Stable isotope turnover of apolipoproteins of high-density lipoproteins in humans.

Amino acid precursors labelled with stable isotopes have been successfully used to explore the metabolism of the apolipoproteins of HDL. Some methodological and mathematical modelling problems remain, mainly related to amino acid recycling in a plasma protein such as apolipoprotein A-I with a long residence time (the reciprocal of the fractional catabolic rate) of 4-5 days. Apolipoprotein A-I, apolipoprotein E, and apolipoprotein A-IV in triglyceride-rich lipoproteins (containing chylomicrons, VLDL, and remnants) exhibit more complex kinetics. The small amounts of apolipoprotein A-I and of apolipoprotein A-IV in the triglyceride-rich lipoproteins have a residence time similar to that of the apolipoprotein A-I of HDL. In contrast, the apolipoprotein E in triglyceride-rich lipoproteins has been found to have an average residence time of 0.11 days. Diets low in saturated fat and cholesterol, which lower HDL levels, do so by decreasing the secretion of apolipoprotein A-I, with apolipoprotein A-II kinetics unaffected. Individuals with impaired glucose tolerance have a decreased residence time of apolipoprotein A-I but no change in secretion rate or in apolipoprotein A-II kinetics. This suggests a link between insulin resistance and the risk of atherosclerosis. In heterozygous familial hypercholesterolemia, both the fractional catabolic rate and the secretion rate of apolipoprotein A-I are increased, resulting in no change in the plasma level. Stable isotope studies have strengthened the evidence that triglyceride enrichment of HDL increases its catabolism Laboratory.

A study of the metabolism of apolipoprotein B100 in relation to insulin resistance in African American males.

The purpose of this study was to determine the relationship between insulin resistance and apoB100 metabolism in African American males. Fifteen subjects, 33 +/- 7.6 years old, were divided into two groups, insulin-resistant (IR) or insulin-sensitive (IS), based on the sum of the plasma insulin concentrations during an oral glucose tolerance test. The IR group (n = 8) differed significantly from the IS group (n = 7) with respect to body mass index (BMI) (30.1 vs 23.1 kg/m2; P = 0.0003), fasting triglycerides, (118 vs 54 mg/dl, P = 0. 013), and total plasma apolipoprotein B100 (80 vs 59 mg/dl, P = 0.014). Significantly elevated apoB100 levels in the IR group were seen in very low density lipoprotein (VLDL) (5.1 vs 3.4 mg/dl, P = 0.045) and intermediate density lipoprotein (IDL) (18 vs 12 mg/dl, P = 0.017) but not in low density lipoprotein (LDL) (57 vs 46 mg/dl, P = 0.19). Total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein A-I, and blood pressure were not significantly different between the two groups. There was a high correlation between the sum of insulins during the oral glucose tolerance test and the BMI (rho = 0.88, P = 0.0001). In five IR and five IS subjects, apoB100 kinetics were determined in the fasting state using a bolus dose of deuteroleucine and multicompartmental modeling. IR subjects had significantly lower fractional catabolic rates (FCR) in the larger VLDL1 (-70%), the smaller VLDL2 (-71%), and the IDL (-53%) fractions. No significant differences in production rates were observed for any lipoprotein class. There was a significant correlation between the sum of insulins and the FCR of the apoB100 of VLDL1 (rho = -0.65, P = 0.05) and of IDL (rho = -0.85, P = 0.004). The correlation coefficient of the sum of insulins and the FCR of VLDL2 was -0.61 with P = 0.067. We conclude that in this population of African American males, IR is correlated with a decreased FCR of apoB100 in VLDL and IDL and elevated plasma levels of apoB and triglycerides (TG). These changes might be explained by decreased clearance of the TG-rich lipoproteins. We postulate that this may reflect decreased lipoprotein and/or hepatic lipase activity related to insulin resistance and its association with obesity.

Sex differences in African-Americans regarding sensitivity to insulin's glucoregulatory and antilipolytic actions.

OBJECTIVE: The purpose of this study was to determine if there are sex differences in African-Americans regarding the effect of obesity on sensitivity to insulin as a glucoregulatory and antilipolytic hormone. RESEARCH DESIGN AND METHODS: Data from study participants, 127 nondiabetic African-Americans (mean age 32 +/- 4 years), included anthropometric measurements, an oral glucose tolerance test (OGTT), a 2-h euglycemic-hyperinsulinemic clamp, and a fasting triglyceride level. Sensitivity to insulin as a glucoregulatory hormone was determined by M/FFM, where M is the mean glucose infusion rate during the second hour of the clamp and FFM is fat-free mass. Sensitivity to insulin's antilipolytic action was assessed during the OGTT by the percent suppression of free fatty acid (FFA) concentrations between 0 and 120 min. The higher the suppression of FFAs, the greater the sensitivity to insulin's antilipolytic action. RESULTS: The participants were classified by BMI into three groups: nonobese (31 men, 24 women), obese (17 men, 14 women), and severely obese (12 men, 29 women). The women had higher percentages of body fat (P < 0.001), and the men had greater FFM (P < 0.001). The M/FFM values for men versus women in each BMI group were nonobese, 8.8 +/- 2.8 vs. 10.8 +/- 4.4; obese, 7.2 +/- 3.4 vs. 8.5 +/- 3.4; and severely obese, 4.7 +/- 2.1 vs. 6.1 +/- 2.2. The difference between the BMI groups was significant (P < 0.001), as was the difference between men and women (P < 0.01). In addition, there was a significant sex difference in percent suppression of FFAS (P < 0.001). The men and women had similar fasting insulin and FFA concentrations; however, in the men only, the percent suppression of FFA declined with increasing obesity (nonobese, 83 +/- 15%; obese, 73 +/- 18%; and severely obese, 69 +/- 19%; P = 0.02). The women in all three BMI groups had lower FFA levels of 86-88%. CONCLUSIONS: Obese African-American men and women are resistant to insulin as a glucoregulatory hormone, but only obese men are resistant to insulin's antilipolytic action; obese African-American women are sensitive to insulin's antilipolytic action. The combined presence of sensitivity to insulin's antilipolytic action with resistance to insulin's glucoregulatory action in obese African-American women may contribute to their high prevalence of obesity and type 2 diabetes.

The full induction of human apoprotein A-I gene expression by the experimental nephrotic syndrome in transgenic mice depends on cis-acting elements in the proximal 256 base-pair promoter region and the trans-acting factor early growth response factor 1.

To identify molecular factors regulating apo A-I production in vivo, we induced in transgenic mice the experimental nephrotic syndrome, which results in elevated levels of HDL cholesterol (HDL-C), plasma apo A-I, and hepatic apo A-I mRNA. Human (h) apo A-I transgenic mice with different length 5' flanking sequences (5.5 or 0.256 kb, the core promoter for hepatic-specific basal expression) were injected with nephrotoxic (NTS) or control serum. With nephrosis, there were comparable (greater than twofold) increases in both lines of HDL-C, h-apo A-I, and hepatic h-apo A-I mRNA, suggesting that cis-acting elements regulating induced apo A-I gene expression were within its core promoter. Hepatic nuclear extracts from control and nephrotic mice footprinted the core promoter similarly, implying that the same elements regulated basal and induced expression. Hepatic mRNA levels for hepatocyte nuclear factor (HNF) 4 and early growth response factor (EGR) 1, trans-acting factors that bind to the core promoter, were measured: HNF4 mRNA was not affected, but that of EGR-1 was elevated approximately fivefold in the nephrotic group. EGR-1 knockout (EGR1-KO) mice or mice expressing EGR-1 were injected with either NTS or control serum. Levels of HDL-C, apo A-I, and hepatic apo A-I mRNA were lowest in nonnephrotic EGR1-KO mice and highest in nephrotic mice expressing EGR-1. Although in EGR1-KO mice HDL-C, apo A-I, and apo A-I mRNA levels also increased after NTS injection, they were approximately half of those in the nephrotic EGR-1-expressing mice. We conclude that in this model, basal and induced apo A-I gene expression in vivo are regulated by the trans-acting factor EGR-1 and require the same cis-acting elements in the core promoter.

The relationship in African-Americans of sex differences in insulin-mediated suppression of nonesterified fatty acids to sex differences in fasting triglyceride levels.

Insulin is a potent antilipolytic hormone that promotes the deposition of fat and decreases the release of nonesterified fatty acids (NEFA) from adipose tissue. The purpose of this study was to investigate in African-Americans (AAs) sex differences in insulin-mediated suppression of plasma NEFA and fasting triglyceride (TG) levels. Ninety AAs, 44 men and 46 women with a mean age of 34 +/- 8 years were classified by body mass index (BMI) into three groups: non-obese (22 men and 18 women), obese (12 men and 10 women), and severely obese (10 men and 18 women). In each BMI group, women versus men had greater percent body fat (non-obese, 30 +/- 6 v 18 +/- 6, P < .001; obese, 36 +/- 3 v 26 +/- 2, P < .001; and severely obese, 39 +/- 4 v 29 +/- 4, P < .001). An oral glucose tolerance test (OGTT) was performed with fasting TG levels and plasma insulin and NEFA concentrations obtained at 0, 30, 60, and 120 minutes. In women, insulin-mediated NEFA suppression was similar in each of the three BMI groups (non-obese, 85% +/- 14%; obese, 88% +/- 11%; and severely obese, 87% +/- 10%; P = .8). In men, the percent suppression of NEFA declined with increasing obesity (non-obese, 83% +/- 14%; obese, 71% +/- 21%; and severely obese, 68% +/- 16%; P = .04). Changes in NEFA suppression were reflected in the fasting TG levels. TG levels in women were similar in each BMI group (non-obese, 71 +/- 39 mg/dL; obese; 69 +/- 21; severely obese, 79 +/- 30; P = .7). In contrast, fasting TG levels for men were higher in the higher BMI groups. Plasma TG levels in men were 87 +/- 41 mg/dL for obese, 113 +/- 65 for obese, and 169 +/- 81 for severely obese (P = .001). These data demonstrate sex differences in insulin-mediated NEFA metabolism. In AA women, the maintenance of sensitivity to insulin-mediated suppression of NEFA regardless of the degree of obesity may contribute to the normal plasma TG levels. For AA men, the resistance to insulin-mediated suppression of NEFA in the higher BMI categories may allow more NEFA to be released from adipose tissue into the circulation and available to the liver for synthesis into TG-containing lipoproteins.

Lipoprotein metabolism in experimental nephrosis.

In experimental nephrosis, a decrease in plasma albumin resulting from proteinuria causes a decreased in the plasma oncotic pressure. The existence of an osmoreceptor, which responds to the low oncotic pressure and produces a factor(s) that signals the liver to increase the secretion of plasma proteins, is postulated. The hyperlipidemia characteristic of the nephrotic syndrome results primarily from increased hepatic secretion of apolipoproteins and lipoproteins representing the entire density spectrum from VLDL, IDL, and LDL to HDL. Not all plasma proteins and apolipoproteins are affected to the same extent. Increased mRNA levels due to increased transcription have been shown for albumin and apolipoprotein A-1 (apoA-1). The increased secretion of VLDL, the major vehicle for triglyceride transport from the liver, appears to be due mainly to posttranscriptional events possibly related to increased lipogenesis. Once proteinuria begins, the demand for amino acids for albumin and apolipoprotein synthesis by the liver is increased. To meet this demand, protein catabolism in the peripheral tissues is increased. One manifestation of this process is a decrease in lipoprotein lipase which reduces VLDL catabolism, contributing to the sustained elevation of plasma VLDL. The spectacular overproduction of apoA-1 in nephrosis in the rat is accompanied by a decreased fractional catabolic rate (FCR), contributing to the maintenance of high levels of HDL. Urinary loss of HDL and its renal catabolism does not account for the decreased FCR. The reason for the decreased FCR is not known. Work with nephrotic rats overexpressing transgenic human apoA-1 has shown that human A-1 forms smaller HDL3-sized particles, rather than the larger HDL2 of the rat. This may contribute to the failure of HDL levels to increase in the human nephrotic syndrome. High plasma VLDL and LDL with normal or low HDL probably account for the increased incidence of coronary artery disease in the nephrotic syndrome.

Effect of experimental nephrosis on hepatic lipoprotein secretion and urinary lipoprotein excretion in rats expressing the human apolipoprotein A-I gene.

When human apolipoprotein A-I was expressed in transgenic rats, induction of the nephrotic syndrome resulted in plasma A-I levels exceeding 10 mg/ml. Plasma lipids were no higher than in non-transgenic nephrotic rats. To explain this, the livers from four groups of rats were perfused: wild-type controls (WC), high expressor human apoA-I transgenic controls (TrGC), wild-type nephrotics (WN), and high expressor transgenic nephrotics (TrGN). Compared to the WC group, TrGC rats secreted the same amount of d < 1.063 g/ml lipoproteins but 50% more high density lipoprotein (HDL), with a 5-fold increase in total apoA-I output due to human apoA-I. Compared to the WC group, nephrosis in the WN rats caused a 2-fold increase in both d < 1.063 g/ml lipoproteins and HDL secretion with a 4.6-fold increase in rat apoA-I output. Compared to the TrGC group, nephrosis in the TrGN rats did not increase d < 1.063 g/ml lipoprotein secretion, but caused a 50% increase in HDL secretion and a 6-fold increase in human apoA-I output. The hepatic levels of mRNA for apoB and for HMG-CoA reductase, as well as the degree of apoB mRNA editing, were unchanged. Examination of the perfusate HDL by electron microscopy revealed spherical particles averaging 30 nm in diameter in the WC and WN rats and 17 and 20 nm in the TrGC and TrGN rats. Urinary HDL particles from the TrGN rats did not contain rat apoA-I and averaged 8.2 nm versus 11 nm in the WN rats. We conclude that the size of the nascent HDL, and subsequently of the mature HDL, is determined by the primary structure of apoA-I. In the TrGN rats, the heterogeneous mature HDL has a population of smaller human HDL which is more readily lost in the urine, accounting for the failure of plasma HDL levels to rise above those in TrGC rats. The fact that plasma triglyceride levels in TrGN rats were also not increased may relate to the failure of hepatic apoB secretion to increase, which in turn may have been due to saturation of the protein synthetic capacity by human apoA-I production.

Gender differences in insulin-induced free fatty acid suppression: studies in an African American population.

This study of African Americans (AA) was designed to investigate gender differences in insulin-induced free fatty acid (FFA) suppression. Sixty AA (34 women, mean age 34 +/- 7.6 years, and 26 men, mean age 30 +/- 2.9 years) participated. All subjects had an oral glucose tolerance test (OGTT). Nineteen women and 18 men also underwent a euglycemic hyperinsulinemic clamp (IC) study. Plasma insulin and FFA concentrations were obtained during both tests at 0, 60, and 120 min. While there was no gender difference in body mass index (P = 0.21), women had greater percent body fat (P < 0.001) calculated by the Siri formula. There was no gender difference in fasting FFA levels, but during the OGTT, women compared to men had significantly greater FFA suppression. Both nonobese and obese women suppressed FFA concentration by 88%, and nonobese and obese men suppressed FFA concentration by 80 and 66% respectively. This gender difference in FFA suppression was significant (P = 0.001) and independent of obesity and insulin concentration. During the IC studies, there were no gender or obesity differences in FFA suppression, with women and men suppressing FFA levels by 87-89% (P = 0.7). Fasting insulin concentrations were higher in obese vs. nonobese (P = 0.03), but fasting FFA concentrations were not different (P = 0.15). For nonobese and obese females, fasting FFA levels were 0.55 +/- 0.24 and 0.44 +/- 0.26 mEq/L, respectively, and for nonobese and obese males, 0.45 +/- 0.2 and 0.35 +/- 0.18 mEq/L, respectively. In women, development of obesity may be enhanced by greater sensitivity to insulin-induced FFA suppression as measured during an OGTT. To detect gender differences in FFA metabolism, the OGTT is superior to the IC. The lack of elevation in fasting FFA levels in obese AA women and men has not been reported in other racial groups and may indicate a greater adipocyte sensitivity to insulin in AA.

Insulin sensitivity, lipids, and blood pressure in young American blacks.

The purpose of this study was to determine whether insulin resistance was linked with alterations in plasma lipids in adult young blacks with borderline hypertension. Ninety-four American blacks participated (46 men, 48 women, age range 28 to 33 years). Within this group of 94 subjects, there were 60 normotensive (Nt) subjects and 36 subjects with borderline hypertension (BHt), defined as blood pressure > 135/85 mm Hg. None of the subjects were diabetic or receiving antihypertension medication. All participants had blood pressure and anthropometric measurements, a fasting lipid profile, an oral glucose tolerance test, and a euglycemic hyper-insulinemic clamp. Insulin-stimulated glucose utilization (M), determined by insulin clamp, was significantly lower in the BHt subjects compared with the Nt subjects (men, Nt 6.91 +/- 0.62 versus BHt 5.54 +/- 0.65; women, Nt 5.97 +/- 0.47 versus BHt 3.79 +/- 0.38 mg.kg-1.min-1, P = .006). When M was corrected for adiposity and expressed in milligrams per kilogram of fat free mass (M'), the difference between Nt and BHt remained significant (P = .006). There was a significant correlation of M' with systolic blood pressure (r = .393, P < .0001), HDL-C (r = .382, P < .0001), triglyceride level (r = 308, P < .001), apolipoprotein A-I (r = .190, P = .033), and apolipoprotein B stepwise multiple linear regression analysis, HDL-C emerged as the most significant lipid component in the model for insulin resistance. These data suggest that in American blacks with mild hypertension, the risk for cardiovascular disease may be augmented in the presence of insulin resistance.

Overexpression of human apolipoprotein A-I in transgenic rats and the hyperlipoproteinemia associated with experimental nephrosis.

Hyperlipoproteinemia contributes both to kidney disease progression and the development of atherosclerosis. Elevated high density lipoprotein cholesterol and apolipoprotein A-I (apoA-I) serum levels are independent factors protective against the atherosclerotic process. We examined the effects in a transgenic rat model of human apoA-I expression on the hyperlipoproteinemia and edema after puromycin aminonucleoside-induced nephrosis in three groups of animals: low line (TgR[hAI]low, human plasma apoA-I = 16.0 mg/dl); high line (TgR[hAI]high, 284 mg/dl); and non-transgenic litter mates (TgR[hAI]non). Nephrosis increased total plasma apoA-I levels 2-fold in TgR[hAI]non rats (75 vs. 162 mg/dl) and 4-fold in the TgR[hAI]low (97 vs. 458 mg/dl) and TgR[hAI]high rats (356 vs. 1,346 mg/dl). In both transgenic lines, this increase was due mainly to elevations of serum human apoA-I. The hepatic steady-state levels of rat apoA-I mRNA increased 5- to 7-fold in all three groups, while human apoA-I mRNA levels increased 21- and 65-fold in the low and high expressing groups, respectively, indicating a different degree of responsiveness of the rat and human genes. While nephrotic TgR[hAI]non and TgR[hAI]low rats showed severe hyperlipoproteinemia and edema, much lower levels of edema and of serum triglycerides, phospholipids, and cholesterol were seen in the TgR[hAI]high group. Urinary excretion of apoA-I, phospholipids, and cholesterol was significantly increased in the TgR[hAI]high group, indicating this as one possible mechanism for the relatively lower serum levels of these lipids. We conclude that the human apoA-I gene is responsive to nephrosis and that human apoA-I-transgenic rats with this syndrome provide an animal model for the study of human high density lipoprotein and apoA-I metabolism.

Kinetic evidence for both a fast and a slow secretory pathway for apolipoprotein A-I in humans.

The kinetics of apolipoproteins A-I and A-II were examined in human subjects using leucine tracers administered intravenously. High density lipoproteins were separated and apoA-I and A-II were isolated. The specific activity or enrichment data for these apolipoprotein were analyzed by mathematical compartmental modeling. In 11 of 14 subjects studied with a bolus-injected [3H]leucine tracer, in 3 subjects studied similarly with [3H]leucine, and in one subject studied by primed dose, constant infusion of [3H]leucine, a rapidly turning-over apoA-I fraction was resolved. A similar component was observed in 7 of 10 studies of apoA-II. The apoA-I data were analyzed using a compartmental model (Zech, L.A. et al. 1983. J. Lipid Res. 24: 60-71) modified to incorporate plasma leucine as a precursor for apoprotein synthesis. The data permitted resolution of two apoA-I pools, one, C(2), turned-over with a residence time of less than 1 day, the other, C(1), a slowly turning-over pool, appeared in plasma after a delay of less than half a day. C(1) comprised the predominant mass of apoA-I and was also the primary determinant of the residence time of apoA-I. Although the mass of the fast pool, C(2), was considerably less than that of C(1), because of its rapid turnover, the quantities of apoA-I transported through this fast pathway were 2- to 4-fold greater. These kinetic studies indicate that apoA-I is secreted into both fast and slowly turning-over plasma pools. The latter is predominantly measured with radioiodinated apoA-I tracers. The data can be analyzed by postulating either separate input pathways to each of the pools or by assuming the fast pool is the precursor to the slow pool. Thus, apoA-I could be initially secreted as a family of particles that are rapidly cleared from plasma, and a portion of this apoprotein then reappears in a slowly turning-over pool that constitutes the major mass of apoA-I. The physiologic identity of these kinetically distinct apoA-I species is unknown; however, the fast pool of apoA-I demonstrated in these studies is strikingly similar to that seen in subjects with Tangier disease who lack the slow pool.

Characterization of a bile salt-dependent cholesteryl ester hydrolase activity secreted from HepG2 cells.

HepG2 cells and medium were assayed for cholesteryl ester hydrolase (CEH) activity in the presence and absence of sodium cholate. Although bile salt-dependent CEH activity was measured in the medium at 6 to 96 h (up to 4500 pmol/h per mg cell protein), there was very little activity detected in the corresponding cell homogenates (less than 70 pmol/h per mg cell protein). Activity in the medium was expressed only in the presence of trihydroxy bile salts and was maximal at 40 mM cholate and pH 7.5. Incubation of HepG2 cells with brefeldin A resulted in an 80 to 90% inhibition of secretion of the bile salt-dependent CEH activity, while only inhibiting total protein secretion by 42%. Bile salt-dependent CEH activity could also be detected in rat liver perfusates. Although there was measurable activity in all of 14 livers analyzed (47 +/- 10 and 53 +/- 17 nmol/h per g liver per h perfusion during two 5-min collections after 15 and 30 min of perfusion, respectively), it did not correlate with the activity found in corresponding liver homogenates, as only four livers had detectable bile salt-dependent CEH activity. These results provide evidence for the secretion of a bile salt-dependent CEH activity, from both a hepatic cell line and the intact liver, that has similar properties to the enzyme previously isolated from rat liver homogenates and rat pancreas.

Use of [15N]glycine in the measurement of apolipoprotein B synthesis in perfused rat liver.

Rat livers were perfused with [15N]glycine and unlabeled sodium benzoate by the single-pass technique via the portal vein or in retrograde fashion via the inferior vena cava. Perfusate [15N]hippurate enrichment was significantly greater than that of hepatic free glycine from 15 to 90 min, regardless of the direction of the perfusion. This result implies that differential labeling by periportal versus perivenous hepatocytes is not likely. When fasted animals were compared to those fed a chow diet or a sucrose-enriched diet, the labeling ratio of medium hippurate/hepatic free glycine decreased by only 9% in spite of a 5-fold decrease in the concentration of intrahepatic free glycine. Administration of nembutal to the intact animal significantly increased the enrichment of medium hippurate by 24% but did not affect the enrichment of the hepatic free glycine. We conclude that the difference between hippurate and free glycine enrichment is related to intracellular compartmentation of glycine transport. We suggest that measurement of the enrichment of hippurate after the administration of [15N]glycine with benzoate in intact animals or human subjects can therefore be used to estimate the enrichment of the intracellular precursor pool of glycine with a correction factor that does not vary appreciably under fed or fasted conditions. When uniformly labeled deuteroglycine was used as the tracer, enrichment of hepatic free glycine was decreased fivefold compared with [15N]glycine. Isotopic enrichments of apoBH and apoBL from the d less than 1.063 g/ml lipoprotein fraction isolated from the perfusion medium between 30 and 90 min averaged 3.7 and 4.1% excess, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of fish oil, safflower oil and palm oil on fatty acid oxidation and glycerolipid synthesis in rat liver.

Studies were conducted to explore the mechanisms by which dietary fish oil decreases hepatic triglyceride secretion. Forty-five rats (15/group) were fed purified diets containing 10% fat as either fish oil, safflower oil or palm oil for 10 d. Plasma triglyceride concentration was lowest in the fish oil-fed group followed by the groups fed safflower oil and palm oil. The liver's capacity to oxidize fatty acids was assessed by assays of mitochondrial and peroxisomal beta-oxidation pathways in whole homogenates. Additionally, key enzymatic activities in the biosynthesis of triglyceride (diacylglycerol acyltransferase, phosphatidate hydrolysis) and phosphatidylcholine (CTP:phosphocholine cytidylyltransferase) were assayed. Compared with those fed palm oil the fish oil-fed animals showed 25% greater mitochondrial beta-oxidation but this difference was not statistically significant (P = 0.1). Fish oil feeding led to 45% greater (P less than 0.05) peroxisomal beta-oxidation. Diacylglycerol acyltransferase activity was unaffected by the type of dietary fat and slightly (13%) but significantly (P less than 0.02) lower cytidylyltransferase activity due to fish oil feeding was observed. More strikingly, both fish oil and safflower oil diets significantly lowered phosphatidate hydrolysis by 37 and 22%, respectively, compared with the palm oil diet. This activity directly correlated (r = 0.68; P less than 0.001) with plasma triglyceride concentration. Thus, dietary fish oil might suppress triglyceride secretion by decreasing glycerolipid synthesis, an effect mediated by changes in one or more enzymes involved in phosphatidate catabolism.

Macrophages from nephrotic rats regulate apolipoprotein E biosynthesis and cholesterol content independently.

The effects of the nephrotic syndrome in rats on the cholesterol content and the biosynthesis of apolipoprotein E (apoE) by resident peritoneal macrophages have been investigated. Since the nephrotic syndrome has been associated with an increased risk of coronary atherosclerosis, we hypothesized that macrophages from nephrotic rats would accumulate cholesterol and undergo transformation into foam cells, with a concomitant increase in apoE biosynthesis. The nephrotic syndrome was induced in rats with puromycin aminonucleoside. Peritoneal macrophages exposed in vivo for 7-21 d to ascites fluid derived from plasma containing sixfold elevations of lipoproteins did not accumulate unesterified or esterified cholesterol. Nevertheless, immunoprecipitation assays after incubation of the isolated cells with [35S]methionine, or immunoblot analysis of the incubation medium demonstrated a 2.6-fold increase in apoE secretion compared with normal macrophages. This increase was accompanied by 5- to 10-fold increases in cellular apoE messenger RNA as determined by quantitative solution hybridization assay. Peritoneal macrophages cultured from nephrotic rats during the period of hypercholesterolemia also showed distinct and highly reproducible morphologic changes. The dissociation between apoE biosynthesis and macrophage cholesterol content provides new insight into the response of peritoneal macrophages in vivo to endogenous hyperlipemia.

Isolation of nascent high-density lipoprotein from rat liver perfusates by immunoaffinity chromatography: effects of oleic acid infusion.

Immunoaffinity chromatography on a column of rabbit IgG anti-rat apolipoprotein (apo) A-I covalently bonded to agarose was used to isolate nascent high-density lipoprotein (nHDL) from recirculated perfusates of rat livers. After passage through the affinity column, the bound material was eluted with sodium thiocyanate and analyzed for apolipoproteins and lipids. The protein content was 52% and the lipid composition was 37% triglyceride, 40% phospholipid, and 23% cholesterol. Apolipoproteins E and A-I each comprised approximately one third of the total, and very little apo B was detectable as judged by SDS-PAGE analysis. The affinity-isolated particles were therefore similar in composition to the major apo A-I:apo E-rich subfraction of nHDL isolated by ultracentrifugation in earlier work. It is concluded that the apo E in this class of nHDL (containing both apo E and apo A-I) is present in the secreted particle and is not a consequence of a loss of apo E from very-low-density lipoprotein (VLDL) during ultracentrifugation. The high triglyceride content in the virtual absence of apo B confirms and extends previous analyses and reinforces the conclusion that nHDL particles are enriched in triglyceride compared to plasma HDL. The inclusion of 4% albumin in the perfusion medium did not significantly change the total triglyceride output of 115 micrograms/g liver/h, but it decreased the triglyceride output isolated by anti-apo A-I affinity chromatography from 3.2 to 0.48 micrograms/g liver/h. The addition of oleic acid complexed to albumin increased the total triglyceride output by 70% and that associated with the immunoaffinity column increased from 0.48 to 2.7 micrograms/g liver/h.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of (C13)arginine incorporation into apolipoprotein B-100 in very low density lipoproteins and low density lipoproteins in normal subjects using (13C)sodium bicarbonate infusion and isotope ratio mass spectrometry.

We describe a new stable isotope technique for the in vivo study of hepatic plasma protein synthesis in humans. The method involves the infusion of (13C)sodium bicarbonate for 1 h and the measurement of the isotopic enrichment of (13C)arginine in newly synthesized apolipoprotein B of very low density lipoproteins (VLDL-apoB) and low density lipoproteins (LDL-apoB) in blood samples taken over a 5-6 h period from the commencement of the infusion. Isotope ratio mass spectrometry was utilized to measure 13CO2 enrichment following hydrolysis of these proteins and conversion of the guanidinium carbon of arginine in the hydrolysate to carbon dioxide by sequential incubation with arginase and urease. The method is capable of measuring isotopic enrichment as low as 0.001 at.% excess (APE) with a precision of 1.2%. In both subjects studied, the (13C)arginine of VLDL-apoB reached enrichments of 0.2 APE and that of the arginine of LDL-apoB, 0.03 APE. Incorporation of labeled arginine into LDL-apoB was demonstrable at 60-90 min. The new technique is safe and is applicable to the study of the hepatic biosynthesis of a wide range of plasma proteins.

Effects of eicosapentaenoic and docosahexaenoic acids on apoprotein B mRNA and secretion of very low density lipoprotein in HepG2 cells.

Oleic acid (18:1n-9, OA), docosahexaenoic acid (22:6n-3, DHA), or eicosapentaenoic acid (20:5n-3, EPA) was added to HepG2 cells at a concentration of 1 mM in a 5:1 or 2:1 molar complex with bovine serum albumin (BSA), and this was incubated for 3 hours. The incorporation of 3H-glycerol into cellular and medium triglyceride (TG), and the mass of TG were measured. The effects of these fatty acids on the secretion of very low density lipoprotein (VLDL) apolipoprotein B (apo B) were estimated from the incorporation of 3H-leucine into the medium apo B in comparison to cells incubated with fatty acid-poor albumin. The secretion of human albumin by the cells was also estimated by immunochemical precipitation of the labeled albumin. In addition, the intracellular levels of apo B messenger ribonucleic acid (mRNA) were measured by the dot-blot hybridization technique. Relative to control cells incubated with BSA, OA (complexed to BSA at a 5:1 molar ratio) stimulated TG synthesis and secretion sevenfold. Compared to OA, EPA was 24% less effective for both processes, whereas DHA inhibited only the secretion of TG (-43%). The secretion of VLDL apo B was not affected by OA, but was decreased 31% by EPA and 54% by DHA. When the molar ratio of fatty acid complexed to albumin was changed to 2:1, similar results were obtained with respect to TG production. The levels of apo B mRNA relative to actin mRNA were not significantly altered by any of the fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of nascent high density lipoprotein subfractions from perfusates of rat liver.

Nascent high density lipoprotein (HDL) (1.063 less than d less than 1.21 g/ml) was isolated from recirculating rat liver perfusates and separated by heparin-Sepharose chromatography into a nonretained fraction (NR) and a fraction (R) that eluted with 0.5 M NaCl. Fractions NR and R contained 70% and 30% of the nascent HDL protein, respectively. ApoB-containing particles were removed from fraction R by chromatography on concanavalin A. The protein composition of fractions NR and R was 40% and 29%, respectively. Fraction NR contained 25% apoA-I, 11% apoA-IV, 24% apoE, and 38% apoC. Fraction R contained primarily apoE (81% of total protein). The lipid composition of NR and R, respectively, was: triglyceride 44% and 26%, phospholipid 41% and 57%, cholesterol 8% and 13%, and cholesteryl ester 7% and 4%. Fractions NR and R had molecular weights of 400,000 and 860,000, respectively, as calculated from the Stokes radius. Negative staining electron microscopy indicated that both fractions consisted mainly of spherical particles (260-280 A) but some stacked disks were seen in fraction R. Livers perfused by the single-pass technique produced fractions NR and R in the same ratio as livers perfused by recirculation. The apolipoprotein compositions were similar to those in the recirculating perfusion; however, both fractions NR and R had more triglyceride (greater than 50% of total lipid). An HDL fraction was also isolated from liver perfusates by a combination of molecular sieve and heparin-Sepharose affinity chromatography. This HDL contained triglyceride but no apoB, indicating that triglyceride-rich HDL particles are not an artifact of ultracentrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of triglyceride-rich nascent rat hepatic high density lipoproteins.

Nascent high density lipoprotein (HDL) and nascent very low density lipoprotein (VLDL) were isolated from rat livers that had been perfused with [3H]glycerol to label the triglyceride. When injected into intact rats, the labeled HDL-triglyceride disappeared as rapidly as the VLDL-triglyceride, with only 10% of the injected label remaining in the plasma after 30 min. The protein moiety of nascent HDL was labeled with [35S]methionine in a similar fashion and the labeled nascent HDL was separated into nonretained (NR) and retained (R) fractions by heparin-Sepharose affinity chromatography. When injected into rats, 55% of the injected label in nascent fraction NR and 72% of that in nascent fraction R was recovered from plasma at 30 min, compared to only 10% of the triglyceride label from unfractionated nascent HDL, indicating dissociation of triglyceride and apolipoprotein clearance. The plasma decay curves for both triglyceride and protein were biexponential. By 5 min, 15% of the 35S label remaining in plasma represented apoE and apoC that had been transferred from nascent HDL fractions NR and R to the d less than 1.063 g/ml fraction of plasma. Plasma HDL was labeled in vivo with [35S]methionine, separated into fractions NR and R, and the clearance of the two plasma HDL fractions was compared with that of the corresponding nascent HDL fractions. Except for a faster rate of removal of the nascent HDL fractions during the first 5 min, the serum decay curves were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)