Molecular and metabolic basis for the metabolic disorder normotriglyceridemic abetalipoproteinemia.

We have previously described a disorder, normotriglyceridemic abetalipoproteinemia, that is characterized by the virtual absence of plasma low density lipoproteins and complete absence of apoB-100, but with apparently normal secretion of triglyceride-rich lipoproteins containing apoB-48. The patient's plasma lipoproteins were shown on polyacrylamide gels and by antibody mapping to have a new truncated apoB variant, apoB-50, circulating along with her apoB-48. We have found this individual to be homozygous for a single C-to-T nucleotide substitution at apoB codon 2252, which produces a premature in-frame stop codon. Thus, this is a rare example of homozygous hypobetalipoproteinemia. Electron photomicrographs revealed that the diameters of particles in the d less than 1.006 g/ml lipoprotein fraction, in both the postprandial and postabsorptive state, are bimodally distributed. The molar ratio of apoE to apoB in these particles is 3.5:1, similar to normal VLDL. The plasma LDL interval contains both spherical and cuboidal particles. Autologous reinfusion of labeled d less than 1.006 g/ml lipoproteins showed exponential disappearance from plasma, with an apparent half-removal time of 50 min, somewhat slower than for normal chylomicrons but within the normal range for VLDL. The calculated production rate for apoB was within the normal range in this subject. A very small amount of label was found briefly in the IDL fraction, but none at any time in LDL or HDL. Therefore, because LDL particles that contain apoB-50 lack the putative ligand domain of the LDL receptor, we conclude that the very low level of LDL is due to the rapid removal of the abnormal VLDL particles before their conversion to LDL can take place.

Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.

Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by single-strand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-->Cys mutation, caused by a C-->T transition at nucleotide 10800. One unrelated 59-yr-old man of Italian ancestry was found with the same mutation after screening 1,560 individuals. He had coronary heart disease, a TC of 310 mg/dl, and an LDL-C of 212 mg/dl. A total of eight individuals were found with the defect in the families of the two patients. They had an age- and sex-adjusted TC of 240 +/- 14 mg/dl and LDL-C of 169 +/- 10 mg/dl. This compares with eight unaffected family members with age- and sex-adjusted TC of 185 +/- 12 mg/dl and LDL-C of 124 +/- 12 mg/dl. In a dual-label fibroblast binding assay, LDL from the eight subjects with the mutation had an affinity for the LDL receptor that was 63% that of control LDL. LDL from eight unaffected family members had an affinity of 91%. By way of comparison, LDL from six patients heterozygous for the Arg3500-->Gln mutation had an affinity of 36%. The percentage mass ratio of the defective Cys3531 LDL to normal LDL was 59:41, as determined using the mAb MB19 and dynamic laser light scattering. Thus, the defective LDL had accumulated in the plasma of these patients. Using this mass ratio, it was calculated that the defective Cys3531 LDL particles bound with 27% of normal affinity. Deduced haplotypes using 10 apoB gene markers showed the Arg3531-->Cys alleles to be different in the two kindreds and indicates that the mutations arose independently. The Arg3531-->Cys mutation is the second reported cause of familial ligand-defective apoB.

Effects of hormones and pyruvate on the rates of secretion of very-low-density lipoprotein triacylglycerol and cholesterol by rat hepatocytes.

The secretion of very-low-density lipoprotein (VLDL) triacylglycerol and cholesterol was determined under various conditions in hepatocytes prepared from rats maintained on a controlled lighting and feeding schedule. The rate of lipogenesis in hepatocytes prepared from rats during the feeding period was 2-3-fold higher than that in cells prepared immediately before the animals had access to food. However, there were no corresponding changes in the rates of secretion of triacylglycerol and cholesterol. Pyruvate alone stimulated triacylglycerol secretion but had no effect on the secretion of cholesterol. Despite its stimulation of lipogenesis, insulin suppressed the secretion of both triacylglycerol and cholesterol. This effect on triacylglycerol secretion was more pronounced when lipogenesis was enhanced in the presence of pyruvate. Thus, insulin may act to alleviate hypertriglyceridaemia, which may arise during periods of increased hepatic lipogenesis. The inhibitory effect of glucagon on cholesterol secretion was much less pronounced than that on the secretion of triacylglycerol. The inhibitory effects of glucagon were reversed by pyruvate on cholesterol secretion differed according to whether glucagon was present or absent. These results suggest that the rate of hepatic VLDL triacylglycerol secretion is not necessarily coupled to the rate of lipogenesis in the liver; nor is there any obligatory coupling between the output of triacylglycerol and cholesterol associated with VLDL.

Effects of oleate and compactin on the metabolism and secretion of cholesterol and cholesteryl ester by rat hepatocytes.

Incubation of rat hepatocytes with oleate for a period of 1 h gave rise to a decrease in the total (esterified plus unesterified) cholesterol associated with very-low-density lipoprotein (VLDL). This effect was no longer apparent after longer incubation periods. The rate of cholesterol biosynthesis decreased during the first hour of incubation in the presence of oleate. After longer incubation periods, however, more cholesterol was synthesised in the presence of oleate than in its absence. The extracellular presence of oleate gave rise to a 2-fold increase in the concentration of cellular cholesteryl ester. Under these conditions cholesteryl ester contributed a larger proportion of the total cholesterol secreted with the VLDL. The cholesteryl ester associated with VLDL was derived predominantly from cholesteryl ester synthesised intracellularly. Inhibition of cholesterol synthesis with compactin did not significantly alter the rate of secretion of VLDL-cholesterol. Newly synthesised non-esterified cholesterol equilibrated with the bulk of pre-existing cellular cholesterol before secretion with the VLDL. This was true irrespective of the rate of endogenous cholesterol synthesis.

Metabolism of hydroxy sterols by rat liver.

5 alpha-[16-3H]lanost-8-ene-3 beta,15 alpha-diol and 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol were both extensively metabolised by rat liver enzymes in vitro. Quantitatively, the most important product in both cases was a more polar compound, tentatively identified as a 5 alpha-lanost-8-enetriol. In addition, 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol gave rise to the corresponding 3 beta,15beta-diol diester, whilst with 5 alpha-[16-3H]lanost-8-ene-3 beta,15 alpha-diol only the 3 beta-hydroxyl group was esterified. The enzymes involved may normally be responsible for metabolising spontaneously produced non-enzymic oxidation products of dietary or cellular cholesterol. High concentrations of 5 alpha-[16-3H]lanost-8-ene-3 beta,15 beta-diol stimulated ester formation. With both substrates, carbon monoxide inhibited formation of the polar sterol metabolite but stimulated ester formation. Under all conditions, cholesterol was a relatively minor metabolic product of either of the 5 alpha-lanost-8-ene-3 beta,15-diols.

Effect of drugs, peptide hormones and lipogenic precursors on the relative incorporation of [3H]H2O and carbon into hepatic cholesterol.

Measurement of the weight of desmosterol produced during its biosynthesis in the presence of tritiated water and triparanol has permitted a direct determination of the relative flux of carbon and tritium (the H/C ratio) into sterol in hepatocytes. The H/C ratio increased with time of incubation irrespective of the nutritional state of the donor animals. This increase was more marked in hepatocytes from starved animals. Pyruvate and lactate increased, and glucagon decreased, the sterol H/C ratio. Addition of pyruvate to incubations containing glucagon resulted in a 32-67% increase in the H/C ratio depending upon nutritional status. Insulin had no effect whilst (-)-hydroxycitrate decreased the ratio by 25%.

Elevated baseline triglyceride levels modulate effects of HMGCoA reductase inhibitors on plasma lipoproteins.

BACKGROUND: The response in levels of very-low-density (VLDL) and low-density (LDL) lipoproteins varies substantially among hyperlipidemic patients during treatment with HMGCoA reductase inhibitors. Apolipoprotein E genotype and gender are known to contribute to the regulation of steady state levels of plasma lipoproteins. This study explores the effect of these and other potential determinants of the response of VLDL and LDL to treatment with reductase inhibitors. METHODS: Using mixed linear statistical models, the response of lipoprotein lipid values was studied in 142 hyperlipidemic individuals who were treated with reductase inhibitors. Patients received one or more of the following drugs individually for a total of 623 treatment observations: lovastatin, pravastatin, simvastatin, or atorvastatin. For evaluation of the effects of treatment in the aggregate, actual doses were expressed as equivalent doses of atorvastatin, using factors based on random assignment comparisons in 16 reported studies. The analysis factors considered were apolipoprotein E genotype, baseline average triglycerides >170 mg/dL (vs less), and gender. RESULTS: Presence of an apo epsilon4 allele was associated with a trend toward greater reduction of triglyceride levels and a diminished ability of the reductase inhibitors to reduce LDL cholesterol levels. Gender had only minimal effect on the response of either LDL cholesterol or triglycerides. However, the effect of elevated baseline triglycerides on the response of both triglycerides and LDL cholesterol was striking and was exerted in opposite directions. The triglyceride-lowering effect of reductase inhibitors was greater in patients with initial triglyceride levels above 170 mg/dL (P=0.0001). The effect was even greater in patients with initial triglyceride levels over 250 mg/dL (P=0.015). Conversely, for LDL cholesterol levels, elevated baseline triglycerides were associated with a significantly decreased response to the drugs (P=0.0015). CONCLUSIONS: These findings indicate that baseline triglyceride levels are an important predictor of response of plasma lipoproteins to HMGCoA reductase inhibitors, perhaps reflecting fundamental differences in mechanism underlying the hyperlipidemic phenotype.

Association of endothelial lipase Thr111Ile polymorphism with proliferative retinopathy in type 2 diabetes patients.

AIM: Our previous study demonstrated that the endothelial lipase (EL) C.584C>T polymorphism (rs2000813, p.Thr111Ile) was significantly associated with diabetic retinopathy (DR). The present work was conducted to see if this specific variant of the EL gene was more specifically linked to the severity of DR. METHODS: This retrospective cohort study was based on a review of the institutional charts of 287 type 2 diabetes patients (mean age = 59.7 years; mean BMI = 29.0 kg/m(2); mean HbA1c=8.4%) genotyped for the EL C.584C>T polymorphism (rs2000813, p.Thr111Ile). The stage of DR was also determined for each genotype (CC, CT, TT). RESULTS: On univariate analysis, the minor allele homozygote TT variant was significantly associated with severe DR (OR: 4.3; 95% CI: 1.4, 13.1) compared with the major CC homozygote. No significant result was found for the CT heterozygote. Multivariate analysis revealed an increased risk for TT homozygotes to present with severe non-proliferative DR (OR: 8.09; 95% CI: 1.23, 53.1) or proliferative DR. Other associations were not significant. CONCLUSION: Minor allele homozygosity for this EL variant (c.584C>T) could be a significant risk factor for developing severe, sight-threatening disease due to proliferative DR. Further prospective studies of this EL polymorphism in a larger population sample are needed to confirm these results.

Association of endothelial lipase Thr111Ile polymorphism with lipid metabolism and microvascular complications in type 2 diabetic patients.

AIM: Endothelial lipase (EL) is a key enzyme in lipid metabolism, and a polymorphism in the EL gene may be a candidate for modulating lipid parameters in type 2 diabetic (T2D) patients. METHODS: In 396 T2D patients (age: 59.5 ± 10.7 years; BMI: 28.9 ± 5.3 kg/m(2); HbA(1c): 8.2 ± 1.9%), the c.584C>T polymorphism (rs2000813, p.Thr111Ile) was studied in 225 men (frequency of c.584T: 0.351) and 171 women (frequency of c.584T: 0.304). Patients' metabolic parameters, and macrovascular and microvascular complications, were assessed at baseline and at follow-up (mean: 4.2 years). RESULTS: Patients who were homozygous for the minor allele displayed modestly decreased low-density lipoprotein (LDL) cholesterol and raised apolipoprotein B at baseline, and raised systolic blood pressure and high-density lipoprotein (HDL) cholesterol on follow-up. Homozygosity for the minor allele was significantly associated with frequency of retinopathy (P=0.025), with TT homozygous patients more likely to have diabetic retinopathy (OR: 3.505; 95% CI: 1.491-8.239) both initially and at follow-up. CONCLUSION: The c.584C>T EL polymorphism is associated with a higher risk of diabetic retinopathy that could be linked to modifications in HDL-cholesterol metabolism and blood pressure levels.

The role of substrate supply in the regulation of cholesterol biosynthesis in rat hepatocytes.

1. Compactin, (-)-hydroxycitrate and dexamethasone gave rise to a decrease in the rate of cholesterol production in hepatocytes from fed rats by interfering with the flow of substrate into the sterol biosynthetic pathway. The cells responded to the deficit of biosynthetic sterol by increasing the activity of hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase). 2. Compactin and (-)-hydroxycitrate gave similar results in hepatocytes from rats starved for 24 h but in this case dexamethasone had no significant effect. 3. Exogenous oleate interferes with the production of carbohydrate-derived acetyl-CoA and also gives rise initially to opposing effects on the rate of sterol synthesis and HMG-CoA reductase activity. Over a longer period, however, oleate itself was capable of replacing carbohydrate as the major source of carbon for sterol synthesis. 4. The increase in HMG-CoA reductase activity observed when liver cells were incubated in the presence of compactin, (-)-hydroxycitrate or oleate could be partially reversed by the simultaneous presence of glucagon. 5. Under some physiological conditions, a deficiency of biosynthetic cholesterol or of a related precursor may lead to an increase in the activity of HMG-CoA reductase.

The relationship between the rate of hepatic sterol synthesis and the incorporation of [3H]water.

The true rate of sterol synthesis in liver cells was determined by measurement of the weight of desmosterol produced over a given time period during incubations in the presence of triparanol. The simultaneous presence of tritiated water (3H2O) during the incubations permitted a direct observation of the weight of tritium incorporated into a given mass of newly synthesized sterol. The incorporation of tritium per atom of sterol carbon (H/C ratio) was lower than some previously reported values and suggests that a sizeable proportion of the reducing equivalents (NADPH) required for sterol synthesis arises via the pentose phosphate pathway. The H/C ratio changed significantly with length of the incubation period. The value of the ratio was also dependent upon whether the acetyl-CoA units utilized for sterol synthesis were derived predominantly from a carbohydrate or a fatty acid source.

Utilization of endogenous and exogenous sources of substrate for cholesterol biosynthesis by isolated hepatocytes.

The rates of cholesterol biosynthesis in isolated rat hepatocytes were determined by using a method based on measurement of the rate of formation of desmosterol (cholesta-5,24-dien-3beta-ol), which accumulates during inhibition of cholesterogenesis by the drug triparanol. Incubation of cells from normal or 24h-starved animals in a medium containing albumin, glucose, amino acids and acetate as the only organic constituents led to an accelerating rate of sterol formation during the earlier stages of a 6h incubation period. The contribution of exogenously added acetate (initial concentration 3.34mm) to sterol synthesis in both types of cells reached an early maximum and then continually declined. Exogenously added pyruvate and lactate were more efficient sources of sterol carbon than was acetate. Exogenous glucose even at relatively high concentrations (11.1mm) was incapable of providing more than 6% of the total sterol carbon. Although the proportion of total sterol carbon supplied from exogenous acetate increased with increasing concentrations of the extracellular substrate, the rates of total sterol synthesis in both types of cell remained unchanged. Similar observations were made when lactate or pyruvate was the cholesterogenic precursor in normal cells. These studies suggest that, although exogenous substrates were capable of expanding an intracellular pool of cholesterol precursor, the normal supply of intermediary metabolites was not rate-limiting for cholesterogenesis.

Measurement of the absolute rates of cholesterol biosynthesis in isolated rat liver cells.

Triparanol [2-(4-chlorophenyl)-1-(4-diethylaminoethoxyphenyl)-1-p-tolylethanol] at a concentration of 2 micronm has no effect on the overall conversion of [2=14C]acetate into C27 sterols by isolated liver cells. In the presence of triparanol, however, the formation of radioactive cholesterol is inhibited by 85-90% and the balance of radioactivity appears in the C27 sterol desmosterol (cholesta-5,24-dien-3beta-ol). The very small weights of desmosterol which accumulate under these conditions were, as a routine, quantitatively converted into the heptafluorobutyrate 3-enol ester of cholesta-4,24-dien-3-one. This derivative has a high electron-capturing capability, a property that enables extremely small quantities (less than 0.25pmol) of the material to be accurately measured by gas chromatography with electron-capture detection. Measurements of the mass and specific radioactivity of the newly biosynthesized desmosterol formed in the presence of triparanol provides an accurate assessment of the amount of cholesterol that would be synthesized by the liver cells in the absence of the drug.

Regulation of hepatic very-low-density lipoprotein secretion in rats fed on a diet high in unsaturated fat.

Rats were fed ad libitum on either a standard, high-carbohydrate, chow diet or a similar diet supplemented with 15% unsaturated fat (corn oil). Hepatocytes were prepared either during the dark phase (D6-hepatocytes) or during the light phase (L2-hepatocytes) of the diurnal cycle. In hepatocytes from rats fed on the unsaturated-fat-containing diet, secretion of very-low-density lipoprotein (VLDL) triacylglycerol was inhibited to a greater extent in the D6- than in the L2-hepatocytes. Plasma non-esterified fatty acid concentrations were elevated to the same extent at both D6 and L2 in the unsaturated-fat-fed animals. The secretion of VLDL esterified and non-esterified cholesterol was relatively insensitive to changes in the unsaturated-fat content of the diet. This resulted in proportionate increases in the content of these lipid constituents compared with that of triacylglycerol in the nascent VLDL. There was also an increase in the ratio of esterified to non-esterified cholesterol in the nascent VLDL produced by hepatocytes of the unsaturated-fat-fed animals. In the D6-hepatocytes from the unsaturated-fat-fed animals, the decrease in the secretion of VLDL triacylglycerol could not be reversed by addition of exogenous oleate (0.7 mM) to the incubation medium. In contrast, addition of a mixture of lactate (10 mM) and pyruvate (1 mM) stimulated both fatty acid synthesis de novo and the rate of VLDL triacylglycerol secretion. Secretion of esterified and non-esterified cholesterol also increased under these conditions. Insulin suppressed the secretion of VLDL triacylglycerol and cholesteryl ester under a wide range of conditions in all types of hepatocyte preparations. Non-esterified cholesterol secretion was unaffected. In hepatocytes prepared from the fat-fed animals, these effects of insulin were more pronounced at D6 than at L2. Glucagon also inhibited VLDL lipid secretion in all types of hepatocyte preparations. The decrease in cholesterol secretion was due equally to decreases in the rates of secretion of both esterified and non-esterified cholesterol.

Diurnal variations in the effects of an unsaturated-fat-containing diet on fatty acid and cholesterol synthesis in rat hepatocytes.

Rats were fed ad libitum on either a standard high-carbohydrate diet, or a standard diet supplemented with 15% corn oil. Hepatocytes were prepared either during the light phase (L2-hepatocytes) or during the dark phase (D6-hepatocytes) of the diurnal cycle. In hepatocytes from rats fed on the fat-containing diet, fatty acid synthesis (lipogenesis) was suppressed to a much greater extent at D6 than at L2. The magnitude of the increase in plasma-free fatty acid concentration was similar at the two times of day. The rate of cholesterol synthesis was also significantly suppressed in the D6- but not in the L2-hepatocytes. This differential inhibition resulted in the abolition of the normal diurnal rhythm of cholesterogenesis. The initial activity of 3-hydroxy-3-methylglutaryl-CoA reductase in hepatocytes was also suppressed by corn-oil feeding at D6 but not at L2. In D6-hepatocytes, the inhibitory effect of the high-fat diet on the conversion of lactate into cholesterol and fatty acids was greater than that on total carbon flux into these substances for all endogenous sources. Despite this, under these conditions a high concentration of lactate and pyruvate resulted in a several-fold stimulation of total carbon flux into fatty acids. In hepatocytes prepared at L2, fat-feeding had little effect on the degree of stimulation of lipogenesis by insulin or inhibition by glucagon. However, at D6, fat-feeding blunted the response of lipogenesis to both these hormones.

Diurnal changes in the rate of cholesterogenesis in hepatocytes from fed and starved rats: effects of precursors and pancreatic hormones in vitro.

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) varied with a diurnal periodicity in hepatocytes prepared at different times from rats accustomed to a controlled feeding and lighting schedule. The rates of sterol synthesis varied in a similar manner but the maximum rate was not synchronous with maximum HMG-CoA reductase activity. The diurnal increase in HMG-CoA reductase activity and sterol synthesis rate started before food was offered to donor animals. Neither insulin nor glucagon had any effect on the diurnal pattern of hepatic sterol synthesis in vitro. Pyruvate inhibited sterol synthesis in hepatocytes prepared during the feeding period but had no effect at other times of day. When food was withheld from donor animals at the beginning of the normal feeding period both HMG-CoA reductase activity and the rate of sterol synthesis rapidly decreased. During this period neither insulin nor lipogenic substrates, alone or in combination, were able to restore the rates of sterol synthesis to normal values. In hepatocytes prepared from animals starved for a longer period (43 h) the decrease in the activity of HMG-CoA reductase was much less than that in the rate of sterol synthesis. In contrast to hepatocytes from fed or short-term-starved animals, the rate of sterol synthesis in these hepatocytes could be increased by glucose or pyruvate.

Changes in the sensitivity of lipogenesis in rat hepatocytes to hormones and precursors over the diurnal cycle and during longer-term starvation of donor animals.

In rat hepatocytes freshly isolated from donor rats at different times of the day, the rates of lipogenesis (de novo fatty acid synthesis) varied with a diurnal periodicity. The maximal rate occurred approximately 5 hr after the end of the normal 8-hr feeding period and at this time was four- to fivefold higher than the minimum rate which occurred midway through the feeding period. A similar diurnal pattern of change persisted even when the supply of lipogenic substrate, present in the medium as pyruvate, was not limiting. Although insulin stimulated the basal rates of lipogenesis to different relative extents in hepatocytes isolated at different times of the day, in absolute terms the hormone had little effect on the overall pattern of change during the diurnal cycle. The presence of pyruvate protected lipogenesis against inhibition by glucagon. The degree of protection varied over the diurnal cycle. During the early stages of starvation (up to 24 hr) there was a continuous decline in the rate of hepatocyte lipogenesis, irrespective of whether insulin and/or lipogenic substrate (pyruvate) were available or not. After this time the decline in the rate of lipogenesis was much less rapid. Seventeen hr after removal of food from donor rats, a point was reached beyond which pyruvate was incapable of supporting the maximum basal rate of lipogenesis which occurred during the normal diurnal cycle of fed rats. After this time lipogenesis in the presence of pyruvate was inhibited by glucagon to a much greater relative extent than that observed during feeding. The results suggest that variations in the rate of lipogenesis over the diurnal cycle and during the first 24 hr of starvation could not be accounted for entirely by fluctuations in substrate availability. In contrast, changes which occurred subsequent to this (up to 43 hr of starvation) could be eliminated when lipogenic substrate was made more abundant. Longer periods of starvation were marked by a relative increase in the ability of glucagon to prevent the substrate-induced stimulation of lipogenesis.

Evidence that changes in hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activity are required partly to maintain a constant rate of sterol synthesis.

The effects of insulin, glucagon, pyruvate, and lactate on the rate of sterol synthesis and 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity were determined in hepatocytes obtained at different times of the day from rats maintained on a controlled lighting and feeding schedule. In hepatocytes from animals killed immediately before the start of the feeding period (D0 hepatocytes), the initially low activity of HMG-CoA reductase increased during incubation while that in hepatocytes prepared 6 h later (D6 hepatocytes) remained constantly high. The rates of sterol synthesis followed similar patterns of change. In both D0 and D6 cells, insulin stimulated HMG-CoA reductase but had little or no effect on the rates of sterol synthesis. In both types of cell preparation glucagon maximally suppressed HMG-CoA reductase activity at a concentration of 10(-7) M, but there was relatively little change in the rates of sterol synthesis. Both pyruvate and lactate mitigated the glucagon-mediated inhibition of HMG-CoA reductase. Each of these lipogenic precursors alone suppressed the rate of sterol synthesis in a dose-dependent manner. These changes were more apparent in the simultaneous presence of insulin and were greater in the D0 compared to the D6 hepatocytes. In the presence of lactate or pyruvate, the activity of HMG-CoA reductase was elevated, and the increase was greater when insulin was simultaneously present. In general, changes in the rate of fatty acid synthesis were positively correlated with changes in the activity of HMG-CoA reductase. These observations suggest that the latter changes are required to compensate for variations in the availability of simple precursors for sterol synthesis.

The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects.

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.