Uptake of chylomicron remnants by the native LDL receptor in human monocyte-derived macrophages.

Human chylomicron remnants were taken up by cultured human monocyte-derived macrophages. Competition studies using 125I-labeled and unlabeled lipoproteins demonstrated that the remnant particles were not taken up by the modified LDL (acetyl LDL) receptor in these cells, which also contain a receptor for native LDL. The data thus suggest that the apolipoprotein E- and B-containing remnant particles are mainly taken up through an extra-hepatic E and B receptor (the classical LDL receptor pathway) in macrophages as is the case in cultured human skin fibroblasts.

Cell density dependent uptake of LDL in cultured rat hepatocytes.

An inverse relationship between low-density lipoprotein uptake and cell density was observed in rat hepatocyte monolayers incubated with lipoprotein-deficient serum. This was also true for cell association, binding and degradation of low-density lipoproteins. Compactin stimulated cell association and degradation of low-density lipoproteins both at low and high concentrations. Insulin, on the other hand, had no consistent effect on low-density lipoprotein cell association or degradation.

Cell-density-dependent uptake of chylomicron remnants in rat hepatocyte monolayers. Effects of compactin and mevalonic acid.

In rat hepatocytes cultured in lipoprotein-deficient serum, the uptake and degradation of chylomicron remnant cholesteryl ester per mg cell protein varies inversely with cell density. Compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase, stimulates the uptake at all cell densities. Mevalonic acid, on the other hand, can suppress a significant part of the remnant uptake. Chylomicron remnant uptake in hepatocyte cultures can thus be influenced by factors known to regulate the apolipoprotein-BE receptor activity.

Uptake and degradation of rat chylomicron remnants, produced in vivo and in vitro, in rat hepatocyte monolayers.

1. The uptake of small and large chylomicrons in rat hepatocyte monolayer cultures was compared to the uptake of chylomicron remnants prepared either in vitro with pure milk lipoprotein lipase or in hepatectomized rats. 2. Small chylomicrons (Sf less than 400) markedly inhibited remnant uptake and were taken up more efficiently than large ones (Sf greater than 400), indicating that size may be an important factor for the rate of uptake. The Lineweaver-Burk analysis of the data indicated that the V values for the uptake of both small chylomicrons (Sf less than 400) and of remnants prepared either in hepatectomized rats or in vitro was significantly higher than for chylomicrons with Sf greater than 400, whereas the Km values for the different particles did not differ significantly. 3. Preincubation of chylomicrons with serum caused marked changes in their apolipoprotein composition. A loss of apolipoprotein A-I and an increase in apolipoprotein E content was observed by scanning of SDS-polyacrylamide gels. Th preincubation decreased, however, the subsequent uptake of the chylomicrons. In contrast, the uptake of remnants prepared in vivo, or in vitro with serum present, exceeded that of remnants prepared in vitro with albumin or fetal calf serum as the fatty acid acceptor. 4. The data thus indicate that both the decrease in size and the changes in the particle surface during lipolysis with serum present are likely to contribute to the differences seen in the rate of uptake between native chylomicrons and remnants in hepatocyte monolayers.

Regulation of apolipoprotein A-I synthesis in lymph-drained rats.

The effect of lymph diversion on plasma apolipoprotein A-I levels was studied. In lymph fistula rats apolipoprotein A-I levels in plasma stayed constant in spite of a loss of an equivalent of one-half plasma pool of apolipoprotein A-I per day through the lymph fistula. This indicates that synthesis of apolipoprotein A-I increases or that catabolism of apolipoprotein A-I decreases in a compensatory manner as intestinal apolipoprotein A-I is diverted. By using incorporation of [3H]leucine into newly synthesized apolipoprotein A-I it was shown that 2.6-times as much [3H]leucine was incorporated into apolipoprotein A-I in thoracic duct drained animals compared to controls. In experiments in which 125I-labeled HDL was injected intravenously into rats, it was shown that catabolism of HDL and apolipoprotein A-I was not decreased in lymph-drained rats. These data thus suggest that an increased synthesis of apolipoprotein A-I occurs when the intestinal contribution of apolipoprotein A-I diminishes. This is probably due to an increase in liver protein synthesis.

Uptake of chylomicron remnants causes cholesterol accumulation in cultured human arterial smooth muscle cells.

Chylomicron remnants (Sf greater than 100) were prepared by treating human chylomicrons (Sf greater than 400) with human post heparin plasma. Chylomicron remnants recovered after 70-80% of chylomicron triacylglycerol was hydrolyzed, suppressed LDL-receptor activity and increased cell cholesterol esterification to the same extent as did LDL when added to cultured human arterial smooth muscle cells at an equal cholesterol concentration. Cell cholesterol mass increased 36% after incubation with 25 micrograms LDL cholesterol/ml and 35% with 25 micrograms chylomicron-remnant cholesterol/ml. Addition of 30 microM chloroquine plus LDL or chylomicron remnants further increased cholesterol content of cells (74% and 87%, respectively) and caused a significant rise in cell esterified cholesterol (344% and 369%, respectively). Cholesterol content per unit of apolipoprotein B mass of remnants was 2-3-fold higher than that of LDL. Therefore, if lipoprotein particles were added at equivalent apolipoprotein B mass chylomicron remnants increased cell cholesterol content and cholesterol esterification and suppressed LDL receptor activity significantly more than did LDL. This suggests that an additional determinant, presumably apolipoprotein E, is important for receptor recognition of chylomicron remnants. These results may be relevant to the delivery of chylomicron-derived cholesterol to arterial cells proposed as a feature of atherogenesis.

Regulation of chylomicron remnant uptake in the human hepatoma cell-line Hep G2. Role of the low-density lipoprotein receptor.

Uptake and degradation of chylomicron remnants by the human hepatoma cell line Hep G2 was studied. Mesenteric lymph was collected from rats and injected into hepatectomized rats to obtain chylomicron remnants. This remnant preparation was taken up and catabolized by Hep G2 cells. The uptake process was dependent on cell growth and was regulated by compactin (a HMG-CoA reductase inhibitor) which suppresses cholesterol synthesis and by mevalonolactone, which enhances cholesterol synthesis. A monoclonal anti LDL receptor antibody blocked binding of chylomicron remnants to Hep G2 cells to a degree, which was comparable to but generally lower than the suppression of low-density lipoprotein binding. The results thus indicate that in Hep G2 cells, chylomicron remnant uptake is regulated, similarly to low-density lipoprotein uptake and that a significant part of the remnant uptake is mediated through the LDL receptor.

Microcalorimetric investigation of metabolism in rat hepatocytes cultured on microplates and in cell suspensions.

In the present work, heat production rate in rat hepatocytes has been measured by use of thermopile heat conduction calorimeters. Both hepatocytes cultured in monolayers on microplates and hepatocytes in suspensions were used for microcalorimetric measurements. The highest heat production rate was found in newly cultured cells; thereafter, a gradual decrease was noted. After 1 day of culture, metabolic activity had reached a steady state that lasted about 4 days. A cell-density dependence of heat production was found, both in cell suspensions and in cultured hepatocytes on microplates. Higher cell concentration in the calorimeter ampoule was accompanied by decreasing heat production per cell. The heat output recorded for hepatocytes cultured on microplates (25 X 10(3) cells) was found to be 0.327 +/- 0.13 nW per cell after 24-48 h. Addition of sodium azide and sodium fluoride to tissue culture medium reduced heat production rate in cultured hepatocytes by 60 and 20%, respectively. Recording of heat production with the present calorimetric technique is relatively simple and fast, and offers the possibility to perform measurements in small samples of cultured hepatocytes on microplates, thus allowing long-term as well as repeated measurements on the same cell population.

Troglitazone upregulates LDL receptor activity in HepG2 cells.

The aim of this in vitro study was to investigate the effect of troglitazone, a new oral antidiabetic agent, on LDL catabolism. HepG2 cells, which are cells from a well-differentiated cell line of hepatoma cells, were cultured and used to study LDL catabolism. Different concentrations of troglitazone, all within the therapeutic range for humans, were incubated in culture medium with 125I-labeled LDL to measure cell-associated and degraded 125I-LDL. Troglitazone increased cell-associated and degraded 125I-LDL by approximately 30%. We also investigated if this effect occurred through a LDL receptor-mediated pathway or a non-LDL receptor pathway. By using dextran sulfate, a substance known to release bound LDL from its receptor, we found that troglitazone upregulated LDL receptor activity by approximately 35%. In addition, we found that troglitazone increased the expression of the LDL receptor mRNA. The effect of troglitazone was comparable with that of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, fluvastatin, with troglitazone having an upregulatory effect similar to that of fluvastatin. Insulin within human physiological concentrations also increased LDL receptor activity. We found that troglitazone and insulin had an additive effect on LDL catabolism. Also, the effect of troglitazone on LDL catabolism was studied in the presence of cyclosporine, an immunosuppressant drug that reduces LDL catabolism mainly by decreasing LDL receptor activity. The results showed that troglitazone can compensate for the reduced LDL receptor activity induced by cyclosporine, but that cyclosporine had a residual effect on the action of troglitazone. Thus troglitazone enhanced LDL binding, cell association, and degradation by increasing LDL receptor mRNA expression, with a subsequent increase in LDL receptor activity.

Magnesium deficiency in IDDM related to level of glycosylated hemoglobin.

Magnesium and potassium were analyzed in plasma, erythrocytes, and urine collected during 24 h and in muscle biopsies from 25 subjects with insulin-dependent, type I diabetes mellitus (IDDM). Magnesium was also measured in mononuclear cells. The results were compared with those of 28 healthy controls, and were also correlated with the degree of diabetic control as estimated by analysis of the level of glycosylated hemoglobin (HbA1c). Subjects with IDDM had significantly lower muscle (P less than 0.01) and plasma (P less than 0.001) concentrations of magnesium compared with those of healthy controls. The HbA1c levels correlated significantly with the concentrations of magnesium in muscle (r = -0.62, P less than 0.001), plasma (r = -0.62, P less than 0.001), and mononuclear cells (r = -0.47, P less than 0.05). The results indicate that some patients with IDDM have lowered contents of magnesium in striated muscular and/or plasma, and that those parameters are dependent on the degree of diabetic control.

Polymyxin B complexes with and cationizes low density lipoproteins. The cause of polymyxin B-induced enhancement of endocytotic catabolism of low density lipoproteins.

We previously reported that polymyxin B (PMB) enhances cellular catabolism of low density lipoproteins (LDLs) through a non-LDL receptor-mediated endocytotic pathway. These data were obtained mainly by using Hep G2 cells, a well differentiated human hepatoma cell line. In the current study, we explore the mechanisms of PMB-mediated endocytotic catabolism of LDL. We found that PMB enhanced LDL catabolism also in homozygous familial hypercholesterolemia fibroblasts, thereby establishing that PMB-mediated cellular catabolism of LDL does not involve LDL receptors. By using [14C]sucrose, and ligands for the asialoglycoprotein (ASGP) receptors, possibilities were excluded that PMB enhances cellular endocytosis of LDL, by inducing a general increase of cellular pinocytic activity or by causing endocytosis of LDL via the ASGP receptors in Hep G2 cells. We further show, by using polymyxin B coupled Sepharose 4B (PMB-Sepharose 4B) beads, that PMB binds to LDL to form a complex. This binding was tight, and changes in pH and salt concentrations had no significant effect on the binding, but unlabelled LDL competed with 125I-LDL to bind to PMB-Sepharose 4B. Urea and endotoxins decreased this binding, suggesting that PMB binds to LDL at least partially through hydrophobic interactions. Agarose gel electrophoresis of PMB-LDL indicates that PMB cationizes LDL. In conclusion, PMB binds to LDL to form a PMB-LDL complex presumably through interactions between lipid groups. This endows LDL with positive charges, which enhances LDL binding to negatively charged cell membranes, and such bound LDL is rapidly internalized through absorptive endocytosis.

Estrogen-induced increase in uptake of cholesterol-rich very low density lipoproteins in perfused rabbit liver.

A nonrecirculating rabbit liver perfusion system was developed to test whether estrogen increases hepatic uptake of radio-iodinated normal and/or cholesterol-rich very low density lipoproteins (VLDL, d less than 1.006 g/ml) from cholesterol-fed rabbits. When equal concentrations of VLDL protein from normal rabbits and from cholesterol-fed rabbits were perfused together through the same liver, there was a selectively higher (1.4-fold) uptake of cholesterol-rich VLDL. These particles were rich in apolipoprotein E, and the radioactivity bound to this apolipoprotein was selectively removed by the perfused normal rabbit liver relative to its uptake of apolipoproteins B and C. When livers from estrogen-treated rabbits were perfused under identical conditions as normal livers and with the same lipoproteins, the uptake of cholesterol-rich VLDL was increased by 76%, compared with 21% for normal VLDL.

Enhancement of low density lipoprotein catabolism by non-steroidal anti-inflammatory drugs in cultured HepG2 cells.

Several clinical studies have shown that different types of non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the cholesterol content of atherosclerotic blood vessels. The mechanism of this reduction is not established. One possibility is that NSAIDs affect low density lipoprotein (LDL) catabolism. In this study, we investigated the effect of the NSAIDs, indomethacin, flufenamic acid, ibuprofen, acetaminophen, and also acetylsalicylic acid on LDL binding, cell-association and degradation in cultured hepatoma HepG2 cells. LDL was labelled with 125I to study LDL catabolism. Furthermore, dextran sulphate, a substance that is known to release bound LDL from its receptors, was used to study LDL receptor activity. Reverse transcription-polymerase chain reaction was used to study the messenger RNA (mRNA) of LDL receptor. Our results show that flufenamic acid, indomethacin, and to a lesser extent ibuprofen, and acetaminophen increase LDL binding, cell-association, and degradation. Flufenamic acid was most potent and increased LDL catabolism by 50-70%, whereas acetylsalicylic acid had only a modest effect. Also, flufenamic acid and indomethacin were both found to increase the synthesis of mRNA of the LDL receptor with a subsequent increase of LDL receptor protein. We also investigated the effect of indomethacin on LDL binding in the presence of the 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase inhibitor, fluvastatin. We found that both indomethacin and fluvastatin had an additive up-regulatory effect on LDL receptor activity. In addition the effect of flufenamic acid on cell-associated LDL was examined in the presence of cyclosporine, which is known to decrease LDL catabolism. The results show that flufenamic acid can restore the inhibitory effect of cyclosporine. The study thus shows that NSAIDs enhance LDL catabolism due to increased synthesis of the mRNA for LDL receptor protein. This action might contribute to the lipid-lowering effect of NSAIDs.

Low prevalence of anti-neutrophil cytoplasmic antibodies in ulcerative colitis patients with long-term remission.

OBJECTIVES: In spite of a strong positive association between ulcerative colitis and the presence of perinuclear anti-neutrophil cytoplasmic antibodies (p-ANCAs), the immunogenetic significance of these antibodies remains unclear. We studied patients with quiescent disease to clarify whether ANCAs are present even in the absence of inflammation. DESIGN: The prevalence of ANCAs was estimated blindly in 137 patients with ulcerative colitis, 128 of whom had quiescent disease with a mean duration of complete clinical and biochemical remission of 14 years. For comparison, we studied sera from 110 patients with Crohn's disease, 27 of whom had a low or intermediate grade of inflammatory activity. The mean duration of complete remission in these patients was 8.5 years. METHODS: ANCAs were detected using indirect immunofluorescence and enzyme-linked immunosorbent assays (ELISAs). RESULTS: Only 13 (9%) of 137 patients with ulcerative colitis had ANCAs (5% had p-ANCAs). Three patients had previously undergone colectomy. In patients with Crohn's disease, ANCAs were observed in 17 of 110 patients (15%, 6% had p-ANCAs). Fifteen of these patients had colonic disease. CONCLUSION: In patients with ulcerative colitis free from inflammation for prolonged periods of time, ANCAs occurred less frequently than has previously been reported. Patients with Crohn's disease had the expected frequency of ANCA positivity, which for colonic Crohn's disease was comparable to that found in patients with ulcerative colitis. These findings suggest that the titre of ANCAs decreases with time in inactive disease and may be undetectable with conventional assays after several years of complete remission.

Upregulation of low density lipoprotein receptor activity by tumor necrosis factor, a process independent of tumor necrosis factor-induced lipid synthesis and secretion.

It has been shown that tumor necrosis factor (TNF) rapidly upregulates expression of the low density lipoprotein (LDL) receptors on Hep G2 cells and acutely stimulates hepatic lipid synthesis and secretion in vivo. It may thus be possible that TNF-induced expression of LDL receptors is secondary to a decrease in cellular cholesterol content caused by TNF-stimulated lipid secretion. In order to know whether TNF upregulates LDL receptors by depletion of the cellular cholesterol content, the present experiments were designed to study the temporal relationship between TNF-stimulated expression of LDL receptor activity and TNF-induced changes in lipid synthesis and secretion in an in vitro setting by using Hep G2 cells (a highly differentiated human hepatoma cell line) as a hepatocyte model. Hep G2 cells were incubated with TNF (usually 2.5 nmol/L) for certain periods, and LDL receptor activity was evaluated by measuring [125I]LDL binding at 4 degrees C; lipid synthesis and secretion were assayed by measuring [3H]glycerol incorporation into triglycerides and phospholipids as well as [14C]acetate incorporation into cholesterol. We found that a 30-h exposure of the cells to TNF was needed for the effect of TNF to be seen on lipid synthesis and secretion as measured by incorporation of [3H]glycerol into triglycerides and phospholipids, whereas TNF rapidly (in several hours) upregulated LDL receptor activity. TNF stimulated triglyceride synthesis, but did not stimulate phospholipid synthesis. On the other hand, TNF stimulated phospholipid secretion, but did not stimulate triglyceride secretion. Exposure of the cells to TNF for 16 or 24 h neither decreased cholesterol synthesis nor stimulated cholesterol secretion as measured by [14C]acetate incorporation into cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein receptor mediated metabolism of [14C]arachidonic acid labeled chylomicron remnants by Hep G2 cells.

During lipolysis of chylomicron triacylglycerol by lipoprotein lipase, arachidonic acid (AA) esters are hydrolyzed at a slower rate than the predominant 16-18 carbon fatty acid esters. The further metabolism of the AA that is hereby enriched in the chylomicron remnant acylglycerols has not been investigated. In the present study, we examined the low density lipoprotein (LDL) dependent and independent metabolism of [14C]AA present in chylomicron remnants in the human hepatoma cell line Hep G2. Mesenteric duct cannulated rats were fed [14C]AA and [3H]cholesterol in corn oil, and the chyle obtained was injected intravenously into hepatectomized rats to form chylomicron remnants labeled with [14C]AA in the triacylglycerol (TG) and with 3H in the cholesteryl ester portion. The remnants were then incubated with Hep G2 cells. The uptake of [14C]AA within 2-4 h was similar to that of [3H]cholesteryl ester. After uptake into the cells, [14C]AA was preferentially incorporated into phospholipids, a high proportion being found in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. [14C]AA and [3H]cholesteryl ester uptake were influenced to similar extents by factors unknown to regulate the LDL receptor and by an anti-LDL receptor antibody. Addition of compactin thus increased the uptake of [14C]AA by 50% in 4 h and mevalonolactone decreased the uptake by 86%. Using an anti-LDL receptor antibody, 25.0% of [3H]cholesterol/cholesteryl ester and 37.7% of [14C]AA binding to the cells at 4 degrees C were blocked. There was no lipolysis of [14C]TG or [14C]diacylglycerol by lipase secreted into the medium during incubations.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactulose--a potential drug for the treatment of inflammatory bowel disease.

Lactulose is a drug mainly used as a laxative and for the treatment of porto-systemic encephalopathy. Following oral administration, intact lactulose reaches the colon, where it is split by bacteria, leading to a reduction in faecal pH and creating intestinal conditions beneficial to Lactobacillus acidophilus and inhibitory to coliform bacteria, bacteroides, Salmonella and Shigella. It was shown that lactulose therapy clears faecal salmonella and shigella species and reduces the prevalence of urinary-tract infection and respiratory tract infections. Oral administration of lactulose abolishes and prevents systemic endotoxemia of gut origin. Therefore lactulose may be used for treatment of inflammatory bowel disease as bacteria and bacterial endotoxin have an important role in the pathogenesis of this disease.

Measurements of magnesium in mononuclear cells.

In this study analysis of magnesium in mononuclear cells was compared to that in muscle in patients and healthy controls. Mononuclear cell concentrations of magnesium expressed as nmol/mg cell protein correlated significantly with muscle biopsy concentrations of magnesium (r = 0,68; p less than 0,001). Hence, we suggest that analyzing of magnesium in mononuclear cells could be an easy and convenient way to estimate tissue magnesium.

Cost-Effectiveness of Budesonide Controlled Ileal Release (CIR) Capsules as Maintenance Therapy versus No Maintenance Therapy for Ileocaecal Crohn's Disease in Sweden.

A decision-analytic model was designed to estimate the associated costs and outcomes of maintenance therapy for Crohn's disease with budesonide controlled ileal release (CIR) capsules (Entocort((R)) capsules, Astra Draco, Lund, Sweden) versus no maintenance therapy. A third-party payer perspective was adopted to compare the direct costs associated with the medication and healthcare resource use for each therapy over a period of 12 four-week cycles. The costs of routine patient care and the consequences of failure, in terms of relapses, acute therapies, hospitalisations and surgery, were included. The outcome was measured as the average number of days in remission per patient per 12-cycle period. Based on the assumptions in the model, the results show that budesonide CIR capsules are associated with a reduction of 16.6 (26%) days in relapse, i.e. a 6% increase in days in remission, over a one-year period compared with no maintenance therapy. Direct healthcare costs are increased by 6% or Swedish kronor (SEK) 1673 ($US1 ~ SEK7.60). Overall, the model shows that there are substantial (non-drug associated) cost offsets from using budesonide CIR capsules as maintenance therapy in Crohn's disease. These cost offsets, in addition to improvements in patients' well-being and quality of life, indicate that maintenance therapy is cost effective compared with no maintenance therapy. The cost per added day in remission is relatively modest (SEK101 ~ $US13). If indirect costs are added to the calculation, it is realistic to argue that a net saving to society would be most likely.

[Enteroscopy valuable in obscure small bowel disease. Chances are good to discover curable conditions]. / Enteroskopi av värde vid oklar tunntarmssjukdom. Chansen stor att hitta behandlingsbara tillstånd.

Indications for enteroscopic examination of the proximal small bowel are expanding, above all in cases of gastro-intestinal bleeding of obscure origin. Of 66 patients examined enteroscopy revealed new and unforeseen diagnoses in about half of them, such as angiodysplasia and erosions (15 per cent of cases each). Former as well as ongoing bleeding was treated with electro cautery, bicap. In four cases the need for blood transfusion ceased. Ulcers, neoplasia and varices were also diagnosed. 16 out of 36 pathologic lesions were located within reach of an ordinary gastroscope, in spite of the patients being selected through repeated normal upper and lower endoscopic examinations. This emphasises the need for better quality assurance in routine endoscopic examinations.