Peroxisome proliferator-activated receptor alpha deficiency modifies glucose handling by isolated mouse adipocytes.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a transcription factor that regulates enzymes involved in fatty acid (FA) utilisation. PPARalpha null mice have recently been demonstrated to have increased whole-body glucose turnover in vivo. This has been attributed to increased glucose uptake by adipose tissue, but the impact of PPARalpha deficiency on the characteristics of glucose handling by isolated adipocytes ex vivo is unknown. To determine directly the impact of PPARalpha deficiency on adipocyte glucose handling, thereby excluding any influence of humoral/neuronal factors, we examined total glucose metabolism as well as glucose disposition towards alternative fates in epididymal adipocytes isolated from wild-type and PPARalphanull mice. Total glucose metabolism (oxidation, incorporation into FA and glycerol moieties of triglyceride (TAG) and conversion to lactate) was measured under basal conditions (low glucose) and 'stimulated lipogenic' conditions (high glucose + insulin). Adipocytes from PPARalpha null mice had higher rates of glucose metabolism under both basal and stimulated lipogenic conditions, with increased glucose utilisation both for oxidation and entry into the synthesis of the FA and glycerol components of lipid. In particular, the capacity of adipocytes from PPARalpha-deficient mice to utilise glucose for synthesis of the glycerol backbone of TAG was greatly enhanced under stimulated (high glucose + insulin) conditions. The increased use of glucose for the glycerol moiety of adipocyte TAG may therefore contribute to, and provide explanation for, enhanced glucose turnover in PPARalpha null mice.

Fasting-induced increases in aquaporin 7 and adipose triglyceride lipase mRNA expression in adipose tissue are attenuated by peroxisome proliferator-activated receptor alpha deficiency.

OBJECTIVE: To investigate the impact of peroxisome proliferator-activated receptor alpha deficiency on gene expression of adipose triglyceride lipase and the glycerol transporter aquaglyceroporin 7 in white adipose tissue in the fed and fasted states in relation to glycerol release by isolated adipocytes. MEASUREMENTS: Studies using wild-type and peroxisome proliferator-activated receptor alpha null mice. Hormone and metabolite concentrations, real-time polymerase chain reaction (PCR), basal and stimulated adipocyte lipolysis, estimated by glycerol release. RESULTS: Peroxisome proliferator-activated receptor alpha deficiency blocked the increase in aquaglyceroporin 7 transcript level and attenuated the increase in adipose triglyceride lipase transcript level in white adipose tissue elicited by fasting. Fasting glycerol levels were lower in peroxisome proliferator-activated receptor alpha null than wild-type mice, despite increased mobilization of adipocyte fat reserves in vivo as indicated by reduced adipose tissue masses (three distinct depots) and a significantly lower epididymal adipocyte diameter. Basal net glycerol release was unchanged but beta-adrenergic-stimulated net glycerol release was higher with isolated adipocytes from fasted peroxisome proliferator-activated receptor alpha null mice compared with those of fasted wild-type mice. CONCLUSION: Peroxisome proliferator-activated receptor alpha deficiency prevents effects of fasting to increase adipocyte aquaglyceroporin 7 gene expression, and influences the regulation of inter-tissue glycerol flux after fasting via lowered adipocyte aquaglyceroporin 7 expression. Lowered gene expression of adipose triglyceride lipase and aquaglyceroporin 7 in peroxisome proliferator-activated receptor alpha null mice is not limiting for adipose triglyceride breakdown in vivo during fasting.

Deficiency of PPARalpha disturbs the response of lipogenic flux and of lipogenic and cholesterogenic gene expression to dietary cholesterol in mouse white adipose tissue.

PPARalpha-deficiency in mice fed a high-carbohydrate, low-cholesterol diet was associated with a decreased weight of epididymal adipose tissue and an increased concentration of adipose tissue cholesterol. Consumption of a high (2% w/w) cholesterol diet resulted in a further increase in the concentration of cholesterol and a further decrease in epididymal fat pad weight in PPARalpha-null mice, but had no effect in the wild-type. These reductions in fat pad weight were associated with an increase in hepatic triacylglycerol content, indicating that both PPARalpha-deficiency and cholesterol altered the distribution of triacylglycerol in the body. Adipose tissue de novo lipogenesis was increased in PPARalpha-null mice and was further enhanced when they were fed a cholesterol-rich diet; no such effect was observed in the wild-type mice. The increased lipogenesis in the chow-fed PPARalpha-null mice was accompanied paradoxically by lower mRNA expression of SREBP-1c and its target genes, acetyl-CoA carboxylase and fatty acid synthase. Consumption of a high-cholesterol diet increased the mRNA expression of these genes in the PPARalpha-deficient mice but not in the wild-type. De novo cholesterol synthesis was not detectable in the adipose tissue of either genotype despite a relatively high expression of the mRNA's encoding SREBP-2 and 3-hydroxy-3-methylglutaryl Coenzyme A reductase. The mRNA expression of these genes and of the LDL-receptor in adipose tissue of the PPARalpha-deficient mice was lower than that of the wild-type and was not downregulated by cholesterol feeding. The results suggest that PPARalpha plays a role in adipose tissue cholesterol and triacylglycerol homeostasis and prevents cholesterol-mediated changes in de novo lipogenesis.

A dominant negative human peroxisome proliferator-activated receptor (PPAR){alpha} is a constitutive transcriptional corepressor and inhibits signaling through all PPAR isoforms.

Several missense mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor (PPAR)gamma have been described in subjects with dominantly inherited severe insulin resistance associated with partial lipodystrophy, hypertension, and dyslipidemia. These mutant receptors behave as dominant-negative inhibitors of PPARgamma signaling when studied in transfected cells. The extent to which such dominant-negative effects extend to signaling through other coexpressed PPAR isoforms has not been evaluated. To examine these issues further, we have created a PPARalpha mutant harboring twin substitutions, Leu459Ala and Glu462Ala, within the ligand binding domain (PPARalpha(mut)), examined its signaling properties, and compared the effects of dominant-negative PPARalpha and PPARgamma mutants on basal and ligand-induced gene transcription in adipocytes and hepatocytes. PPARalpha(mut) was transcriptionally inactive, repressed basal activity from a PPAR response element-containing promoter, inhibited the coactivator function of cotransfected PPAR-gamma coactivator 1alpha, and strongly inhibited the transcriptional response to cotransfected wild-type receptor. In contrast to PPARgamma, wild-type PPARalpha failed to recruit the transcriptional corepressors NCoR and SMRT. However, PPARalpha(mut) avidly recruited these corepressors in a ligand-dissociable manner. In hepatocytes and adipocytes, both PPARalpha(mut) and the corresponding PPARgamma mutant were capable of inhibiting the expression of genes primarily regulated by PPARalpha, -gamma, or -delta ligands, albeit with some differences in potency. Thus, dominant-negative forms of PPARalpha and PPARgamma are capable of interfering with PPAR signaling in a manner that is not wholly restricted to their cognate target genes. These findings may have implications for the pathogenesis of human syndromes resulting from mutations in this family of transcription factors.

Synthesis and function of hepatic very-low-density lipoprotein.

Most of the triacylglycerol (TAG) utilized for the assembly of very-low-density lipoprotein (VLDL) in the secretory apparatus of the hepatocyte is mobilized by lipolysis of the cytosolic TAG pool, followed by re-esterification. The lipases involved include arylacetamide deacetylase and/or triacylglycerol hydrolase. Some of the re-esterified products of lipolysis gain access to an apolipoprotein-B-rich VLDL precursor to form mature VLDL. Some, however, are returned to the cytosolic pool in a process that is stimulated by insulin and inhibited by microsomal triacylglycerol transfer protein (MTP). Phospholipids also contribute to VLDL TAG in a process which involves ADP-ribosylation factor-1 (ARF-1)-mediated activation of phospholipase D. The temporary storage of TAG in the liver, followed by its mobilization and secretion as VLDL, form part of a process by which the liver protects vulnerable body tissues from excess lipotoxic non-esterified ('free') fatty acids in the plasma.

Role of peroxisome proliferator-activated receptor-alpha in the mechanism underlying changes in renal pyruvate dehydrogenase kinase isoform 4 protein expression in starvation and after refeeding.

The pyruvate dehydrogenase complex (PDC) occupies a strategic role in renal intermediary metabolism, via partitioning of pyruvate flux between oxidation and entry into the gluconeogenic pathway. Inactivation of PDC via activation of pyruvate dehydrogenase kinases (PDKs), which catalyze PDC phosphorylation, occurs secondary to increased fatty acid oxidation (FAO). In kidney, inactivation of PDC after prolonged starvation is mediated by up-regulation of the protein expression of two PDK isoforms, PDK2 and PDK4. The lipid-activated transcription factor, peroxisome proliferator-activated receptor-alpha (PPAR alpha), plays a pivotal role in the cellular metabolic response to fatty acids and is abundant in kidney. In the present study we used PPAR alpha null mice to examine the potential role of PPAR alpha in regulating renal PDK protein expression. In wild-type mice, fasting (24 h) induced marked up-regulation of the protein expression of PDK4, together with modest up-regulation of PDK2 protein expression. In striking contrast, renal protein expression of PDK4 was only marginally induced by fasting in PPAR alpha null mice. The present results define a critical role for PPAR alpha in renal adaptation to fasting, and identify PDK4 as a downstream target of PPAR alpha activation in the kidney. We propose that specific up-regulation of renal PDK4 protein expression in starvation, by maintaining PDC activity relatively low, facilitates pyruvate carboxylation to oxaloacetate and therefore entry of acetyl-CoA derived from FA beta-oxidation into the TCA cycle, allowing adequate ATP production for brisk rates of gluconeogenesis.

Insulin inhibits the maturation phase of VLDL assembly via a phosphoinositide 3-kinase-mediated event.

LY 294002 (80 micromol/L), an inhibitor of phosphoinositide 3-kinase, was used to investigate the involvement of this enzyme in the insulin-mediated regulation of very low density lipoprotein (VLDL) apolipoprotein B (apoB) output from cultured rat hepatocytes. Newly synthesized apoB was pulse-labeled with [(35)S]methionine and was then allowed to assemble, via an intermediate precursor stage, into mature VLDL during subsequent chase periods. Brefeldin A (BFA, 0.2 microgram/mL) was used to discriminate between the role of insulin in the regulation of the early, compared with the later, events of VLDL assembly, including apoB degradation. Insulin (78 nmol/L), when present during the pulse-labeling and subsequent chase periods, inhibited the secretion of apoB-100 and apoB-48 as VLDL by 53% and 56%, respectively. Degradation of both was concomitantly increased. Secretion of high density lipoprotein apoB, derived from VLDL precursors, was relatively unaffected under these conditions, as was the net synthesis of apoB-100 and apoB-48. The presence of BFA during the pulse-labeling period and subsequent chase period prevented the maturation of VLDL in the insulin-treated and the non-insulin-treated cells. BFA was then removed, allowing the maturation of VLDL to proceed. Removal of insulin at this stage reversed the overall inhibitory effect of insulin. Furthermore, when insulin remained present during this period, the simultaneous presence of LY 294002 also reversed the inhibitory effect of insulin on VLDL apoB output and abolished the increase in apoB degradation. The results suggest that insulin signaling via phosphoinositide 3-kinase inhibited the maturation phase of VLDL assembly by preventing bulk lipid transfer to a VLDL precursor, thus enhancing the degradation of apoB. There was no inhibition of the conversion of newly synthesized apoB into the VLDL precursor form.

Microsomal membrane-associated apoB is the direct precursor of secreted VLDL in primary cultures of rat hepatocytes.

Brefeldin A (BFA) added to primary cultures of rat hepatocytes, at a concentration of 0.2 microg/ml, prevented the assembly of newly synthesized apolipoprotein B (apoB) into mature, secretory VLDL but did not prevent the secretion of apoB as denser particles (HDL apoB), or of albumin. The unassembled apoB remained associated with the membranes of the cellular microsomal fraction. There was no effect of BFA on the removal of apoB from the lumen of these vesicles. VLDL apoB formed only a minor component of the total apoB in the microsomal lumen. Higher (5 microg/ml) concentrations of BFA were required to prevent the secretion of HDL apoB and albumin. Under these conditions apoB accumulated in the microsomal lumen, as well as in the membranes of these vesicles. Again, apoB VLDL formed only a minor proportion of the total lumenal apoB. ApoB-48 VLDL and apoB-100 VLDL assembly could be restored by removing BFA from the medium. This reactivation of VLDL assembly was accompanied by an increased removal of apoB from the microsomal membranes, but there was no detectable increase in the small quantity of VLDL apoB that was recovered from the microsomal lumen. In the absence of BFA, during pulse-chase experiments the pattern of change in the specific radioactivity of microsomal membrane apoB was similar to that of the secreted VLDL apoB whereas that of the lumenal apoB resembled that of the secreted HDL apoB. The results suggest that membrane-associated apoB is the main direct precursor of secreted VLDL apoB in primary cultures of rat hepatocytes and that VLDL assembly does not involve primarily microsomal lumenal apoB as an intermediate.

Characterization of the rodent genes for arylacetamide deacetylase, a putative microsomal lipase, and evidence for transcriptional regulation.

In the current study, we have determined the cDNA and the genomic sequences of the arylacetamide deacetylase (AADA) gene in mice and rats. The AADA genes in the rat and mouse consist of five exons and have 2.4 kilobases of homologous promoter sequence upstream of the initiating ATG codon. AADA mRNA is expressed in hepatocytes, intestinal mucosal cells (probably enterocytes), the pancreas and also the adrenal gland. In mice, there is a diurnal rhythm in hepatic AADA mRNA concentration, with a maximum 10 h into the light (post-absorptive) phase. This diurnal regulation is attenuated in peroxisome proliferator-activated receptor alpha knockout mice. Intestinal but not hepatic AADA mRNA was increased following oral administration of the fibrate, Wy-14,643. The homology of AADA with hormone-sensitive lipase and the tissue distribution of AADA are consistent with the view that AADA plays a role in promoting the mobilization of lipids from intracellular stores and in the liver for assembling VLDL. This hypothesis is supported by parallel changes in AADA gene expression in animals with insulin-deficient diabetes and following treatment with orotic acid.

The life and work of Dermot Hedley ("Derek") Williamson (1929-1998).

Derek Williamson's scientific career spanned the "Golden Age" of research into metabolic regulation, to which he made an important and sustained contribution. Derek joined Hans Krebs' laboratory at Sheffield University in 1946 and moved to Krebs' MRC Unit in Oxford in 1960. He elaborated an enzymic method for the determination of acetoacetate and 3-hydroxybutyrate [Williamson, Mellanby and Krebs, Biochem. J. (1962) 82, 90-96], which opened up the field of ketone body metabolism and its regulation and became a Citation Classic. Another Citation Classic followed [Williamson, Lund and Krebs, Biochem. J. (1967) 103, 514-527]. He moved with Krebs to the Metabolic Research Laboratory at the Radcliffe Infirmary in 1967, where he blossomed, formulating his ideas about the integrated regulation of metabolic pathways, particularly with regard to fatty acid oxidation, lipid synthesis and ketone body metabolism. His success was illustrated by more than 200 publications. Derek implanted and nurtured a sense of the excitement of scientific discovery in his colleagues and students, and he worked hard to provide a friendly, supportive and encouraging environment. Many lives have been enriched by the privilege of working with him.

Disturbances in the normal regulation of SREBP-sensitive genes in PPAR alpha-deficient mice.

Peroxisome proliferator-activated receptor alpha (PPAR alpha)-null mice were used to investigate the nature of the relationship between the normal circadian rhythm of hepatic PPAR alpha expression and the expression of the lipogenic and cholesterogenic sterol regulatory element-binding protein (SREBP)-regulated genes, acetyl-CoA carboxylase, fatty acid synthase (FAS), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAR). The expression of FAS and HMG-CoAR varied rhythmically over the diurnal cycle in the normal mice, with patterns that were the opposite of that of PPAR alpha. The diurnal variation of lipogenic and cholesterogenic gene expression was attenuated or abolished in the PPAR alpha-null mice. This resulted in decreased expression compared with normal mice, but only during the dark phase of the cycle, when food intake was high. The diurnal variation in hepatic fatty acid and cholesterol synthesis was also abolished in the PPAR alpha-null animals and the variations in the concentration of plasma triacylglycerol, nonesterified fatty acids, and cholesterol were all attenuated. The failure of HMG-CoAR expression to increase during the feeding period in the PPAR alpha-null mice was associated with a decrease in hepatic nonesterified cholesterol content and an increase in cholesteryl ester compared with normal mice. There was no defect in the downregulation of hepatic HMG-CoAR mRNA in response to dietary cholesterol in the PPAR alpha-null mice. Under these conditions, hepatic PPAR gamma expression increased in both the control and PPAR alpha-deficient mice. The results suggest that PPAR alpha-deficiency disturbs the normal circadian regulation of certain SREBP-sensitive genes in the liver, but does not affect their response to dietary cholesterol. -- Patel, D. D., B. L. Knight, D. Wiggins, S. M. Humphreys, and G. F. Gibbons. Disturbances in the normal regulation of SREBP-sensitive genes in PPAR alpha-deficient mice. J. Lipid Res. 2001. 42: 328--337.

The effect of peroxisome-proliferator-activated receptor-alpha on the activity of the cholesterol 7 alpha-hydroxylase gene.

Cholesterol 7 alpha-hydroxylase (Cyp7a1) plays a central role in the regulation of bile acid and cholesterol metabolism, and transcription of the gene is controlled by bile acids and hormones acting through a complex interaction with a number of potential steroid-hormone-binding sites. Transcriptional activity of the human CYP7A1 gene promoter transfected into HepG2 cells was decreased in a concentration-dependent manner by co-transfection with an expression vector for peroxisome-proliferator-activated receptor-alpha (PPAR alpha). This effect was augmented by 9-cis-retinoic acid receptor-alpha (RXR alpha) and activators of PPAR alpha to give a maximum inhibition of approx. 80%. The region responsible for this inhibition contained a site known to bind hepatocyte nuclear factor 4 (HNF4), and mutation of this site greatly decreased the effect. Co-expression of HNF4 increased promoter activity and decreased the effect of PPAR alpha. Gel-mobility-shift assays failed to detect any binding of PPAR alpha/RXR alpha dimers to any regions of the promoter containing potential binding sites. Also the hepatic abundance of Cyp7a1 mRNA in mice in which the PPAR alpha gene was disrupted was the same as in normal mice, both during the dark phase, when the animals were feeding, and during the light phase, when mRNA abundance was greatly increased. Cholesterol feeding produced the same increase in hepatic Cyp7a1 mRNA abundance in PPAR alpha-null animals as in normals. It is concluded that, whereas PPAR alpha can affect CYP7A1 gene transcription in vitro through an indirect action, probably by competing for co-factors, this is unlikely to be a major influence on Cyp7a1 activity under normal physiological conditions.

Decreased hepatic expression of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein in aging rats is associated with delayed clearance of chylomicrons from the circulation.

Aging in both humans and rats is associated with the development of insulin resistance and the ensuing alterations in the plasma lipoprotein profile. In this study, young (2 months) and old (15 months) Sprague-Dawley (SD) rats were used to investigate age-related alterations in the chylomicron clearance pathway. Clearance from the blood of an intravenously injected bolus of 14C-labeled cholesterol ester (CE) and 3H-labeled triacylglycerol (TAG) lymph chylomicrons was markedly delayed in the old rats (P < .05). Hepatic expression of the two principal receptors of chylomicron remnant removal, the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP), was determined by ligand blotting and immunoblotting. The old rats expressed 43%+/-7% of the level of LDL receptor in the young animals (P < .05) and 45%+/-16% of the corresponding level of LRP (P < .05). The results suggest that the delayed clearance of chylomicron remnants in this animal model of aging and insulin resistance is due, at least in part, to a decrease in the hepatic expression of LDL receptor and LRP.

Effect of dietary fish oil on the sensitivity of hepatic lipid metabolism to regulation by insulin.

The contribution of dietary fat content and type to changes in the sensitivity of hepatic lipid metabolism to insulin was studied in primary hepatocyte cultures from donor rats maintained on a low-fat diet (LF), or on diets enriched in olive oil (OO) or fish oil (FO). The higher rate of fatty acid oxidation in hepatocytes from the FO-fed group was resistant to the inhibitory effects of insulin observed in hepatocytes from the other groups. Insulin stimulation of fatty acid incorporation into triglyceride (TG) was also less pronounced in hepatocytes from the FO-fed group than in those from the OO-fed group but there was no difference in the stimulatory effect of insulin on fatty acid incorporation into phospholipid (PL) in these two groups. In the case of fatty acid incorporation into both PL and TG, hepatocytes from the LF group were refractory to stimulation by insulin. At each concentration of insulin, hepatocytes from the FO-fed group secreted less very low density lipoprotein (VLDL) TG than those from the other groups. However, the absolute suppression of VLDL TG secretion by insulin was similar irrespective of the diet of the donor animals.We conclude that chronic consumption of a particular type of dietary fat does not affect the insulin sensitivity of the major pathways of hepatic lipid metabolism in a consistent manner.

Mobilisation of triacylglycerol stores.

Triacylglycerol (TAG) is an energy dense substance which is stored by several body tissues, principally adipose tissue and the liver. Utilisation of stored TAG as an energy source requires its mobilisation from these depots and transfer into the blood plasma. The means by which TAG is mobilised differs in adipose tissue and liver although the regulation of lipid metabolism in each of these organs is interdependent and synchronised in an integrated manner. This review deals principally with the mechanism of hepatic TAG mobilisation since this is a rapidly expanding area of research and may have important implications for the regulation of plasma very-low-density lipoprotein metabolism. TAG mobilisation plays an important role in fuel selection in non-hepatic tissues such as cardiac muscle and pancreatic islets and these aspects are also reviewed briefly. Finally, studies of certain rare inherited disorders of neutral lipid storage and mobilisation may provide useful information about the normal enzymology of TAG mobilisation in healthy tissues.

Co-ordination of hepatic and adipose tissue lipid metabolism after oral glucose.

The integration of lipid metabolism in the splanchnic bed and in subcutaneous adipose tissue before and after ingestion of a 75 g glucose load was studied by Fick's principle in seven healthy subjects. Six additional subjects were studied during a hyperinsulinemic euglycemic clamp. Release of non-esterified fatty acids (NEFA) from adipose tissue and splanchnic NEFA extraction followed a similar time-course after oral glucose, and there was a highly significant relationship between adipose tissue NEFA release and splanchnic NEFA uptake. There was no immediate inhibition of splanchnic very low density lipoprotein (VLDL)-triacylglycerol (TAG) output when plasma insulin levels increased after glucose. Adipose tissue extraction of VLDL-TAG tended to vary in time in a manner similar to splanchnic VLDL-TAG output and the two were significantly related. The area-under-curves (AUC) for splanchnic extraction of NEFA was significantly lower than that for output of VLDL, implying depletion of hepatic TAG stores during the experiment. In the hyperinsulinemic clamp experiments, there was on average suppression of splanchnic VLDL-TAG output although between-person variability was marked. This suppression could be explained by a very low supply of NEFA during the clamp. We conclude that there is an integrated pattern of metabolism in splanchnic and adipose tissues in the postabsorptive and post-glucose states. Flux of NEFA from adipose tissue drives splanchnic NEFA uptake. Splanchnic VLDL-TAG secretion appears to be regulated by a number of factors and in turn controls TAG extraction in adipose tissue. Insulin does not seem to play a key role in the acute regulation of hepatic VLDL metabolism under these particular conditions in vivo.

Inactivation of microsomal triglyceride transfer protein impairs the normal redistribution but not the turnover of newly synthesized glycerolipid in the cytosol, endoplasmic reticulum and Golgi of primary rat hepatocytes.

The requirements for microsomal triglyceride transfer protein (MTP) during the turnover and transfer of glycerolipids from intracellular compartments into secretory very low-density lipoprotein (VLDL) were studied by pre-labelling lipids with [(3)H]glycerol and [(14)C]oleate in primary cultures of rat hepatocytes. The intracellular redistribution of pre-labelled glycerolipids was then compared at the end of subsequent chase periods during which the MTP inhibitor BMS-200150 was either present or absent in the medium. Inhibition of MTP resulted in a decreased output of VLDL triacylglycerol (TAG) and a delayed removal of labelled TAG from the cytosol and from the membranes of the smooth endoplasmic reticulum (SER), the cis- and the trans-Golgi. Inactivation of MTP did not decrease the bulk lipolytic turnover of cellular TAG as reflected by changes in its [(3)H]glycerol:[(14)C]oleate ratios. However, a larger proportion of the resultant TAG fatty acids was re-esterified and remained with the membranes of the various subcellular fractions rather than emerging as VLDL. The effects of BMS-200150 on the pattern of phospholipid (PL) mechanism and redistribution suggested that inhibition of MTP prevented the normal lipolytic transfer of PL-derived fatty acids out of the SER, cis- and trans-Golgi membrane pools. Finally, changes in the (14)C specific radioactivities of the cytosolic and membrane pools of TAG suggested that inhibition of MTP prevented a normal influx of relatively unlabelled fatty acids into these pools during the chase period.

Manipulation of cholesterol and cholesteryl ester synthesis has multiple effects on the metabolism of apolipoprotein B and the secretion of very-low-density lipoprotein by primary hepatocyte cultures.

Inhibition of esterified and non-esterified cholesterol synthesis by lovastatin in primary rat hepatocytes suppressed the net synthesis and very-low-density lipoprotein (VLDL) secretion of apolipoprotein B (apoB)-48 and apoB-100. Lovastatin did not alter the rates of apoB-48 and apoB-100 post-translational degradation. 25-Hydroxycholesterol, which inhibited non-esterified cholesterol synthesis but increased the synthesis of cholesteryl ester, showed differential effects on the metabolism of apoB-48 and apoB-100. Whereas the secretion of apoB-48 VLDL was suppressed there was no effect on the secretion of apoB-100 VLDL. The post-translational degradation of apoB-48, but not of apoB-100, was enhanced by 25-hydroxycholesterol. The net synthesis rates of apoB-48 and apoB-100 were unaffected by 25-hydroxycholesterol. The inhibitory effect of lovastatin alone on the net synthesis of apoB-48 and apoB-100 was reversed by the simultaneous presence of 25-hydroxycholesterol, suggesting a role for newly synthesised cholesteryl ester. Prevention of the reversal effect by the acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor YM 17E supported this interpretation. In the presence of lovastatin, restoration of the net synthesis of apoB by 25-hydroxycholesterol was not accompanied by an increased VLDL output of apoB-48 and apoB-100. However, under these conditions there was an increased post-translational degradation of apoB-48 and apoB-100. These results suggest that interference with intracellular cholesterol and cholesteryl ester metabolism interrupts VLDL assembly at sites of both apoB net synthesis and post-translational degradation.

Suppression of cytosolic triacylglycerol recruitment for very low density lipoprotein assembly by inactivation of microsomal triglyceride transfer protein results in a delayed removal of apoB-48 and apoB-100 from microsomal and Golgi membranes of primary rat hepatocytes.

Cellular apoB in primary rat hepatocyte cultures was pulse-labeled with [(35)S]methionine for 1 h. Cells were then chased with excess unlabeled methionine for periods of up to 16 h in the presence or absence of BMS-200150, an inhibitor of microsomal triglyceride transfer protein (MTP). The secretion of apoB-48-VLDL was more sensitive to MTP inhibition than was apoB-100-VLDL. Inhibition of MTP had no inhibitory effect on the secretion of denser particles (apoB-48 HDL and apoB-100 HDL). BMS-200150 delayed the net removal of newly synthesized apoB-48 and apoB-100 from the microsomal and Golgi membranes, but not from the corresponding lumenal compartments. Only minor proportions of the microsomal lumen apoB-48 and apoB-100 (12-16% and 17-19%, respectively) were present as VLDL irrespective of whether MTP was inactivated or not. The HDL fraction contained most of the lumenal apoB-48 (67-73%) and a somewhat smaller proportion of apoB-100 (44-47%). The remainder of the lumenal apoB was associated with the IDL/LDL fraction. These proportions were unaffected by MTP inactivation. Excess labeled apoB which accumulated in the membranes in the presence of BMS-200150 was degraded. Inhibition of MTP prevented the removal of pre-synthesized triacylglycerol (TAG) from the hepatocytes as apoB-VLDL. Under these conditions intracellular TAG accumulated mainly in the cell cytosol, but also, to a lesser extent, in the microsomal membranes. The results suggest that inactivation of MTP inhibits a pathway of VLDL assembly which does not involve the bulk lumenal compartments of the microsomes. Suppression of this pathway ultimately prevents the net transfer of cytosolic TAG into mature apoB-VLDL.