Project description:In contrast to water-soluble fuels such as glucose or ketone bodies, the use of lipids as an energy source for tissues has required the development of complex structures for their transport through the aqueous plasma. In the case of endogenously synthesized triacylglycerol this is achieved by the assembly and secretion of hepatic VLDL which provides the necessary stability in an aqueous medium. An essential component of this assembly process is apo B. Dietary changes which require an increase in hepatic VLDL secretion appear to be accompanied by increases in the availability of functional apo B. Interesting questions relate to: (a) the intracellular site(s) of triacylglycerol association with apo B, and (b) the mechanism(s) by which the availability of functional apo B at this site responds to metabolic and hormonal signals which reflect dietary status and, thus, the need to secrete triacylglycerol. As regards the latter, although in some cases changes in apo B synthesis occur in response to VLDL secretion hepatic apo B mRNA levels appear to be quite stable in vitro. Intracellular switching of apo B between the secretory and degradative pathways may be important in controlling VLDL assembly and post-translational modifications of the apoprotein may also play a role by influencing its ability to bind to triacylglycerol. Transport is not the only problem associated with the utilization of a concentrated energy source such as triacylglycerol and the complex problems of waste product disposal and recycling have to be dealt with. In the case of triacylglycerol, potentially toxic waste products include atherogenic remnants and LDL. The overall problem, then, in the long-term, involves the development of a 'safe' means of utilizing triacylglycerol and this requirement accounts for much of the complexity of plasma lipoprotein metabolism. In this area, the rat could teach the human a few tricks. One of these appears to be the utilization of hepatic apo B48 rather than apo B100 for VLDL assembly in response to increases in the extrahepatic utilization of hepatically synthesized triacylglycerol. Under these conditions, the remnants of hepatic triacylglycerol utilization by peripheral tissues are cleared from the plasma much more readily via a process which seems to involve the cycling of more triacylglycerol back to the liver than that which occurs in humans. The means by which this is achieved, though, are obscure and may involve a chylomicron remnant receptor, the nature of which, itself, remains controversial.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:When rat hepatocytes were cultured for 24 h in the absence of exogenous fatty acid, the amount of very-low-density lipoprotein (VLDL) triacylglycerol (TAG) secreted (114 +/- 14 micrograms/mg of cell protein) could not be accounted for by the mass of TAG lost from the cells (29 +/- 6.1 micrograms/mg of cell protein) during this period (n = 12). Of the balance (85 +/- 14 micrograms/mg; 94 +/- 15 nmol/mg), a maximum of only 37 nmol/mg of cell protein of TAG could be accounted for by fatty acids synthesized de novo. When labelled exogenous oleate (initial concentration, 0.75 nM) was present in the culture medium, the net gain in cellular plus VLDL TAG (253 +/- 38 micrograms/mg of cell protein per 24 h) was greater than that contributed by the exogenous fatty acid (155 +/- 18.2 micrograms/mg of cell protein, n = 5). Again, the balance (98.8 +/- 18.2 micrograms/mg of cell protein per 24 h) was too great to be accounted for by fatty acid synthesis de novo. In experiments in which cellular glycerolipids were prelabelled with [9, 10(n)-3H]oleic acid, following removal of the labelled fatty acid, there was a net increase in labelled cellular plus VLDL TAG over the next 24 h. That cellular phospholipids are the source of a substantial part of the excess TAG synthesized is supported by the following evidence. (1) The loss of prelabelled cellular phospholipid during culture was greater than could be accounted for by secretion into the medium. (2) During culture of cells prelabelled with 1,2-di-[l-14C]palmitoyl phosphatidylcholine, a substantial amount of label was secreted as VLDL TAG. (3) In pulse-chase experiments, the kinetics of labelled phospholipid turnover were consistent with conversion into a non-phospholipid pool. The enzymology involved in the transfer of phospholipid fatty acids into TAG is probably complex, but the present results suggest that this pathway may represent an important route by which extracellular fatty acids are channelled into VLDL TAG.

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Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.

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