Regulation of bile acid metabolism in biliary atresia: reduction of FGF19 by Kasai portoenterostomy and possible relation to early outcome.

BACKGROUND: Fibroblast growth factor 19 (FGF19) is produced in the small intestine and is involved in suppression of hepatic bile acid (BA) synthesis. FGF19 is also expressed in the liver and serum levels are elevated in adults with cholestatic liver disease. This may reflect a rescue mechanism to dampen liver injury caused by increased intrahepatic BAs. OBJECTIVES: To examine circulating FGF19 at early stages of biliary atresia and at short-term follow-up post-Kasai portoenterostomy (KPE) in relation to noncholestatic infants. The relationship between FGF19, BAs and markers for BA synthesis and hepatic gene expression of factors involved in BA metabolism were also evaluated. METHODS: Liver tissue, portal and peripheral blood samples were obtained from fifteen patients at KPE; additional blood was collected 4-6 months after surgery. Two control groups were included; to examine possible changes related to surgery and to compare FGF19 in biliary atresia to noncholestatic infants. RESULTS: Circulating FGF19 levels correlated to its hepatic gene expression at time of KPE in biliary atresia and levels were elevated compared to noncholestatic infants. At follow-up, FGF19 levels were markedly reduced, and the decline coincided with reductions in bilirubin and conjugated chenodeoxycholic acid and with increased levels of the BA synthesis marker C4. CONCLUSION: Elevated circulating FGF19 in biliary atresia is of hepatic origin and reduced following KPE. Changes in serum FGF19 may reflect the level of restoration of the enterohepatic circulation, and this warrants further long-term studies on the role of FGF19 in the cholestatic liver.

Asynchronous rhythms of circulating conjugated and unconjugated bile acids in the modulation of human metabolism.

BACKGROUND AND OBJECTIVES: Bile acids (BAs) traversing the enterohepatic circulation (EHC) influence important metabolic pathways. By determining individual serum BAs in relation to markers of metabolic activity, we explored how diurnal variations in their EHC relate to hepatic metabolism in normal humans. METHODS: Serum BAs, fibroblast growth factor 19 (FGF19), lipoproteins, glucose/insulin and markers of cholesterol and BA syntheses were monitored for 32 h in 8 healthy males. Studies were conducted at basal state and during initiation of cholestyramine treatment, with and without atorvastatin pretreatment. Time series cross-correlation analysis, Bayesian structural model and Granger causality test were applied. RESULTS: Bile acids synthesis dominated daytime, and cholesterol production at night. Conjugated BAs peaked after food intake, with subsequent FGF19 elevations. BA synthesis was reduced following conjugated BA and FGF19 peaks. Cholestyramine reduced conjugated BAs and FGF19, and increased BA and cholesterol production; the latter effects attenuated by atorvastatin. The relative importance of FGF19 vs. conjugated BAs in this feedback inhibition could not be discriminated. Unconjugated BAs displayed one major peak late at night/early morning that was unrelated to FGF19 and BA synthesis, and abolished by cholestyramine. The normal suppression of serum triglycerides, glucose and insulin observed at night was attenuated by cholestyramine. CONCLUSIONS: Conjugated and unconjugated BAs have asynchronous rhythms of EHC in humans. Postprandial transintestinal flux of conjugated BAs increases circulating FGF19 levels and suppresses BA synthesis. Unconjugated BAs peak late at night, indicating a non-postprandial diurnal change in human gut microflora, the physiological implications of which warrants further study.

Treatment with the natural FXR agonist chenodeoxycholic acid reduces clearance of plasma LDL whilst decreasing circulating PCSK9, lipoprotein(a) and apolipoprotein C-III.

BACKGROUND: The natural farnesoid X receptor (FXR) agonist chenodeoxycholic acid (CDCA) suppresses hepatic cholesterol and bile acid synthesis and reduces biliary cholesterol secretion and triglyceride production. Animal studies have shown that bile acids downregulate hepatic LDL receptors (LDLRs); however, information on LDL metabolism in humans is limited. METHODS: Kinetics of autologous 125 I-LDL were determined in 12 male subjects at baseline and during treatment with CDCA (15 mg kg-1 day-1 ). In seven patients with gallstones treated with CDCA for 3 weeks before cholecystectomy, liver biopsies were collected and analysed for enzyme activities and for specific LDLR binding. Serum samples obtained before treatment and at surgery were analysed for markers of lipid metabolism, lipoproteins and the LDLR modulator proprotein convertase subtilisin/kexin type 9 (PCSK9). RESULTS: Chenodeoxycholic acid treatment increased plasma LDL cholesterol by ~10% as a result of reduced clearance of plasma LDL-apolipoprotein (apo)B; LDL production was somewhat reduced. The reduction in LDL clearance occurred within 1 day after initiation of treatment. In CDCA-treated patients with gallstones, hepatic microsomal cholesterol 7α-hydroxylase and HMG-CoA reductase activities were reduced by 83% and 54%, respectively, and specific LDLR binding was reduced by 20%. During treatment, serum levels of fibroblast growth factor 19 and total and LDL cholesterol increased, whereas levels of 7α-hydroxy-4-cholesten-3-one, lathosterol, PCSK9, apoA-I, apoC-III, lipoprotein(a), triglycerides and insulin were reduced. CONCLUSIONS: Chenodeoxycholic acid has a broad influence on lipid metabolism, including reducing plasma clearance of LDL. The reduction in circulating PCSK9 may dampen its effect on hepatic LDLRs and plasma LDL cholesterol. Further studies of the effects of other FXR agonists on cholesterol metabolism in humans seem warranted, considering the renewed interest for such therapy in liver disease and diabetes.

Acute caloric restriction counteracts hepatic bile acid and cholesterol deficiency in morbid obesity.

BACKGROUND: Bile acid (BA) synthesis is regulated by BA signalling in the liver and by fibroblast growth factor 19 (FGF19), synthesized and released from the intestine. In morbid obesity, faecal excretion and hepatic synthesis of BAs and cholesterol are strongly induced and caloric restriction reduces their faecal excretion considerably. We hypothesized that the high intestinal food mass in morbidly obese subjects promotes faecal excretion of BAs and cholesterol, thereby creating a shortage of both BAs and cholesterol in the liver. METHODS: Ten morbidly obese women (BMI 42 ± 2.6 kg m-2 ) were monitored on days 0, 3, 7, 14 and 28 after beginning a low-calorie diet (800-1100 kcal day-1 ). Serum was collected and liver size and fat content determined. Synthesis of BAs and cholesterol was evaluated from serum markers, and the serum levels of lipoproteins, BAs, proprotein convertase subtilisin/kexin type 9 (PCSK9), insulin, glucose and FGF19 were monitored. Fifty-four nonobese women (BMI <25 kg m-2 ) served as controls. RESULTS: At baseline, synthesis of both BAs and cholesterol and serum levels of BAs and PCSK9 were elevated in the obese group compared to controls. Already after 3 days on a low-calorie diet, BA and cholesterol synthesis and serum BA and PCSK9 levels normalized, whereas LDL cholesterol increased. FGF19 and triglyceride levels were unchanged, and liver volume was reduced by 10%. CONCLUSIONS: The results suggest that hepatic BAs and cholesterol are deficient in morbid obesity. Caloric restriction rapidly counteracts these deficiencies, normalizing BA and cholesterol synthesis and circulating PCSK9 levels, indicating that overproduction of cholesterol in enlarged peripheral tissues cannot explain this phenotype. We propose that excessive food intake promotes faecal loss of BAs and cholesterol contributing to their hepatic deficiencies.

Muricholic bile acids are potent regulators of bile acid synthesis via a positive feedback mechanism.

OBJECTIVE: Bile acid (BA) synthesis is regulated by negative feedback end-product inhibition, initiated by farnesoid X receptors (FXRs) in liver and gut. Studies on cholic acid (CA)-free Cyp8b1(-/-) mice have concluded that CA is a potent suppressor of BA synthesis. Cyp8b1(-/-) mice have increased BA synthesis and an enlarged BA pool, a phenotype shared with bile-duct-ligated, antibiotics-administered and with germ-free mice. Studies on such mice have concluded BA synthesis is induced due to reduced hormonal signalling by fibroblast growth factor (FGF)15 from intestine to liver. A mutual finding in these models is that potent FXR-agonistic BAs are reduced. We hypothesized that the absence of the potent FXR agonist deoxycholic acid (DCA) may be important for the induction of BA synthesis in these situations. DESIGN: Two of these models were investigated, antibiotic treatment and Cyp8b1(-/-) mice and their combination. Secondary BA formation was inhibited by ampicillin (AMP) given to wild-type and Cyp8b1(-/-) mice. We then administered CA, chenodeoxycholic acid (CDCA) or DCA to AMP-treated Cyp8b1(-/-) mice. RESULTS: Our data show that the phenotype of AMP-treated wild-type mice resembles that of Cyp8b1(-/-) mice with fourfold induced Cyp7a1 expression, increased intestinal apical sodium-dependent BA transporter expression and increased hepatic BA levels. We also show that reductions in the FXR-agonistic BAs CDCA, CA, DCA or lithocholic acid cannot explain this phenotype; instead, it is likely due to increases in levels of α- and ß-muricholic BAs and ursodeoxycholic acid, three FXR-antagonistic BAs. CONCLUSIONS: Our findings reveal a potent positive feedback mechanism for regulation of BA synthesis in mice that appears to be sufficient without endocrine effects of FGF15 on Cyp7a1. This mechanism will be fundamental in understanding BA metabolism in both mice and humans.

Influence of growth hormone on circulating fibroblast growth factor 21 levels in humans.

OBJECTIVE: Findings from animal studies indicate that growth hormone (GH) may stimulate the production of the putative metabolic regulator fibroblast growth factor 21 (FGF21). We investigated whether circulating FGF21 levels are altered in patients with GH deficiency and characterized how levels of this growth factor are influenced by acute and long-term administration of GH, and the potential relationship between FGF21 and nonesterified fatty acids (NEFAs). DESIGN AND SETTING: GH-deficient patients (n = 9) were studied prior to and during 1 year of replacement with GH. Healthy subjects (n = 8) received an intravenous bolus of GH with or without concomitant oral glucose. Healthy subjects and patients with heterozygous familial hypercholesterolaemia (n = 23) were monitored following increasing doses of GH for 3 weeks. The main outcome measures were serum FGF21 and NEFA levels. Studies were performed at two academic centres. RESULTS: GH-deficient patients had FGF21 levels within the normal range, and GH replacement did not influence circulating FGF21 or NEFA concentrations. Acute GH administration to healthy control subjects did not change FGF21 levels, whereas an oral glucose load increased serum FGF21 by 25% and reduced NEFA levels by 48%. Similar effects were seen on administration of glucose together with GH. However, FGF21 levels increased dose dependently up to 3.7-fold in control subjects treated with GH for 3 weeks; simultaneously NEFA levels were increased by 47%. CONCLUSIONS: GH is not critical for the maintenance of basal serum FGF21 levels in humans, but circulating FGF21 levels increase following administration of GH to healthy individuals. There is no correlation between plasma NEFA and circulating FGF21 levels.

Pronounced variation in bile acid synthesis in humans is related to gender, hypertriglyceridaemia and circulating levels of fibroblast growth factor 19.

BACKGROUND: Bile acid (BA) synthesis is essential in cholesterol and lipid homoeostasis. METHODS: Serum samples from 435 normal and 23 cholecystectomized subjects were obtained after overnight fasting and assayed for markers of BA and cholesterol synthesis, as well as cholesterol absorption. We determined whether BA synthesis was related to fibroblast growth factor 19 (FGF19; a circulating metabolic regulator that is thought to inhibit BA synthesis), gender, age and serum lipids. RESULTS: Bile acid synthesis varied more than 9-fold in normal individuals and was 29% higher in men than in women. Whilst low-density lipoprotein cholesterol increased with age, BA and cholesterol synthesis were stable. BA production was positively correlated with serum triglycerides (TGs), and 35% of individuals with a high level (>95th percentile) of BA synthesis had hypertriglyceridaemia (HTG) (>95th percentile). Serum FGF19 levels varied by 7-fold in normal individuals and were related inversely to BA synthesis but were not related to gender, plasma lipids or history of cholecystectomy. CONCLUSIONS: Bile acid synthesis has a wide inter-individual variation, is lower in women than in men and is correlated positively with serum TGs. High BA production is frequently linked to HTG. Age-related hypercholesterolaemia is not associated with changes in BA or cholesterol production, nor to an increase in cholesterol absorption. In humans, the circulating level of FGF19 may regulate hepatic BA production under fasting conditions.

Stimulation of rat hepatic low density lipoprotein receptors by glucagon. Evidence of a novel regulatory mechanism in vivo.

We studied the influence of glucagon on hepatic LDL receptors and plasma lipoproteins in rats. A dose-dependent (maximum, threefold) increase in LDL-receptor binding was evident already at a dose of 2 x 4 micrograms, and detectable 3 h after injection; concomitantly, cholesterol and apolipoprotein (apo) B and apoE within LDL and large HDL decreased in plasma. LDL receptor mRNA levels were however unaltered or reduced. Hepatic microsomal cholesterol was increased and the enzymatic activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in hepatic microsomes were reduced. Insulin alone increased receptor binding and receptor mRNA levels twofold, but plasma cholesterol was unchanged and plasma apoE and apoB increased. Administration of insulin to glucagon-treated animals reduced the LDL-receptor binding to control levels and apoB appeared in LDL particles. Estrogen treatment increased LDL-receptor binding and mRNA levels five- and eightfold, respectively. Combined treatment with glucagon and estrogen reduced the stimulation of LDL-receptor mRNA levels by 80% although LDL-receptor binding was unchanged. Immunoblot analysis showed that glucagon increased the number of hepatic LDL receptors. We conclude that glucagon induces the number of hepatic LDL receptors by a mechanism not related to increased mRNA levels, suggesting the presence of a posttranscriptional regulatory mechanism present in the liver in vivo.

Growth hormone and bile acid synthesis. Key role for the activity of hepatic microsomal cholesterol 7alpha-hydroxylase in the rat.

Growth hormone (GH) has an important role in the regulation of hepatic LDL receptor expression and plasma lipoprotein levels. This investigation was undertaken to characterize the effects of GH on hepatic cholesterol and bile acid metabolism in the rat. In hypophysectomized (Hx) rats, the activities of the rate-limiting enzymes in cholesterol and bile acid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and cholesterol 7alpha-hydroxylase (C7alphaOH), were reduced by 71 and 64%, respectively. HMG CoA reductase mRNA levels were reduced by 37%, whereas C7alphaOH mRNA was increased by 81%. LDL receptor expression was reduced by 18% in Hx rats, without any change in the LDL receptor mRNA levels. Although the normal diurnal variation of C7alphaOH activity was preserved in Hx rats, the activity of C7alphaOH was much reduced both at midday and midnight. Total hepatic cholesterol was increased by 14% in Hx animals whereas microsomal cholesterol was unchanged. The rate of cholesterol esterification was enhanced (by 38%) in liver microsomes from Hx rats. Stepwise hormonal substitution of Hx rats showed that GH, but not thyroid hormone or cortisone, was essential to normalize the enzymatic activity of C7alphaOH. GH also normalized the altered plasma lipoprotein pattern in Hx rats, and increased the fecal output of bile acids. The latter effect was particularly evident when GH was combined with cortisone and thyroid hormone. Also in normal rats, GH stimulated C7alphaOH activity. In conclusion, GH has an essential role to maintain a normal enzymatic activity of C7alphaOH, and this, at least in part, explains the effects of GH on hepatic cholesterol metabolism. GH is also of critical importance to normalize the altered plasma lipoprotein pattern in Hx rats.

Regulation of rat hepatic low density lipoprotein receptors. In vivo stimulation by growth hormone is not mediated by insulin-like growth factor I.

Growth hormone (GH) has an important role in the regulation of hepatic LDL receptor expression and plasma lipoprotein levels. This investigation was undertaken to evaluate if these effects of GH on hepatic LDL receptors are direct or mediated by insulin-like growth factor I (IGF-I). Two models were studied in which substitution with GH is important for the regulation of hepatic LDL receptors: hypophysectomized rats receiving high-dose ethynylestradiol or challenge with dietary cholesterol. The hypophysectomized rats were hormonally substituted by infusion with dexamethasone and L-thyroxine, and either GH or IGF-I. In both models, GH was essential for maintaining normal expression of LDL receptors. In contrast, despite fully normalized plasma levels, IGF-I did not support the expression of hepatic LDL receptors. Analysis of plasma lipoproteins revealed that substitution with GH, but not with IGF-I, reduced LDL and intermediate density lipoproteins. In addition, determination of hepatic mRNA levels for apo B-100 and apo B-48 indicated that GH may be more effective than IGF-I in the promotion of apo B mRNA editing. In conclusion, GH has specific effects on hepatic LDL receptor expression and plasma lipoprotein levels that are not mediated by IGF-I.

Effects of estrogen on low density lipoprotein metabolism in males. Short-term and long-term studies during hormonal treatment of prostatic carcinoma.

To characterize the effects of estrogen treatment on the metabolism of LDL we studied six males with metastatic prostatic carcinoma before and during the initiation of therapy; a repeated study was performed in five participants after 3-6 mo of treatment. The fractional catabolic rate (FCR) of autologous 125I-LDL was calculated both from elimination curves of plasma radioactivity and from urine/plasma (U/P) radioactivity ratios. Within 1-2 d of onset of estrogen therapy a more rapid decay of plasma radioactivity occurred, and FCR measured from U/P ratios increased by 20%. Concomitantly, LDL cholesterol levels decreased by 16%. After 3-6 mo of treatment FCR determined by both techniques was almost doubled, and LDL cholesterol was reduced by 34%. This occurred despite a 29% increase in the calculated synthesis rate of LDL. Tissue culture studies demonstrated that the receptor affinity of LDL isolated from patients on long-term estrogen therapy was reduced. We conclude that a profound increase in LDL catabolism is induced through administration of pharmacological doses of estrogen in males, and hypothesize that this is the consequence of an increased expression of hepatic LDL receptors. This enhanced catabolism of LDL leaves LDL particles in plasma with lower affinity for the LDL receptor.

Hepatic cholesterol metabolism and resistance to dietary cholesterol in LXRbeta-deficient mice.

The nuclear oxysterol-receptor paralogues LXRalpha and LXRbeta share a high degree of amino acid identity and bind endogenous oxysterol ligands with similar affinities. While LXRalpha has been established as an important regulator of cholesterol catabolism in cholesterol-fed mice, little is known about the function of LXRbeta in vivo. We have generated mouse lines with targeted disruptions of each of these LXR receptors and have compared their responses to dietary cholesterol. Serum and hepatic cholesterol levels and lipoprotein profiles of cholesterol-fed animals revealed no significant differences between LXRbeta(-/-) and wild-type mice. Steady-state mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, and squalene synthase were increased in LXRbeta(-/-) mice compared with LXRbeta(+/+) mice, when fed standard chow. The mRNA levels for cholesterol 7alpha-hydroxylase, oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase, and sterol 27-hydroxylase, respectively, were comparable in these strains, both on standard and 2% cholesterol chow. Our results indicate that LXRbeta(-/-) mice - in contrast to LXRalpha(-/-) mice - maintain their resistance to dietary cholesterol, despite subtle effects on the expression of genes coding for enzymes involved in lipid metabolism. Thus, our data indicate that LXRbeta has no complete overlapping function compared with LXRalpha in the liver.

Atheroprotective effect of adjuvants in apolipoprotein E knockout mice.

Strategies aimed at treating atherosclerosis by immunization protocols are emerging. Such protocols commonly use adjuvants as non-specific stimulators of immune responses. However, adjuvants are known to modify various disease processes. The aim of this study was to determine whether adjuvants alter the development of atherosclerosis. We performed immunization protocols in apolipoprotein E knockout mice (E degrees ) following chronic administration schedules commonly employed in experimental atherosclerosis. Our results point out a dramatic effect of several adjuvants on the development of atherosclerosis; three of the four adjuvants tested reduced lesion size. The Alum adjuvant, which is the adjuvant currently used in most vaccination protocols in humans, displayed a strong atheroprotective effect. Mechanisms accounting for atheroprotective effect of Freund's adjuvants included their capacity to increase both Th2 responses and anti-MDA-LDL IgM titers, and/or to impose atheroprotective lipoprotein profiles. The present study indicates that adjuvants have potent atheromodulating capabilities, and thus, implies that the choice of adjuvant is crucial in long-term immunization protocols in experimental atherosclerosis.

Budesonide treatment is associated with increased bile acid absorption in collagenous colitis.

BACKGROUND: Bile acid malabsorption is frequent in collagenous colitis and harmful bile acids may play a pathophysiological role. Glucocorticoids increase ileal bile acid transport. Budesonide have its main effect in the terminal ileum. AIMS: To evaluate whether the symptomatic effect of budesonide is linked to increased uptake of bile acids. METHODS: Patients with collagenous colitis were treated with budesonide 9 mg daily for 12 weeks. Prior to and after 8 weeks of treatment, the (75)SeHCAT test, an indirect test for the active uptake of bile acid-s, measurements of serum 7alpha-hydroxy-4-cholesten-3-one, an indicator of hepatic bile acid synthesis, and registration of symptoms were performed. RESULTS: The median (75)SeHCAT retention increased from 18% to 35% (P < 0.001, n = 25) approaching the values of healthy controls (38%). The 7alpha-hydroxy-4-cholesten-3-one values decreased significantly among those with initially high synthesis (from 36 to 23 ng/mL, P = 0.04, n = 9); however, for the whole group the values were not altered (19 ng/mL vs. 13 ng/mL, P = 0.23, N.S., n = 19). CONCLUSION: The normalization of the (75)SeHCAT test and the reduction of bile acid synthesis in patients with initially high synthetic rate, suggests that the effect of budesonide in collagenous colitis may be in part due to decreased bile acid load on the colon.

Long-term GH treatment of GH-deficient adults: comparison between one and two daily injections.

The physiological pulsatile secretion of GH in humans might be important for the metabolic effects of GH. In the treatment of GH-deficient (GHD) patients, the most common regimen is a single sc injection at bedtime. It has not been completely established if this is the optimal mode of GH administration during long-term GH treatment. The aim of the present study was to evaluate the metabolic effects of two different GH replacement regimens comparing one to two daily injections. Eight men and six women, 42-78 yr old, with verified severe GHD, participated. Patients were matched for gender, age and body mass index (BMI) and were randomized to GH therapy (one or two injections daily) for 12 months. GH doses were individually titrated to IGF-I levels of age-matched controls. IGF-I, glucose, insulin, oral glucose tolerance test (OGTT), cholesterol, triglycerides, lipoproteins, including size fractionation with fast performance liquid chromatography, BMI and body composition were analyzed. After 12 months the median GH dose was 0.45 mg (range 0.25-0.50 mg) in both groups. Body fat had decreased by 20% (p<0.05) in the group receiving one daily GH injection. There were no differences between the two treatment groups in indices of carbohydrate or lipid metabolism. The administration of GH divided into two daily doses offered no major advantage as compared to the more convenient single injection in the evening. The GH-induced reduction in body fat occurred independently from changes in serum lipids.

Delivery of aclacinomycin A to human glioma cells in vitro by the low-density lipoprotein pathway.

The present study shows that a human malignant glioma cell line (U-251 MG) accumulates and degrades low-density lipoprotein (LDL) by a saturable, high-affinity process (Km approximately equal to 5 micrograms/ml). Accumulation and degradation could be enhanced by preincubating the cells in a lipoprotein-deficient medium. The LDL degradation rate was highest when the cells were proliferating rapidly. An aclacinomycin A:LDL complex containing 150 to 450 drug molecules per LDL particle could be obtained by incubating LDL with a large excess of aclacinomycin A at 40 degrees. When the glioma cells were incubated with the aclacinomycin A:LDL complex, cellular drug accumulation was dependent on the LDL receptor activity. There are four reasons for drawing this conclusion. (a) U-251 MG cells with high LDL receptor activity accumulated more drug than U-251 MG cells with low LDL receptor activity. (b) U-251 MG cells accumulated more drug than a mutant fibroblast line (GM 1915) lacking LDL receptor activity. (c) Aclacinomycin A accumulation was increased when U-251 MG cells were incubated in the presence of chloroquine, an agent that inhibits LDL degradation. (d) Aclacinomycin A accumulation was reduced when U-251 MG cells were incubated in the presence of either an excess of native LDL or heparin, which has been demonstrated to inhibit receptor-mediated binding and degradation of LDL. The aclacinomycin A:LDL complex also inhibited growth of the glioma cells. Our results suggest that the glioma cells studied have LDL receptors and that it may be possible to use LDL as a vehicle for lipophilic antineoplastic drugs in order to increase the drug accumulation in tumor cell populations with high LDL receptor activity.

Low density lipoprotein receptor activity in human intracranial tumors and its relation to the cholesterol requirement.

The receptor binding of low density lipoprotein (LDL) was determined in homogenates of surgically removed specimens from primary and metastatic intracranial tumors and in some cases also from surrounding brain. Seventy-one specimens from 63 patients were analyzed. In a subsample of 16 specimens from 13 patients, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was assayed in parallel. The LDL binding in the tumors varied over a wide range. A significantly higher LDL binding activity was found when all tumor samples were compared to brain (P less than 0.05). In the three patients where LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase activities were assayed in both tumor tissue and surrounding brain, it was found that the receptor or the enzyme activity was increased in the tumors. It is suggested that certain intracranial tumors have an increased cholesterol requirement and that this may be fulfilled by an enhanced LDL receptor activity or an increased 3-hydroxy-3-methylglutaryl-CoA reductase activity. The data indicate that the LDL receptor activity may be regulated independently of the reductase in intracranial tumors.

Role of the liver for the receptor-mediated catabolism of low-density lipoprotein in the 17 alpha-ethinylestradiol-treated rat.

Effects of ethinylestradiol on LDL catabolism and plasma cholesterol were studied in the rat. Hormone treatment led to a reduction in plasma cholesterol and to an increased receptor-mediated catabolism of LDL in the liver and the adrenals. The liver was the most important organ accounting for more than 95% of the receptor-mediated tissue accumulation of LDL following estrogen treatment. It is concluded that the reduction in plasma cholesterol during treatment with ethinylestradiol is due to the stimulation of LDL receptors in the liver.

A simple binding assay for the determination of low-density lipoprotein receptors in cell homogenates.

A convenient binding assay has been developed for the determination of low-density lipoprotein (LDL) receptors in homogenates of cultured and freshly-isolated normal and malignant human cells. Cell homogenates were incubated with 125I-labeled LDL and the ligand bound to the homogenate particulates was separated from the unbound ligand by filtration. When the particulates of the homogenates were subsequently incubated with heparin, a fraction of the bound 125I-LDL was released. Previous studies on intact cells have shown that heparin exclusively releases LDL bound to its cell surface receptor. The heparin-sensitive binding of 125I-LDL to cell homogenate particulates represents LDL bound to its cell surface receptor as judged from the following criteria: (a) it was quantitatively similar to the heparin-sensitive binding of 125I-LDL to intact cells, (b) it showed a direct correlation to the receptor-mediated degradation of 125I-LDL by intact cells, (c) no heparin-sensitive binding could be detected in homogenates prepared from normal erythrocytes or from cultured fibroblasts from a patient with homozygous familial hypercholesterolemia (two types of cell lacking LDL receptors), (d) it was dependent on calcium and inhibited by EDTA, (e) it was susceptible to treatment with pronase, and (f) it was heat-labile. The assay developed should be of value in determining the number of LDL receptors in tissues, since it is far less time-consuming and requires less material than currently available methods.

LDL receptors in bovine tissues assayed as the heparin-sensitive binding of 125I-labeled LDL in homogenates: relation between liver LDL receptors and serum cholesterol in the fetus and post term.

The heparin-sensitive binding of 125I-labeled LDL in homogenates of bovine tissues was determined using a membrane filter assay. The binding fulfilled several criteria which have been established for the binding of LDL to its receptor, namely: saturability, dependence on Ca2+, sensitivity to proteolytic destruction and heat sensitivity. The adrenal cortex and the active corpus luteum exhibited the highest binding activity of the 22 different tissues assayed. Tissues from the central nervous system had low binding activity. Livers from fetal animals had higher binding than livers from young and adult animals and the binding of 125I-LDL to fetal liver homogenates showed an inverse correlation to the serum cholesterol levels, indicating that the LDL receptors in fetal liver may play a role in the regulation of the serum cholesterol level in the fetus during gestation. After birth, the binding of 125I-LDL to calf liver homogenates decreased to levels found in adult animals and this was paralleled by an increase of total serum cholesterol, suggesting that the rapid rise in serum cholesterol in mammals observed soon after birth may be caused by a decrease of the receptor-mediated catabolism of LDL in the liver.