NK-104, a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, decreases apolipoprotein B-100 secretion from Hep G2 cells.

Intracellular cholesterol biosynthesis may play a key role in supplying cholesterol (as cholesteryl ester) for the neutral core of very low density lipoprotein (VLDL), thus modulating the secretion of apolipoprotein B-100 (apo B-100) from hepatocytes. The effect of compound NK-104 was studied, a new competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA-reductase), on apo B-100 synthesis and secretion from the human hepatoma cell line Hep G2. Cells were preincubated with NK-104 (0.01-5 microM) in the presence or absence of oleate (0.8 mM). Apo B-100 in the medium was determined by an enzyme-linked immunosorbent assay (ELISA). Incubation of Hep G2 with NK-104 resulted in a marked inhibition of cholesterogenesis (up to 95%), determined as incorporation of [14C]acetate into sterols, and decreased in a dose-dependent manner apo B-100 secretion, both in basal conditions (from 110 to 82 ng/mg cell protein, P < 0.01) and after incubation with oleate (from 227 to 165 ng/mg cell protein, P < 0.01). Density gradient for distribution of apo B-100 secreted, showed that this decrease was essentially due to a reduction of apo B-100 associated with lipoproteins in the density range of low density lipoproteins (LDL). Pulse chase experiment demonstrated that NK-104 did not affect the synthetic rate of apo B-100 but increased intracellular degradation of newly synthesized protein. The compound had only marginal effect on the mass of intracellular triglyceride but significantly decreased intracellular mass of free cholesterol and cholesteryl ester (P < 0.01). It is speculated that the ability of compound NK-104 to decrease apo B-100 secretion from Hep G2 cells is due to a decreased intracellular cholesterol availability.     

Prolonged inhibition of cholesterol synthesis by atorvastatin inhibits apo B-100 and triglyceride secretion from HepG2 cells.

Atorvastatin is a new HMG-CoA reductase inhibitor that strongly lowers plasma cholesterol and triglyceride (TG) levels in humans and animals. Since previous data indicated that atorvastatin has prolonged inhibition of hepatic cholesterol synthesis, we tested whether this longer duration of inhibitory effect on cholesterol synthesis decreased hepatic lipoprotein secretion in vitro. We used the HepG2 hepatoma cell line to: (1) determine the time required until levels of secreted apo B-100 and TG declined significantly, (2) examine the relation to the mass of cellular cholesteryl ester (CE) and (3) test microsomal triglyceride transfer protein (MTP) activity which leads to decreased apo B-100 production. Although atorvastatin significantly inhibited cholesterol synthesis in HepG2 cells regardless of treatment duration (1, 14 or 24 h), it did not inhibit TG synthesis. Apo B-100 and TG secretion were unchanged after 1-h atorvastatin treatment, but declined significantly after 24-h treatment. Atorvastatin treatment also reduced cellular CE mass, exhibiting both time- and dose-dependency. Mevalonolactone, a product of HMG-CoA reductase, attenuated the inhibitory effects of atorvastatin. Atorvastatin strongly reduced mRNA levels of MTP, whereas it did not inhibit MTP activity as measured by TG transfer assay between liposomes. Simvastatin also induced treatment- and time-dependent reductions in apo B-100, whereas the MTP inhibitor BMS-201038 exhibited no time dependency, instead inhibiting this variable even on 1-h treatment. These results indicate that reduced apo B-100 secretion caused by atorvastatin is a secondary result owing to decreased lipid availability, and that atorvastatin's efficacy depends on the duration of cholesterol synthesis inhibition in the liver.     

The isoflavone genistein inhibits copper and peroxyl radical mediated low density lipoprotein oxidation in vitro

Oxidation of low density lipoprotein (LDL) is implicated in the development of atherosclerosis and dietary antioxidants may provide a useful therapy in the prevention of LDL oxidation and atheroma development. The aim of these experiments was to investigate the antioxidant activity of the soybean isoflavone, genistein, in in vitro models of LDL oxidation. Genistein inhibited copper-mediated oxidation of LDL in a concentration-dependent manner by lengthening the time for conjugated diene formation (54.1±5.1 min in control LDL and 107.1±1.8 min with 5 μmol/l genistein, P<0.001) and decreasing the oxidation rate (14.4±1.9 nmol conjugated diene/mg LDL protein/min in control LDL and 7.4±1.1 nmol conjugated diene/mg LDL protein/min with 5 μmol/l genistein, P<0.001). Peroxy radical (azo-initiated) oxidation of LDL was significantly inhibited by 200 μmol/l genistein as indicated by: (i) increase in the time required for malondialdehyde (MDA) formation (7 h incubation compared to 3 h incubation with control LDL), (ii) 32, 44 and 46% decreases in MDA concentration compared to control samples following 3, 4 and 5 h incubation, respectively and (iii) decrease in relative electrophoretic mobility (REM) of LDL. Incorporation of genistein into LDL and its resultant antioxidant activity was also investigated. LDL was isolated from plasma which had been pre-incubated with 25, 50 or 100 μmol/l genistein at 37°C for 24 h. Approximately 3–4% of genistein present in plasma was incorporated into LDL, however copper-mediated oxidation of control LDL and LDL isolated from plasma pre-incubated with genistein was not significantly different.     

Enhanced RAGE-mediated NFkappaB stimulation in inflamed hemodialysis patients

Advanced glycation end-products (AGEs), a group of carbohydrate-derived compounds formed by non-enzymatic glycation and oxidation, are markedly elevated in end-stage renal disease (ESRD) and may be related to both inflammation and oxidative stress. The cellular effects of AGE are largely mediated by their interaction with specific surface receptors, such as RAGE. Measurements of biomarkers of inflammation and oxidative stress were conducted in 7 hemodialysis (HD) patients (5 males) with persistent high-grade inflammation (C-reactive protein [CRP] > 10 mg/L) and 11 HD-patients (6 males) with low-grade inflammation (CRP < 10 mg/L) for at least 6 months. Measured biomarkers for inflammation included hs-CRP, interleukin (IL)-6, white blood cells, neutrophils, S-albumin, peroxisome proliferator-activated receptors (PPAR , β, γ) and nuclear factor κB (NFκB) activity. Markers for oxidative stress were advanced oxidation products (AOPP), myeloperoxidase (MPO)-activity, pentosidine and carboxymethyl lysine (CML). In addition, the effect of increasing doses of CML-modified human serum albumin on NFκB activity was tested in mononuclear cells isolated from each patient. As expected, HD-patients with high-grade inflammation had significantly elevated levels of IL-6 (median 9.2 pg/mL versus 2.5 pg/mL; p < 0.01), MPO-activity (134.5 ± 14.6 ΔOD₆₃₀/(min mg protein) versus 80.5 ± 12.9 ΔOD₆₃₀/(min mg protein); p < 0.05), PPAR-γ (0.65 ± 0.01 OD₆₅₅ versus 0.56 ± 0.01 OD₆₅₅; p < 0.01), and AOPP (269 ± 54 μM versus 163 ± 15 μM; p < 0.05) compared with low-grade inflamed patients. Significant associations were demonstrated between hs-CRP and NFκB (ρ = 0.58; p < 0.05), AOPP (ρ = 0.49; p < 0.05) and PPAR-γ (ρ = 0.62; p < 0.05), respectively. In the patient group with high-grade inflammation, stimulation of mononuclear cells with CML-modified human serum albumin caused a rapid dose-dependent rise (p < 0.0001) in NFκB activity that could be completely blocked by an anti-RAGE antibody. Inflammation and oxidative stress biomarkers are interrelated in ESRD. Inflammatory cell signal pathways, such as NFκB, are activated by CML-modification of proteins via RAGE.     

Apolipoprotein B (B-48) of rat chylomicrons is not a precursor of the apolipoprotein of low density lipoproteins.

We have found that, in chylomicrons from intestinal lymph fistulas in the rat, the sole molecular species of apolipoprotein B (apo B) is B-48. This protein is analogous to the B-48 apoprotein of human chylomicrons. In contrast, preparations of chylomicrons from blood serum are known to contain species of B apolipoproteins of higher molecular weight, presumably due to the presence of hepatogenous lipoproteins. We studied the removal of 125I-labeled apo B-48 of intestinal lymph chylomicrons from blood plasma of rats. The removal of [14C]cholesteryl esters of biologically labeled chylomicrons was unaffected by radioiodination. The labeled cholesteryl esters and apo B-48 disappeared rapidly from the density (P) less than 1.006 g/ml fraction of plasma. In contrast to the apo B of very low density lipoproteins (1.019 less then p less then 1.063 g/ml) after 1 hr. No 125I was found in the B-100 or B-95 apolipoproteins at any time. We conclude that, unlike the species of apo B found uniquely in hepatogenous very low density lipoproteins, the apo B-48 protein of chylomicrons is not a precursor of the B apoprotein of low density lipoproteins.     

Quantitation of apo b in human aortic fatty streaks A comparison with grossly normal intima and fibrous plaques

The content of apolipoprotein B (apo B)-containing lipoproteins was measured in aortic fatty streak lesions of 18 male individuals between the ages of 21 and 67 years, and compared to the values found in adjacent grossly normal intima. Extraction of apo B was accomplished by sequential treatment of aortic tissue homogenates with a standard buffer and one containing the detergent Triton X-100. Mean apo B values (μg per mg tissue dry weight) in fatty streaks were: BUFFER-EXTRACTED = 4.67 ± 0.51, Triton-extracted = 1.88 ± 0.39; while in adjacent grossly normal intima: BUFFER-EXTRACTED = 6.51 ± 0.92, Tritonextracted = 0.37 ± 0.15. Using a paired t-test, buffer-extracted apo B was marginally significantly greater in the adjacent normal intima than fatty streaks (P < 0.05), whereas Triton-extracted apo B was highly significantly greater in fatty streaks than adjacent normal intima (P < 0.0005). When the mean apo B values of these 18 fatty streaks and 23 fibrous plaques from separate cases were compared in a non-paired t-test, buffer-extracted apo B was slightly higher in fatty streaks than fibrous plaques (P < 0.025), whereas Triton-extracted apo B was much higher in fibrous plaques than in fatty streaks (P < 0.0005). The intermediate position of fatty streaks between grossly normal and fibrous plaques with respect to buffer- and Triton-extracted apo B content, gives additional support to the contention that this lesion is an intermediate step in the progression of the grossly normal intima to a fibrous plaque. Assuming this sequence of progression to occur, our results demonstrate a marginally significant decrease in buffer-extracted apo B but a highly statistically significant increase in Triton-extracted apo B with lesion development.     

Effects of a thyromimetic on apolipoprotein B-100 in rats

We have studied the effects of a cardiac sparing thyromimetic, CGS 23425, on postprandial levels of triglycerides, abundance of apolipoprotein B (apo B) protein and hepatic apo B mRNA expression in rats. When compared with control rats, triglyceride clearance was significantly accelerated by treatment with CGS 23425. A full return to baseline values was achieved within 8 h after ingesting a large quantity of fat, as compared to >24 h in control animals. The abundance of apo B-100 protein in CGS 23425-treated hyperlipidemic rats decreased in a dose-dependent manner, but levels of apo B-48 were not significantly affected. Like L-tri-iodothyronine (L-T(3)), treatment with 30 microg/kg CGS 23425 for 6 or 9 days decreased the levels of apo B-100 protein by 80% and 40% respectively. This change was paralleled by a 27% reduction in hepatic apo B-100 mRNA. To investigate a potential mechanism of CGS 23425 action, we measured in vitro apo B mRNA editing activity in hepatocellular extract from control or CGS 23425-treated rats. Treatment with CGS 23425 increased activity of the hepatic apo B-100 editosome, apobec-1. In human hepatoma cells which lack apobec-1 activity, apo B-100 mRNA levels remained the same in cells treated with or without the agent. In summary, these observations show that CGS 23425 decreases the levels of apo B-100 in rats. This action of CGS 23425 involves apo B-100 mRNA editing activity.     

Inhibitory effect of prostaglandin E1 on intimal thickening following photochemically induced endothelial injury in the rat femoral artery

The inhibitory effect of prostaglandin E₁, which has an anti-platelet action and a vasodilating action via intracellular cyclic AMP elevation, was studied on intimal thickening in the rat femoral artery. A segment of the femoral artery was occluded by a platelet and fibrin-rich thrombus due to photochemical reaction between systemically administered Rose Bengal and transluminal green light which causes endothelial injury followed by platelet adhesion and aggregation at the site of photochemical reaction. Three weeks after endothelial injury, intimal thickening occurred at the irradiated site. Prostaglandin E₁ (0.3 μg/kg per min), administered as a continuous infusion 10 min before photochemical reaction significantly (P<0.05) prolonged the time to occlusion of the femoral artery. In a separate experiment, prostaglandin E₁ (0.3 μg/kg per min) administered as a continuous infusion for 7 days just after endothelial injury significantly (P<0.05) inhibited intimal thickening compared with a control group. In cultured rat-derived vascular smooth muscle cells, prostaglandin E₁ produced concentration-dependent inhibition of migration and proliferation, stimulated by platelet-derived growth factor. These results suggest that prostaglandin E₁ may be effective in preventing vascular restenosis after vascular surgery and angioplasty.     

Prevention of focal intimal hyperplasia in rat vein grafts by using a tissue engineering approach

The present study focused on the role of blood flow in the formation of focal intimal hyperplasia in vein grafts, as well as the development of an engineering approach that can be used to eliminate disturbed blood flow and prevent blood flow-related focal intimal hyperplasia. A rat vein graft model was constructed by interposing a jugular vein into the abdominal aorta with end-to-end anastomoses. Locally disturbed flow was identified by analyzing particle streak-lines in methyl salicylate-cleared and perfused vein grafts in vitro with a physiological Reynolds number. At day 10, 20, and 30 after surgery, focal intimal hyperplasia of the vein grafts was examined using a histological approach and the density of -actin positive cells was determined using immunohistological and fluorescent approaches. Results showed that apparent eddy blood flow formed at the proximal, but not at the distal, end of the vein grafts due to graft-host diameter mismatch and local geometric distortions, and was associated with apparent focal intimal hyperplasia. The thickness of the -actin positive layers of the proximal vein grafts was significantly higher than that of the distal grafts (192±27 vs. 94±18 μm, 278±55 vs. 124±20 μm, and 288±24 vs. 131 ±23 μm for day 10, 20, and 30, respectively). The density of the -actin positive cells, however, was similar between the proximal and the distal regions (3569±361 vs. 3285±343 cells/mm², 5540±650 vs. 5376±887 cells/mm², and 5465±791 vs. 5278±524 cells/mm² for day 10, 20, and 30, respectively). When eddy blood flow was eliminated by matching the graft-host diameters using a tissue engineering approach, the average thickness of the -actin positive layers of the proximal (71±15, 86±16, and 85±14 μm for day 10, 20, and 30, respectively) and the distal vein grafts (68±13, 80±14, and 79±13 μm for day 10, 20, and 30, respectively) was reduced significantly. The density of the -actin positive cells was also reduced significantly in the proximal (2946±359, 3261±295, 3472±599 cells/mm² for day 10, 20, and 30, respectively) and in the distal regions (3151±511, 3466±687, 3593±688 cells/mm² for day 10, 20, and 30, respectively). The thickness of the -actin positive layers and the density of the -actin positive cells were not significantly different between the proximal and distal regions of the engineered vein grafts at each observation time. These results suggest that eddy flow may develop in vein grafts and may facilitate the formation of focal intimal hyperplasia, and the vascular tissue engineering approach developed in this study may be used to prevent blood flow-related focal intimal hyperplasia in vein grafts.     

Recent progress in understanding apolipoprotein B

For the past 5 years, investigators from many different laboratories have contributed to a greatly increased understanding of two very important lipid-carrying proteins in plasma--apo B-100 and apo B-48. Apo B-100, an extremely large protein composed of 4,536 amino acids, is synthesized by the liver and is crucial for the assembly of triglyceride-rich VLDL particles. Apo B-100 is virtually the only protein of LDL, a cholesteryl ester-enriched class of lipoproteins that are metabolic products of VLDL. The apo B-100 of LDL serves as a ligand for the LDL receptor-mediated uptake of LDL particles by the liver and extrahepatic tissues. The LDL receptor-binding region of apo B-100 is located in the carboxyterminal portion of the molecule, whereas its lipid-binding regions appear to be broadly dispersed throughout its length. Apo B-48 contains the amino-terminal 2,152 amino acids of apo B-100 and is produced by the intestine as a result of editing of a single nucleotide of the apo B mRNA, which changes the codon specifying apo B-100 amino acid 2,153 to a premature stop codon. Apo B-48 has an obligatory structural role in the formation of chylomicrons; therefore, its synthesis is essential for absorption of dietary fats and fat-soluble vitamins. Both apo B-48 and apo B-100 are encoded on chromosome 2 by a single gene that contains 29 exons and 28 introns. An elevated level of apo B-100 in the plasma is a potent risk factor for developing premature atherosclerotic disease. In the past 3 years, many different apo B gene mutations that affect the concentrations of both apo B and cholesterol in the plasma have been characterized. A missense mutation in the codon for apo B-100 amino aid 3,500 is associated with hypercholesterolemia. This mutation results in poor binding of apo B-100 to the LDL receptor, thereby causing the cholesteryl ester-enriched LDL particles to accumulate in the plasma. This disorder is called familial defective apo B-100, and it is probably a cause of premature atherosclerotic disease. Familial hypobetalipoproteinemia is a condition associated with abnormally low levels of apo B and cholesterol; affected individuals may actually have a reduced risk of atherosclerotic disease.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microsomal triglyceride transfer protein: does insulin resistance play a role in the regulation of chylomicron assembly?

We have previously demonstrated that diabetes is associated with an increase in intestinal microsomal triglyceride transfer protein (MTP) mRNA in both the rat and rabbit models. The present study was designed to investigate the relationship between MTP expression and chylomicron assembly in an insulin resistant non-diabetic animal model. Ten insulin resistant Zucker obese fa/fa rats and ten lean fa/− rats were examined at 8–10 weeks of age. The lymph duct was cannulated and lymph collected for 4 h. Lymph chylomicrons were isolated by ultracentrifugation and their composition determined. RNA was extracted from intestinal mucosa and from the liver. MTP mRNA was measured using the RNase protection assay. Blood sugar in the fatty rats was significantly higher (6.3±1.2 vs. 5.4±0.4 P<0.05) and plasma insulin was almost six times that of the lean rats (P<0.001). Plasma cholesterol and phospholipid but not triglyceride were significantly increased in the obese animals (P<0.01). Obese animals secreted significantly more lymph chylomicron apo B48 (0.05±0.02 vs. 0.02±0.01mg/h P<0.005), triglyceride (9.7±5.3 vs. 3.8±1.9 mg/h P<0.005) and phospholipid (1.5±0.7 vs. 0.4±0.3 mg/h P<0.001). The only difference in the chylomicron particle composition between the two groups was a significant increase in phospholipid (P<0.01). Intestinal MTP mRNA expression was significantly higher in the fatty compared to the lean rats (22.1±9.5 vs. 7.8±5.6 amol MTP mRNA/μg total RNA P<0.001) as was hepatic MTP mRNA expression (6.9±3.5 vs. 3.4±1.5 amol MTP mRNA/μg total RNA, P<0.01). Thus in this animal model of insulin resistance, increased MTP, which was associated with increased chylomicron particle number, may play a crucial role in the development of atherosclerosis.     

Absence of intestinal synthesis of apolipoprotein B-48 in two cases of abetalipoproteinemia

Previous studies have reported that the absence of chylomicron, very-low-density lipoprotein, and low-density lipoprotein in abetalipoproteinemia is a consequence of apoprotein B (apo B) deficiency. Although the absence of apo B from the intestine has been shown by immunofluorescence, the antiserum used was raised against low-density lipoprotein apo B. Therefore, the precise nature of the underlying defect remains unknown, given that the postulated gene mutation could prevent the synthesis of the molecular form of apo B specific for chylomicrons, apo B-48, or produce an unstable aberrant form of apo B particle. This report concerns 2 girls aged 5.5 and 4.75 with well-documented clinical and biological manifestations of the disease in whom there was no immunologically detectable plasma apo B-48 and apo B-100. Their cultured jejunal explants incubated with [14C]palmitate showed slight decrease in the esterification of triglycerides, phospholipids, and cholesteryl esters. However, only traces of triglycerides and small amounts of cholesteryl esters were found in the culture medium in contrast to phospholipids, which were readily exported. Protein synthesis as assessed by [3H]leucine incorporation by explants was normal and only modestly diminished in the fat chylomicronlike fraction floated from the sonicated explants. However, there was no radioactivity at the electrophoretic position of apo B-100 and apo B-48. Immunologic confirmation of the absence of these two apoproteins was obtained by Western blots. These data confirm the hypothesis that in certain cases of abetalipoproteinemia the intestinal defect results from the lack of synthesis of apo B-48.     

Pharmacology of the AC AT Inhibitor Avasimibe (CI‐1011)

Avasimibe is a novel orally bioavailable ACAT inhibitor, currently under clinical development (phase III trials). It was safe when administered to rats, dogs, and humans. In vitro studies in human macrophages demonstrated that avasimibe reduces foam cell formation not only by enhancing free cholesterol efflux, but also by inhibiting the uptake of modified LDL. The concentration‐dependent reduction in cellular cholesteryl ester content in these cells was not accompanied by an increase in intracellular free cholesterol, which is in agreement with a good safety profile for avasimibe. In the liver, avasimibe caused a significant reduction in the secretion of apo B and apo B‐containing lipoproteins into plasma. Avasimibe induced cholesterol 7α‐hydroxylase and increased bile acid synthesis in cultured rat hepatocytes, and its administration to rats did not produce an increase in lithogenicity index of the bile. The hypolipidemic efficacy of the compound was demonstrated in cholesterol‐fed as well as in non‐cholesterol‐fed animals. In these models, plasma cholesterol levels were reduced, mainly due to the decrease in the non‐HDL cholesterol fraction. Clinical data are scarce, but in a study performed in 130 men and women with combined hyperlipidemia and hypoalphalipoproteinemia, avasimibe, 50–500 mg/day, significantly reduced plasma total triglyceride and VLDL‐cholesterol. Although total cholesterol, LDL‐cholesterol, and HDL‐cholesterol were unchanged, it must be stressed that animal data suggest that avasimibe may have direct antiatherosclerotic activity in addition to its cholesterol‐lowering effect. Avasimibe treatment can also contribute to increase plaque stability, as it reduces the accumulation of lipids in the arterial wall, inhibits macrophage infiltration into the media and reduces matrix metalloproteinase expression and activity. Moreover, avasimibe and statins have been shown to have synergistic effects, and the combination therapy may not only inhibit atherosclerotic lesion progression but also induce lesion regression, independently of changes in plasma cholesterol.     

Fasting and postprandial apolipoprotein B-48 levels in healthy, obese, and hyperlipidemic subjects

Apolipoprotein (apo) B-48 is the only specific marker of intestinal lipoproteins. We evaluated a novel enzyme-linked immunosorbent assay (ELISA) standardized with recombinant apo B-48 to measure apo B-48 in plasma and triglyceride-rich lipoproteins (TRLs, density <1.006 g/mL). Coefficients of variation were less than 2.5%. Assay values correlated well (r = 0.82, P < .001) with values obtained by gel scanning of TRLs (n = 75 samples); however, the gel scanning method yielded values that were about 50% lower than ELISA values. About 60% to 70% of apo B-48 was found in TRLs. In 12 healthy subjects, median fasting plasma apo B-48 levels were 0.51 mg/dL and were increased by 121% to 147% in the fed state. In 63 obese subjects, median fasting apo B-48 values were 0.82 mg/dL; and feeding resulted in almost no change in total cholesterol, non–high-density lipoprotein cholesterol, or total apo B values, whereas triglyceride, remnant lipoprotein cholesterol, and apo B-48 levels were significantly higher (P < .05; by +73%, +58%, and +106%), and direct low-density lipoprotein cholesterol and direct high-density lipoprotein cholesterol were significantly lower (P < .001, by −13% and −20%) than fasting values. Relative to controls, 270 hyperlipidemic subjects had significantly higher (P < .001, +115%) fasting total apo B and higher apo B-48 values (P = .06, +37%). Our data indicate that the apo B-48 ELISA tested provides highly reproducible results and is excellent for research studies. Median apo B-48 values in healthy subjects are about 0.5 mg/dL and increase more than 100% in the fed state. Elevated levels are observed in obese and hyperlipidemic subjects.     

Secretion of cholesteryl ester-enriched very low density lipoproteins by the liver of cholesterol-fed rabbits

The output of lipids and lipoproteins by isolated perfused livers of normal-fed and cholesterol-fed rabbits has been examined. There was a comparable output of triglyceride by the livers of both groups, resulting in an accumulation of 40-50 mg triglyceride/liver/2 h in the perfusate in each case. The output of cholesteryl esters, however, was very much greater from the livers of cholesterol-fed (45 mg/liver/2 h) than from normal-fed (3.3 mg/liver/2 h) rabbits. The major lipoproteins in liver perfusates from both groups of animals were very low density lipoproteins (VLDL). In the perfusate of normal livers the VLDL were enriched with triglyceride and depleted of cholesteryl esters when compared with plasma VLDL from normal animals. VLDL in the perfusate of livers from cholesterol-fed rabbits, on the other hand, were markedly enriched with cholesteryl esters; cholesteryl esters accounted for 33% by mass of VLDL from cholesterol-fed livers and only 3.1% of VLDL from normal livers. The cholesteryl esters in the plasma lipoproteins of cholesterol-fed rabbits were relatively enriched with cholesteryl oleate when compared to those in normal plasma. Similarly, cholesteryl oleate predominated in the VLDL in the liver perfusate of the cholesterol-fed animals, consistent with an hepatic acyl CoA/cholesterol acyltransferase origin. Thus, cholesterol-feeding in the rabbit results in a marked increase in the hepatic secretion of cholesteryl esters as a component of VLDL.     

Differential effect of fenofibrate and atorvastatin on in vivo kinetics of apolipoproteins B-100 and B-48 in subjects with type 2 diabetes mellitus with marked hypertriglyceridemia

The specific impact of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors and fibrates on the in vivo metabolism of apolipoprotein (apo) B has not been systematically investigated in patients with type 2 diabetes mellitus with high plasma triglyceride (TG) levels. Therefore, the objective of this 2-group parallel study was to examine the differential effects of a 6-week treatment with atorvastatin or fenofibrate on in vivo kinetics of apo B-48 and B-100 in men with type 2 diabetes mellitus with marked hypertriglyceridemia. Apolipoprotein B kinetics were assessed at baseline and at the end of the intervention using a primed constant infusion of [5,5,5-D(3)]-l-leucine for 12 hours in the fed state. Fenofibrate significantly decreased plasma TG levels with no significant change in plasma low-density lipoprotein cholesterol (LDL-C) and apo B levels. On the other hand, atorvastatin significantly reduced plasma levels of TG, LDL-C, and apo B. After treatment with fenofibrate, very low-density lipoprotein (VLDL) apo B-100 pool size (PS) was decreased because of an increase in the fractional catabolic rate (FCR) of VLDL apo B-100. No significant change was observed in the kinetics of LDL apo B-100. Moreover, fenofibrate significantly decreased TG-rich lipoprotein (TRL) apo B-48 PS because of a significant increase in TRL apo B-48 FCR. After treatment with atorvastatin, VLDL and IDL apo B-100 PSs were significantly decreased because of significant elevations in the FCR of these subfractions. Low-density lipoprotein apo B-100 PS was significantly lowered because of a tendency toward decreased LDL apo B-100 production rate (PR). Finally, atorvastatin reduced TRL apo B-48 PS because of a significant decrease in the PR of this subfraction. These results indicate that fenofibrate increases TRL apo B-48 as well as VLDL apo B-100 clearance in men with type 2 diabetes mellitus with marked hypertriglyceridemia, whereas atorvastatin increases both VLDL and IDL apo B-100 clearance and decreases TRL apo B-48 and LDL apo B-100 PR.     

Effects of a moderate exercise session on postprandial lipoproteins, apolipoproteins and lipoprotein remnants in middle-aged men

Prior moderate exercise reduces postprandial triglyceride concentrations. Its effects on the concentrations, compositions and potential atherogenicity of lipoprotein subfractions were investigated in the present study. Twenty normoglycaemic middle-aged men each underwent two fat tolerance tests (blood taken fasting and for 8 h after a meal containing 80 g fat and 70 g carbohydrate). On the afternoon before one test, subjects performed a 90-min treadmill walk (exercise); no exercise was performed before the control test. Prior exercise significantly reduced postprandial concentrations of chylomicrons (Sf >400) by 28.6% (absolute reduction 14.6 mg dl(-1)), of large VLDL1 (Sf 60-400) by 34.4% (39.7 mg dl(-1)) and of small VLDL2 (Sf 20-60) by 23.0% (9.6 mg dl(-1)). Over 95% of VLDL1 and VLDL2 comprised apolipoprotein (apo) B100-containing particles. Exercise also reduced postprandial remnant-like lipoprotein cholesterol (by 35%) and triglyceride concentrations (by 29%). Postprandial apo CIII/apo B and apo E/apo B ratios in VLDL1 were lower following exercise. Postprandial cholesteryl ester/triglyceride ratios were lower in VLDL1 and VLDL2 and higher in HDL2 following exercise. These data suggest that the effect of prior moderate exercise on VLDL1 is quantitatively greater than its effect on chylomicrons and that, in addition to reducing lipoprotein concentrations, exercise induces compositional changes to lipoprotein species which are likely to influence their metabolism and atherogenicity.     

Serum HDL cholesterol values are associated with apoB-containing lipoprotein metabolism and triglyceride-body fat interrelation in young Japanese men

Serum levels of total cholesterol, high density lipoprotein (HDL) cholesterol, triglyceride, apolipoprotein (apo) AI, ApoB, ApoE and body fat were measured in 226 fasting male Japanese college students aged 18 to 20 years. They were normolipidemic (total cholesterol: 169±31 mg/dl, triglyceride: 56±25 mg/dl) and their HDL cholesterol concentrations were high (61±13 mg/dl). An HDL cholesterol value <35 mg/dl was observed in only one student (0.4%). In contrast, 112 men (49.6%) had an HDL cholesterol level ≥60 mg/dl. Even in this normolipidemic group, as compared with students in a top HDL cholesterol tertile (HDL cholesterol; 75±9 mg/dl), students in a lower HDL cholesterol tertile (HDL cholesterol; 48±5 mg/dl) had significantly increased serum levels of LDL cholesterol (103±30 vs. 91±26 mg/dl), triglyceride (68±30 vs. 45±16 mg/dl) and apoB (83±20 vs 73±17 mg/dl). In addition, they had greater body mass index (23.2±3.6 vs. 20.6±2.5 kg/m²) and greater percent body fat (20.2±6.2 vs. 16.2±4.2%) determined using a bioelectrical impedance analyzer. HDL cholesterol levels were much more strongly related to triglyceride (r=−0.37) than was apoAI (r=−0.13). In stepwise multiple regression analysis in 184 nonsmokers, apoE, apoB and fat mass explained 21% of apoAI variability. Triglyceride in addition to these three parameters explained 41% of HDL cholesterol variability. These results suggest that serum levels of HDL cholesterol are associated with metabolism of apoB-containing lipoproteins as well as triglyceride-body fat interrelationship.     

Improved metabolic control reduces the number of postprandial apolipoprotein B-48-containing particles in type 2 diabetes

Postprandial lipoproteins are raised in diabetes and there is increasing evidence for the atherogenicity of the chylomicron remnant. Increased postprandial cholesteryl ester transfer has also been demonstrated in diabetes and may contribute to the atherogenic lipoprotein profile. The present study examined the effect of improving metabolic control on postprandial lipoproteins in 13 Type 2 diabetic patients. Blood was taken fasting and at 2-h intervals following a high fat, 1100 kcal meal. Patients were brought into good control by intensified dietary advice and oral hyperglycaemic agents or insulin if blood glucose failed to respond. Fasting and postprandial cholesteryl ester transfer protein (CETP) and lecithin:cholesteryl acyltransferase (LCAT) were determined in six patients. Lipoproteins were isolated by sequential ultracentrifugation. Chylomicron and very low density lipoprotein (VLDL) apolipoprotein B-48 and apolipoprotein B-100 were isolated by polyacrylamide gradient gel electrophoresis and quantified by densitometric scanning. CETP and LCAT were determined by an endogenous method which determined cholesterol esterification and transfer between the patients' lipoproteins. There was a significant reduction in postprandial chylomicron apo B-48 (P<0.005), apo B-100 (P<0.0005) and chylomicron cholesterol (P<0.001) following improved diabetic control. The chylomicron lipid/apo B ratio increased with improved control (P<0.01). Postprandial CETP and LCAT were significantly reduced in good control (P<0.01 and P<0.05, respectively) and there were significant changes in HDL composition. The study shows that improvement in metabolic control in Type 2 diabetic patients leads to a reduction in postprandial chylomicron particles and less transfer of cholesterol to apo B-containing lipoproteins.     

Overexpression of glucagon-like peptide-1 receptor in an insulin-secreting cell line enhances glucose responsiveness

Glucagon-like peptide-1 (GLP-1), secreted from intestine in response to food intake, enhances insulin secretion from pancreatic β-cells. In this study, we evaluated the effects of stably transfecting the GLP-1 receptor into an insulinoma cell line, RIN 1046-38, on basal and glucose-mediated insulin secretion and on second messenger pathways involved in insulin secretion. The GLP-1 receptor transfected cells had similar insulin mRNA levels but higher insulin content compared with parental cells. In GLP-1 receptor transfected cells, glucose (0.5 mM)-mediated insulin release was increased compared with parental cells (4.52±0.79 pmol insulin/l per mg protein·h vs. 2.21±0.36 pmol insulin/l per mg protein·h; mean±S.E., n=6, P=0.015, in transfected vs. parental cells, respectively). By hemolytic plaque assay measuring single cell insulin secretion, we observed that in the GLP-1 receptor transfected cells versus parental cells the increased insulin secretion was due to the presence of more glucose-responsive cells as well as more insulin released in response to glucose per cell. Resting intracellular cAMP was higher in the GLP-1 transfected cells (35.96±3.88 vs. 18.6±2.01 nmol/l per mg protein·h; mean±S.E., n=4, P=0.039, in transfected vs. parental cells, respectively). In response to GLP-1, both GLP-1 receptor transfected cells and parental cells showed increased cAMP levels independent of glucose. Resting intracellular calcium was the same in both parental and GLP-1 receptor transfected cells. However, more cells were responsive to glucose in the GLP-1 receptor transfected cells and the calcium transients attained in the presence of glucose developed at a faster rate and reached a higher amplitude than in parental cells. We conclude that having an excess of GLP-1 receptors renders β-cells more sensitive to glucose.