Does pravastatin increase chylomicron remnant catabolism in postmenopausal women with type 2 diabetes mellitus?

OBJECTIVE: We investigated the effects of pravastatin on chylomicron remnant catabolism measured with a 13C stable isotope breath test and plasma apolipoprotein (apo) B-48 and remnant-like particle (RLP)-cholesterol in postmenopausal women with type 2 diabetes mellitus. PATIENTS AND MEASUREMENTS: Nineteen postmenopausal women with type 2 diabetes were randomized to receive 40 mg/day pravastatin or no treatment for 6 weeks followed by a 2-week washout period, and crossed over for a further 6 weeks. Fractional catabolic rate (FCR) of a chylomicron remnant-like emulsion was determined from 13CO2 enrichment in the breath and plasma using isotope-ratio mass spectrometry and multicompartmental modelling. Plasma apo B-48 and RLP-cholesterol concentrations were also measured as static markers of chylomicron remnant metabolism. RESULTS: Pravastatin significantly reduced plasma concentrations of cholesterol (5.9 +/- 0.3 vs. 4.8 +/- 0.2 mmol/l; P < 0.001), low density lipoprotein (LDL)-cholesterol (3.5 +/- 0.2 vs. 2.6 +/- 0.2 mmol/l; P < 0.001), triglyceride (2.1 +/- 0.3 vs. 1.7 +/- 0.2 mmol/l; P = 0.017), non-high density lipoprotein (HDL)-cholesterol (4.4 +/- 0.3 vs. 3.3 +/- 0.2 mmol/l; P < 0.001), lathosterol/total cholesterol ratio (2.6 +/- 0.2 vs. 2.0 +/- 0.3, P = 0.035), apo B-100 (1.1 +/- 0.1 vs. 0.8 +/- 0.1 g/l; P = 0.001), apo B-48 (4.8 +/- 0.9 vs. 3.3 +/- 0.6 mg/l; P = 0.016), and RLP-cholesterol (31.4 +/- 8.2 vs. 18.6 +/- 4.6 mg/dl; P = 0.024). Pravastatin was also associated with an increase in sitosterol/total cholesterol ratio (2.8 +/- 0.3 vs. 3.1 +/- 0.3, P = 0.029). Chylomicron remnant-like emulsion catabolism was not, however, significantly altered by pravastatin estimated by either breath or plasma clearance measurements. CONCLUSIONS: In postmenopausal women, pravastatin decreases plasma concentrations of remnant lipoproteins by a mechanism that may relate chiefly to inhibition of remnant production, but this requires further evaluation.

Chylomicron metabolism.

Chylomicrons are the 'orphans' of the lipoprotein family. Difficulty of measurement has impeded understanding of their metabolism. Plasma concentrations of chylomicrons and chylomicron remnants give no insight into the magnitude of substrate flux through these pathways. A defect in clearance of chylomicron remnants is probably an indication of a more generalized defect in lipoprotein metabolism. Accumulating evidence supports a relationship between abnormalities in the clearance from plasma of chylomicron remnants and accelerated progression of atherosclerosis. Methods using stable isotopes in appropriately formulated emulsions are providing valuable new information.

Chylomicron remnant metabolism studied with a new breath test in postmenopausal women with and without type 2 diabetes mellitus.

OBJECTIVES: The kinetic basis for the effect of type 2 diabetes mellitus (DM) on postprandial lipoproteins has not been fully established. We investigated chylomicron remnant metabolism using a stable isotope breath test and fasting measurements of plasma apolipoprotein (apo) B-48 and apoC-III concentrations in postmenopausal women with and without type 2 DM. PATIENTS: Twenty-four postmenopausal women without DM and 14 postmenopausal women with diet-controlled DM of similar age and body mass index (BMI) were studied in the postabsorptive state. METHODS: The fractional catabolic rate (FCR) of an intravenously injected chylomicron remnant-like emulsion was determined from the appearance of 13CO2 in the breath using isotope-ratio mass spectrometry and multicompartmental modelling. apoB-48, a marker of particle number of intestinal lipoproteins, was determined immunoelectrophoretically. apoC-III was measured by immunoturbidimetric assay. RESULTS: Compared with the nondiabetic women, the women with DM had significantly higher plasma apoB-48 concentration (16.40 +/- 1.18 mg/l vs. 13.0 +/- 0.9 mg/l; mean +/- standard error mean; P = 0.021), higher plasma apoC-III concentration (204.24 +/- 15.18 mg/l vs. 170.74 +/- 10.75 mg/l; P = 0.042) and lower FCR of the chylomicron remnant-like emulsion (0.06 +/- 0.05 pools/h vs. 0.12 +/- 0.02 pools/h; P < 0.001). In the diabetic patients, the FCR of the emulsion was correlated significantly with plasma apoB-48 levels (r = -0.641, P = 0.007) but not with apoC-III levels. CONCLUSIONS: In postmenopausal women, diabetes mellitus appears to decrease the catabolism of chylomicron remnants and result in an accumulation of these particles in plasma. This may chiefly be due to decreased clearance by hepatic receptors related to an effect of insulin resistance. Impairment in the catabolism of chylomicron remnants may contribute to increased risk of atherosclerosis in postmenopausal women with type 2 diabetes mellitus.

Effect of Simvastatin on markers of triglyceride-rich lipoproteins in familial hypercholesterolaemia.

BACKGROUND: We have previously shown elevated fasting plasma concentrations of intestinal remnants, as reflected by apolipoprotein (apo) B-48 and remnant-like particle-cholesterol (RLP-C) in patients with heterozygous familial hypercholesterolaemia (FH). We now investigate the effect of an HMG-CoA reductase inhibitor (simvastatin) on chylomicron remnant metabolism using the measurement of fasting apoB-48 and RLP-C in FH patients after long- and short-term simvastatin therapy and after a wash-out period. We also piloted the response of a breath test, involving the measurement of the fractional catabolic rate (FCR) of an intravenously injected chylomicron remnant-like emulsion labeled with cholesteryl (13)C-oleate. METHODS: Fifteen FH patients were studied after > 6 months 40 mg day(-1) simvastatin treatment (long-term), a wash-out period (4 weeks), and 4 weeks of simvastatin treatment (short-term). Apolipoprotein B-48 was determined by SDS-PAGE and Western blotting/enhanced chemiluminescence and RLP-C by an immunoseparation assay. The FCR of the chylomicron remnant-like emulsion was determined from the appearance of (13)CO(2) in the breath and by multicompartmental mathematical modelling. RESULTS: Both long- and short-term treatment with simvastatin were associated with decreases in the plasma concentration of apoB-48 (P < 0.05) and RLP-C (P < 0.001), but there was no significant change in the FCR of the emulsion. CONCLUSIONS: We suggest that long- and short-term treatments with simvastatin have comparable effects in decreasing the plasma concentration of triglyceride-rich remnants in heterozygous FH, as measured by fasting apoB-48 and RLP-C. The mechanisms for this may involve decreased production of hepatic and possibly intestinal lipoproteins, and/or up-regulation of hepatic receptor clearance pathways, but these changes are apparently not associated with a change in remnant clearance as measured kinetically by the (13)CO(2) breath test.

Factorial study of the effects of atorvastatin and fish oil on dyslipidaemia in visceral obesity.

BACKGROUND: Dyslipidaemia may account for increased risk of cardiovascular disease in central obesity. Pharmacotherapy is often indicated in these patients, but the optimal approach remains unclear. We investigated the effects of atorvastatin and fish oil on plasma lipid and lipoprotein levels, including remnant-like particle-cholesterol and apolipoprotein C-III, in dyslipidaemic men with visceral obesity. METHODS: We carried out a 6-week randomized, placebo-controlled, 2 x 2 factorial intervention study of atorvastatin (40 mg day(-1)) and fish oil (4 g day(-1)) on plasma lipids and lipoproteins in 52 obese men (age 53 +/- 1 years, BMI 33.7 +/- 0.55 kg m(-2)) with dyslipidaemia and insulin resistance. Treatment effects were analysed by general linear modelling. RESULTS: Atorvastatin had significant main effects in decreasing triglycerides (-0.38 +/- 0.02 mmol L(-1), P = 0.002), total cholesterol (-1.89 +/- 0.17 mmol L(-1), P = 0.001), LDL-cholesterol (-1.78 +/- 0.14 mmol L(-1), P = 0.001), remnant-like particle-cholesterol (-0.08 +/- 0.04 mmol L(-1), P = 0.035), apolipoprotein B (-49 +/- 4 mg dL(-1), P = 0.001), apolipoprotein C-III (-12.6 +/- 6.1 mg L(-1), P = 0.044) and in increasing HDL-cholesterol (+0.10 +/0- 0.04 mmol L(-1), P = 0.007). Fish oil had significant main effects in decreasing triglycerides (-0.38 +/- 0.11 mmol L(-1), P = 0.002) and in increasing HDL-cholesterol (+0.07 +/- 0.04 mmol L(-1), P = 0.041). There were no significant changes in weight or insulin resistance during the study. CONCLUSIONS: Atorvastatin and fish oil have independent and additive effects in correcting dyslipidaemia in viscerally obese men. Improvement in abnormalities in remnant lipoproteins may occur only with use of atorvastatin. Combination treatment with statin and fish oil may, however, offer an optimal therapeutic approach for globally correcting dyslipidaemia in obesity.

Preliminary experience with a new stable isotope breath test for chylomicron remnant metabolism: a study in central obesity.

We aimed to investigate the metabolism of chylomicron remnants in the postabsorptive state employing a new stable isotope breath test in centrally obese men without overt hyperlipidaemia. Groups of 12 centrally obese and 12 non-obese men of similar age and with similar plasma cholesterol and triacylglycerol (triglyceride) levels were studied. The catabolism of chylomicron remnants was measured using an intravenous injection of a remnant-like emulsion containing cholesteryl [(13)C]oleate. Isotopic enrichment of (13)CO(2) in breath was determined using isotope-ratio mass spectrometry, and a multi-compartmental model (SAAM II program) was used to estimate the fractional catabolic rate (FCR) of the chylomicron remnant-like particles. The plasma concentrations of low-density lipoprotein (LDL)-cholesterol, non-high-density lipoprotein (HDL)-cholesterol and insulin were significantly higher (P<0.05) in the obese than the control subjects. The obese subjects had significantly lower HDL-cholesterol (P<0.05) and, in particular, a decreased FCR of the remnant-like particles compared with lean subjects (0.061+/-0.014 and 0.201+/-0.048 pools/h respectively; P=0.016). In the obese group, the FCR of remnant-like particles was inversely associated with the waist/hip ratio, and with plasma triacylglycerol, cholesterol, LDL-cholesterol and non-HDL-cholesterol levels. In multiple regression analysis, the waist/hip ratio was the best predictor of the FCR of the emulsion. In conclusion, this new test suggests that postabsorptive chylomicron remnant catabolism is impaired in centrally obese subjects without overt hyperlipidaemia. This defect may be due to the degree of adiposity.

Chylomicron remnant metabolism in familial hypercholesterolaemia studied with a stable isotope breath test.

Chylomicron remnant metabolism was studied using a stable isotope breath test in 25 patients with familial hypercholesterolaemia (FH) (10 homozygotes, 15 heterozygotes), and in 15 normolipidaemic controls. A lipid emulsion mimicking the composition of chylomicron remnants and labelled with cholesteryl (13)C-oleate was injected intravenously; (13)CO(2) was measured subsequently in breath using isotope-ratio mass spectrometry. The fractional catabolic rate (pools/h) of the emulsion, derived from a compartmental model, did not differ significantly among the groups: homozygous FH mean 0.20 (S.E.M. 0.05), heterozygous FH 0.12 (0.02), controls 0.16 (0.03). We suggest that the catabolism of chylomicron remnants from plasma is not impaired in FH and that the hepatic uptake of these particles is not dependent on functional LDL receptors.

Chylomicron remnant metabolism in familial dyslipidemias studied with a remnant-like emulsion breath test.

We have developed a stable isotope breath test for the assessment of chylomicron remnant metabolism and report the results from the breath test in human subjects selected for disorders of chylomicron or remnant metabolism. In type I hyperlipemia, the phenotype is extreme hypertriglyceridemia due to a lack of lipoprotein lipase activity, which causes the failure of remnant formation. The type III dyslipidemia phenotype is caused by the inefficient removal of chylomicron remnants from plasma, generally because of homozygosity for apolipoprotein E2 alleles. The breath test was predicted to be abnormal in type III hyperlipemia, whereas a priori in type I hyperlipemia defective remnant clearance was not anticipated. Subjects were injected with lipid emulsions prepared with a composition similar to normal chylomicron remnants. The emulsions contained cholesteryl ester incorporating the stable nonradioactive isotope (13)C in the fatty acid moiety. End exhalation breath was collected at intervals after intravenous injection of the remnant-like emulsions and analyzed for (13)C enrichment by isotope-ratio mass spectrometry. Compared with the group of normolipemic men, the fractional catabolic rate of remnants measured by the breath test was significantly decreased (P = 0.006) in subjects with type III dyslipidemia. In the group with type I hyperlipemia, the fractional catabolic rate was not different (P = 0.233) from the control group. Therefore, the underlying capacity for remnant catabolism was normal in this group of markedly hypertriglyceridemic subjects. By short-circuiting the step of lipolysis, the remnant-like emulsion breath test provides direct information about remnant clearance and metabolism, which should assist in investigations of postprandial lipid metabolism.

Elevated apolipoprotein B-48 and remnant-like particle-cholesterol in heterozygous familial hypercholesterolaemia.

Apolipoprotein B-48 (apoB-48) is a marker of triglyceride-rich lipoprotein (TRL) remnants of intestinal origin. Chylomicron remnants are causally related to atherosclerosis. We have shown previously that fasting plasma apoB-48 may predict postprandial lipaemia. Remnant-like particle-cholesterol (RLP-C) may also reflect TRL remnants. We aimed to determine whether subjects with heterozygous familial hypercholesterolaemia (FH) had an accumulation of remnants of intestinal origin, as reflected by fasting plasma apoB-48 and RLP-C levels. The fasting plasma concentrations of apoB-48 and RLP-C were measured in 15 subjects with heterozygous FH and 15 age- and sex-matched, normolipidaemic subjects. ApoB-48 was determined using SDS-PAGE and a western blotting/enhanced chemi-luminescence technique. RLP-C was measured using an immuno-separation assay. Serum apolipoprotein B-100 (apoB-100) levels were measured using immunonephelometry; lipids were assayed enzymatically. Compared with controls, FH subjects had significantly elevated plasma concentrations of apoB-48 (29.3 median, 16.7-45.1 mg L-1 range vs. 12.8, 7.3-28.6; P < 0.001) and RLP-C (16.2, 1.5-114.3 mg dL-1 vs. 8.5, 5.0-13.5; P = 0.003), as well as serum total apoB-100 (1.9, 1.3-2.6 g L-1 vs. 1.0, 0.3-1.3; P < 0.001), LDL-cholesterol (8.1, 4.6-10.4 mmol L-1 vs. 3.5, 2.4-4.4; P < 0.001) and triglyceride (1.5, 0.6-5.6 mmol L-1 vs. 1.0, 0.4-1.8; P = 0.018). There was no significant difference in HDL cholesterol. The findings suggest that patients with heterozygous FH have elevated plasma concentrations of TRL remnants, including those of intestinal origin. This may be a consequence of decreased clearance of these particles by the LDL-receptor.

Relative roles of mitochondrial and peroxisomal fatty acid oxidation in the metabolism of chylomicron remnants in rats and mice as assessed by a stable-isotope breath test.

We have developed a stable isotope breath test to trace physiological remnant metabolism. Validity of the test depends on the injected lipid emulsion mimicking chylomicron remnant (CR) clearance and on subsequent metabolism of the emulsion cholesteryl ester (CE). Oxidation of CE fatty acids could involve both mitochondrial and peroxisomal pathways. In the present studies various agents were used to inhibit the binding of remnants, CE hydrolysis or mitochondrial fatty acid oxidation. Treatment of mice with suramin or lactoferrin markedly delayed the clearance and metabolism of remnants as shown by the significantly lower enrichment of 13CO(2) in the breath when compared with untreated mice. In hepatectomized rats injected with remnant-like emulsions, enrichment with 13CO(2) was virtually abolished. Treatment of mice with chloroquine or rats with methyl palmoxirate (an inhibitor of mitochondrial fatty acid oxidation) markedly impaired the recovery of label in the breath. Compared with mice fasted overnight, Intralipid by gavage decreased the breath enrichment with 13CO(2) consistent with competition between endogenous CR and the injected emulsion particles. These findings show that the breath test reliably measures the metabolism of CR and that CE fatty acid is metabolised by mitochondrial pathways.

Relative roles of LDLr and LRP in the metabolism of chylomicron remnants in genetically manipulated mice.

Remnant-like emulsions labeled with cholesteryl [(13)C]-oleate were prepared with lipid compositions similar to remnants derived from triacylglycerol-rich lipoproteins. When injected into the bloodstream of conscious mice, the remnant-like emulsions were metabolized in the liver leading to the appearance of (13)CO(2) in the breath. Previously, using this technique, we found that remnant metabolism was significantly impaired but not completely inhibited in mice lacking low density lipoprotein receptors (LDLr). We have now found in mice with non-functional low density lipoprotein receptor-related protein (LRP) that breath enrichment of (13)CO(2) was significantly decreased, indicating that the LRP also plays an important role in the metabolism of chylomicron remnants (CR). The enrichment of (13)CO(2) in the expired breath was negligible in mice lacking both LDLr and receptor-associated protein (-/-), essential for normal function of LRP. In mice pre-injected with gluthatione S-transferase-receptor-associated protein to block LRP binding, there was a marked inhibition of the appearance of (13)CO(2) in the expired breath of homozygous LDLr-deficient mice, supporting the role of LRP in vivo. Whether or not LDLr were present, in mouse and human fibroblast cells human apoE3 or E4 but not apoE2 were essential for binding of remnant-like emulsions, while lactoferrin and suramin completely inhibited binding. We conclude that in normal mice LDLr are important for the physiological metabolism of CR. When LDLr are absent the evidence supports a role for the LRP in the uptake of CR in liver cells and in fibroblasts, with binding characteristics for CR-associated apoE similar to LDLr.

Antioxidant defenses in rat intestine and mesenteric lymph.

Dietary oxysterols can reach the circulation and this may contribute to atherosclerosis, where lipid oxidation is thought to be important. There is also evidence that, in rats, peroxidized lipids are absorbed and transported into lymph [Aw TY, Williams MW, Gray L. Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH. Am J Physiol 1992; 262: G99-G106], although the method used to detect lipid peroxides lacked specificity. We tested whether intragastric administration of vegetable oils containing triglyceride hydroperoxides (TG-OOH) to rats resulted in detectable lipid hydroperoxides in mesenteric lymph. Using sensitive HPLC with postcolumn chemiluminescence detection, we were unable to detect hydroperoxides of triglycerides, cholesterylesters or phospholipids during the course of lipid absorption, and lymph levels of ascorbate, urate, alpha-tocopherol and ubiquinol-9 did not change significantly. By contrast, we observed a striking reducing activity judged by the efficient reduction of administered ubiquinones-9 and -10 to the corresponding ubiquinols. Exposure of rat lymph and isolated chylomicrons to aqueous peroxyl radicals revealed patterns of antioxidant consumption and lipid hydroperoxide formation similar to those described previously for human extravascular fluids and isolated lipoproteins, respectively. In particular, rates of TG-OOH formation in lymph and chylomicrons were very low to undetectable as long as ascorbate and/or ubiquinols were present, but subsequently proceeded in a chain reaction despite the presence of alpha-tocopherol. These studies demonstrate that rat intestine and mesenteric lymph possess efficient antioxidant defenses against preformed lipid hydroperoxides and (peroxyl) radical mediated lipid oxidation. We conclude that dietary lipid hydroperoxides or postprandial oxidation of lipids are not likely to contribute to these particular forms of oxidized lipids in circulation and aortic tissue.

Lipids in enteral nutrition.

Lipids in enteral nutrition facilitate the presentation of a high energy source with low osmotic impact. Focus has shifted from macronutrients towards the inclusion of special nutrients and growth factors. Recent advances in the design of triacylglycerol lipids with specific structures facilitate the absorption of essential fatty acids of the n-3 series, which provide specific benefits with respect to tissue repair and to the immune system. Enteric formulations containing n-3 lipids are proving to be of value in sustaining seriously ill patients. Information from well-controlled trials is generally consistent in establishing the benefits of formulations containing n-3 lipids.

The metabolism of native and randomized butterfat chylomicrons in the rat is similar.

Reportedly, randomly rearranging the position of fatty acids (FA) in butterfat triacylglycerol (TAG) by interesterification, thereby lowering the proportion of saturated FA in the sn-2 position, reduces its hypercholesterolemic and hypertriglyceridemic properties when fed to humans. The aim of this work was to determine if these reductions in plasma cholesterol and TAG could be explained by an improved rate of clearance from the plasma of chylomicrons composed of randomized butterfat, using a rat model. Acute chylomicron clearance studies demonstrated no differences in fractional clearance rates of cholesteryl esters and TAG from the plasma of rats infused with chylomicrons produced from gastric feeding of either native (NBF) or randomized (RBF) butterfat. Although there was a 14% decrease in the level of saturated FA occupying the sn-2 position of TAG in RBF compared with NBF, this difference became negligible (approximately 5%), following digestion of the fat and subsequent repackaging of TAG into chylomicrons. These observations suggest that the previously observed reduction in hypercholesterolemic properties of randomized butterfat in rat is unlikely to be explained by improved clearance of chylomicron TAG.

Plasma clearance of chylomicrons from butterfat is not dependent on saturation: studies with butterfat fractions and other fats containing triacylglycerols with low or high melting points.

BACKGROUND: Dietary fats influence plasma lipids, and changes in the clearance and metabolism of postprandial lipoproteins can affect atherosclerosis. Butterfat is considered hypercholesterolemic but contains a multitude of constituent fatty acids. OBJECTIVES: We determined triacylglycerol and cholesteryl ester clearances of lymph chylomicrons derived from butterfat, fractions of butterfat, and other dietary fats. METHODS: Radiolabeled lymph chylomicrons resulting from the intestinal absorption of different fats were reinjected into recipient rats to measure plasma clearance. Plasma clearance of [14C]triacylglycerol was used as an indicator of chylomicron lipolysis whereas clearance of [3H]cholesteryl ester was used as an indicator of chylomicron remnant removal. RESULTS: [3H]Cholesteryl ester clearance was slower from chylomicrons derived from a solid, high-saturated-butterfat fraction than from whole butterfat, but clearance of chylomicrons from other fractions did not correlate with the fractions' saturated fatty acid contents. Clearance of cholesteryl esters in chylomicrons derived from cocoa butter, palm oil, and butterfat was slower than clearance of cholesteryl esters in chylomicrons derived from safflower oil. Hepatic uptakes of cholesteryl esters were generally lower for chylomicrons from all butterfat fractions, cocoa butter, and palm oil. CONCLUSIONS: In contrast with minor effects on the lipolysis of chylomicron triacylglycerols, chylomicron remnant removal was strongly influenced by the type of dietary fat, with slower cholesteryl ester clearances for saturated fats with higher melting points. However, remnant removal and hepatic uptake of chylomicrons from whole butterfat and fractions of butterfat were not correlated with fat saturation. The mechanisms of this apparent paradox remain unknown but may be attributable to acyl arrangements in the lipid classes of chylomicrons that influence the association with apolipoproteins and receptors and hence remnant removal.

The diurnal rhythms of cholesterol metabolism and plasma clearance of model chylomicrons: comparison of normal and genetically hypercholesterolemic rats (RICO).

Previous studies showed a slower clearance of cholesterol-labeled lymph chylomicrons in genetically hypercholesterolemic rats (RICO) compared with normocholesterolemic rats. In this study, we compared rates of lipolysis and remnant clearance in RICO versus control normocholesterolemic rats of the same strain (RAIF) or with control Wistar rats, by injecting chylomicron-like lipid emulsions labeled with 14C-triolein to trace lipolysis, and 3H-cholesteryl ester to trace remnant clearance. Our findings showed slower clearance of chylomicron remnants in RICO compared with control RAIF or with control Wistar rats. During the light period, the clearance of lipids from chylomicron-like lipid emulsions injected intravenously was significantly slower in RICO rats compared with normocholesterolemic control rats of the same strain, RAIF. Within the RICO group, clearance of emulsion triolein (TO) was faster during the dark period compared with the light period. In contrast, however, the clearance of the emulsion remnants traced by cholesteryl oleate (CO) was slower during the dark period. This behaviour was not found within the Wistar group, where the clearances of TO and CO were similar in the light and dark period. Hepatic clearance of chylomicron remnants is mediated primarily by the low density lipoprotein (LDL) receptor, the expression of which shows diurnal variation. In both Wistar and RICO rats, the expression of LDL receptors was highest during the dark period. The LDL receptors in hepatic microsomal membranes from RICO rats migrated faster on SDS polyacrylamide gel electrophoresis when compared with normal Wistar and the RAIF. However in hepatic plasma membranes the LDL receptors from RICO and Wistar rats appeared identical after immunoblotting. Furthermore the LDL receptors from RICO and Wistar rats responded similarly to treatment with neuraminidase. An alteration in post-translational processing of the LDL receptor could possibly account for the slower clearance of chylomicron remnants in the RICO.

Chylomicron remnant uptake is regulated by the expression and function of heparan sulfate proteoglycan in hepatocytes.

Chylomicron remnants transport cholesterol from the intestine, and are removed from the circulation principally by the liver. While hepatic receptors, including the low density lipoprotein (LDL) receptor account for endocytosis, heparan sulfate proteoglycans (HSPG) participate in the initial binding of remnants to liver cells. To explore the interactions between HSPG and endocytosis of remnants, in the present study the expression of HSPG was inhibited in HepG2 cells transfected by a synthetic antisense oligodeoxynucleotide SYN5. Immunofluorescent staining by a monoclonal anti-syndecan antibody showed significant reduction in the expression of syndecan in SYN5-treated cells compared with control cells. Remnant binding decreased by about 50-70% in SYN5-transfected cells. Monoclonal antibodies to either heparan sulphate or the LDL receptor decreased binding by about 60-65%. The glycosylation inhibitor beta-nitrophenylxylopyranoside inhibited remnant uptake by 25%, whereas 4-nitrophenyl-beta-D-galactopyranoside had no effect on remnant binding. Heparinase completely abolished binding at appropriate concentrations. Heparitinase was less effective than hep arinase in inhibiting remnant binding. Suramin completely abolished the remnant binding. Poly-arginine, poly-lysine, and protamine all reduced remnant uptake by the cells, as did polybrene, a synthetic polycation, suggesting a role of cation-anion interactions in remnant binding. Brefeldin A, colchicine, and monensin caused the fluorescence associated with remnants to persist within the cells, confirming that blockers of tubulovesicular processes and Golgi function inhibit the intracellular transport and degradation of the remnants. Our results show that remnant binding to liver cells depends on the LDL receptor, on the expression of HSPG core proteins, and on the functionality of heparan sulfate in HSPG.

A 13CO2 breath test to assess the metabolism of triglyceride-rich lipoprotein remnants in mice.

Remnant-like emulsions were prepared with lipid compositions similar to remnants derived from triacylglycerol-rich lipoproteins. When injected into the bloodstream of conscious mice the remnant-like emulsions labeled with cholesteryl[13C]oleate were metabolized in the liver and the appearance of 13CO2 in the breath was measured. In control mice injected with remnant-like emulsions labeled with cholesteryl[13C]oleate, enrichment of 13CO2 in the breath peaked at 45 min and then decreased markedly by 3 h. In apoE-deficient (-/-) mice no enrichment was found and in low density lipoprotein receptor (LDLr)-deficient (-/-) mice the appearance of 13CO2 in the breath was markedly decreased. These findings were consistent with the ability of the breath test to detect defects in remnant metabolism. The breath test was useful in detecting a defect in remnant metabolism in LDLr heterozygote (+/-) mice, in which the appearance of 13CO2 in the breath was less by 45 min but remained elevated for the duration of the experiment when compared with control mice. In hepatic lipase-deficient (-/-) mice no defect in remnant metabolism was found. Under fasting conditions, the enrichment of 13CO2 in the breath after injection of emulsion was markedly increased when compared with fed mice, indicating that the metabolism of the injected remnant-like emulsion was probably competed for by post-prandial particles under fed conditions. Our findings show that a 13C breath test can be used to assess the metabolism of remnants. The test provides a useful and sensitive method for non-invasive testing of remnant metabolism in experimental animals.

Sterol side chain length and structure affect the clearance of chylomicron-like lipid emulsions in rats and mice.

In previous work we found that sterols such as cholesterol were essential for physiological plasma clearance of lipid emulsions mimicking the structure of mammalian triglyceride-rich lipoproteins. In the present study we compared the clearances of emulsions prepared with sterols of varying alkyl chain length (straight chains, n-C3 to n-C7, or branched chains, i-C5 to i-C10) at the C-17 position. Our studies show that the length of the alkyl chain at the C-17 position of sterols markedly affects the removal of remnant particles from the plasma of rats traced by emulsion cholesteryl oleate label. An alkyl chain of 7 carbons or more was needed for normal remnant clearance. Straight and branched chains of similar length were cleared similarly, showing that the presence of a branch at the end of the alkyl chain had no effect on remnant clearance. For side chains of 7 carbons or less, substitution of sterols with an unsaturation in the alkyl chain close to the terminal carbon markedly decreased the clearance of remnants. Triolein label was used to estimate lipolysis of the injected emulsions. Lipolysis was little affected by the structure of the sterol side chain, except that lipolysis was markedly higher with emulsions containing sterols with an alkyl chain having 4 carbon atoms (n-C4) or with an unsaturation in the 4 carbon alkyl chain. We conclude that the length of the alkyl side chain is an important element in the essentiality of cholesterol as a regulator of metabolism of lipid emulsion models of triglyceride-rich lipoproteins.