Influence of physiological changes in endogenous estrogen on circulating PCSK9 and LDL cholesterol.

Pharmacologically increased estrogen levels have been shown to lower hepatic and plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) levels in animals and humans. We hypothesized that physiological changes in estrogen levels influence circulating PCSK9, thereby contributing to the known wide inter-individual variation in its plasma levels, as well as to the established increase in LDL cholesterol (LDL-C) with normal aging. Circulating PCSK9, estradiol, and other metabolic factors were determined in fasting samples from 206 female and 189 male healthy volunteers (age 20-85 years), The mean levels of PCSK9 were 10% higher in females than in males (P < 0.05). PCSK9 levels were 22% higher in postmenopausal than in premenopausal (P < 0.001) females. Within the group of premenopausal females, circulating PCSK9 correlated inversely to estrogen levels, and PCSK9 was higher (305 ng/ml) in the follicular phase than in the ovulatory (234 ng/ml) or the luteal (252 ng/ml) phases (P < 0.05). Changes in endogenous estrogen levels during the menstrual cycle likely contribute to the broad inter-individual variation in PCSK9 and LDL-C in normal females. PCSK9 levels increase in females after menopause but not in men during this phase in life. This likely contributes to why LDL-C in women increases in this period.

Circulating proprotein convertase subtilisin kexin type 9 has a diurnal rhythm synchronous with cholesterol synthesis and is reduced by fasting in humans.

OBJECTIVE: To gain insight into the function of proprotein convertase subtilisin kexin type 9 (PCSK9) in humans by establishing whether circulating levels are influenced by diurnal, dietary, and hormonal changes. METHODS AND RESULTS: We monitored circulating PCSK9 in a set of dynamic human experiments and could show that serum PCSK9 levels display a diurnal rhythm that closely parallels that of cholesterol synthesis, measured as serum lathosterol. In contrast to these marked diurnal changes in cholesterol metabolism, serum low-density lipoprotein (LDL) cholesterol levels remained stable during the diurnal cycle. Depletion of liver cholesterol by treatment with the bile acid-binding resin, cholestyramine, abolished the diurnal rhythms of both PCSK9 and lathosterol. Fasting (>18 hours) strongly reduced circulating PCSK9 and lathosterol levels, whereas serum LDL levels remained unchanged. Growth hormone, known to be increased during fasting in humans, reduced circulating PCSK9 in parallel to LDL cholesterol levels. CONCLUSIONS: Throughout the day, and in response to fasting and cholesterol depletion, circulating PCSK9 displays marked variation, presumably related to oscillations in hepatic cholesterol that modify its activity in parallel with cholesterol synthesis. In addition to this sterol-mediated regulation, additional effects on LDL receptors may be mediated by hormones directly influencing PCSK9.

Importance of proprotein convertase subtilisin/kexin type 9 in the hormonal and dietary regulation of rat liver low-density lipoprotein receptors.

Hormonal or dietary challenge can stimulate hepatic low-density lipoprotein receptor (LDLR) expression through posttranscriptional mechanisms. We here tested whether such observations may be due to regulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). Treatment with glucagon resulted in a 2-fold increase in hepatic LDLR protein expression, whereas its mRNA levels were reduced; this occurred simultaneously with a 70% reduction in PCSK9 expression. Insulin treatment resulted in responses opposite to those seen by treatment with glucagon. Furthermore, high-dose ethinylestradiol treatment reduced PCSK9 expression by half. Finally, feeding of rats with dietary cholesterol reduced PCSK9 expression, resulting in an increased number of hepatic LDLRs despite a reduction of LDLR mRNA levels. Regulation of PCSK9 occurred in part through sterol regulatory element binding protein-2, but changes in this cholesterol-controlled transcription factor could not explain all hormonal effects seen. We conclude that the hormonal and dietary regulation of hepatic LDLRs also involves posttranscriptional regulation by PCSK9. The identification of PCSK9 regulation by these various treatments is important in understanding of the physiological function of this protein and points to new targets for therapeutic treatments to increase hepatic LDLR numbers.

The circulating metabolic regulator FGF21 is induced by prolonged fasting and PPARalpha activation in man.

FGF21 is a critical metabolic regulator, pivotal for fasting adaptation and directly regulated by PPARalpha in rodents. However, the physiological role of FGF21 in man is not yet defined and was investigated in our study. Serum FGF21 varied 250-fold among 76 healthy individuals and did not relate to age, gender, body mass index (BMI), serum lipids, or plasma glucose. FGF21 levels had no diurnal variation and were unrelated to bile acid or cholesterol synthesis. Ketosis induced by a 2 day fast or feeding a ketogenic diet (KD) did not influence FGF21 levels, whereas a 74% increase occurred after 7 days of fasting. Hypertriglyceridemic nondiabetic patients had 2-fold elevated FGF21 levels, which were further increased by 28% during fenofibrate treatment. FGF21 circulates in human plasma and increases by extreme fasting and PPARalpha activation. The wide interindividual variation and the induction of ketogenesis independent of FGF21 levels indicate that the physiological role of FGF21 in humans may differ from that in mice.

Dramatically increased intestinal absorption of cholesterol following hypophysectomy is normalized by thyroid hormone.

BACKGROUND & AIMS: Hypopituitarism is associated with dyslipidemia, and feeding hypophysectomized rats cholesterol induces severe hypercholesterolemia. This study aimed to unravel further how hypophysectomy alters cholesterol and bile acid metabolism. METHODS: Intact and hypophysectomized rats were studied during challenge with dietary cholesterol and ezetimibe and upon hormonal substitution with growth hormone, cortisone, and thyroid hormone. RESULTS: Five findings were established in hypophysectomized rats: (1) The intestinal absorption of cholesterol is doubled. (2) Treatment with ezetimibe abolishes the increases in serum and liver cholesterol. (3) Only thyroid hormone treatment normalizes the increased absorption of cholesterol. (4) The intestinal gene expression of cholesterol transporters NPC1L1 and ABCG5/G8 is unaltered, whereas the hepatic expression of ABCG5/G8 is diminished but strongly stimulated by thyroid hormone. The latter mechanism was supported by measurements of biliary cholesterol and of fecal neutral steroids. (5) The reduced hepatic expression of ABCG5/G8 and Cyp7a1 was normalized by cholesterol feeding, suggesting that other nonestablished mechanisms under pituitary control are important to maintain rats resistant to dietary cholesterol. CONCLUSIONS: The intestinal absorption of dietary cholesterol is under pituitary control largely exerted by thyroid hormone. Hepatic secretion of cholesterol and ABCG5/G8 expression are strongly stimulated in hypophysectomized rats during treatment with thyroid hormone.

Age-induced hypercholesterolemia in the rat relates to reduced elimination but not increased intestinal absorption of cholesterol.

Plasma cholesterol increases in normal aging in both rodents and humans. This is associated with reduced elimination of cholesterol as bile acids (BAs) and decreased receptor-mediated clearance of plasma LDL, changes that can be reversed by treatment with growth hormone (GH). The level of intestinal absorption of cholesterol may also contribute to the development of hypercholesterolemia. In this study, we investigated whether cholesterol absorption increases with age and whether any such age-related change could be influenced by treatment with GH or ezetimibe (EZE). Male rats aged 6 and 18 mo were studied with and without GH or EZE treatment. BA synthesis was reduced and plasma cholesterol was increased in the old animals, whereas cholesterol absorption was unaltered. Cholesterol absorption was not altered by GH treatment but was reduced by EZE in both groups of animals. Hepatic LDL receptors (LDLRs), scavenger receptor class B type 1, and proprotein convertase subtilisin/kexin type 9 serine protease (PCSK9) transcripts were unchanged in old animals. GH treatment induced LDLRs, PCSK9 transcripts, and BA synthesis. We conclude that the age-induced hypercholesterolemia in the rat and its reversal by GH treatment relates to altered degradation of cholesterol in the liver and is not due to changes in cholesterol absorption.

Increased activity of hepatic microsomal triglyceride transfer protein and bile acid synthesis in gallstone disease.

UNLABELLED: A strong interrelationship exists between the regulation of bile acid (BA) metabolism and hepatic very low density lipoprotein (VLDL) production. We have recently shown that BA synthesis is increased in gallstone disease. We investigated the activity of hepatic microsomal triglyceride transfer protein (MTTP) as a surrogate of VLDL production, BA synthesis, and mRNA expression levels of proteins that regulate fatty acid (FA) metabolism in the liver of gallstone (GS) patients compared with GS-free patients. Twenty-seven volunteers subjected to elective surgery; 9 were GS-free and 18 with GS agreed to have a liver biopsy. We quantified by a fluorescence assay the activity of MTTP and by quantitative reverse-transcription PCR (RT-PCR) the mRNA content of hepatic MTTP and genes that regulate hepatic sterol and FA metabolism. Plasma was assayed for lathosterol and 7alpha-hydroxy-4-cholesten-3-one. Liver histology was normal in GS and GS-free patients. Serum VLDL triglycerides and apoB were significantly increased in GS. Hepatic triglycerides tripled in GS (P<0.001) compared with GS-free. MTTP activity increased 70% (P<0.001). Serum lathosterol and hepatic cholesterol concentrations, and mRNA expressions of MTTP, CD36, and FABP1 were similar in GS-free and GS patients. Hepatic mRNA expression of hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and 3-hydroxyl-3-methyl-glutaryl-CoA synthase (HMGS) were significantly decreased--40% and 27%, respectively--in GS. Serum 7alpha-hydroxy-4-cholesten-3-one was 75% higher, and mRNA expression of CYP7A1 was increased sevenfold (P<0.001) in GS. CONCLUSION: Hepatic MTTP activity and BA synthesis are increased in GS. Results suggest that hepatic VLDL production and trafficking of BA are increased in gallstone patients.

Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption.

The etiology of most cases of idiopathic bile acid malabsorption (IBAM) is unknown. In this study, a Swedish family with bile acid malabsorption in three consecutive generations was screened for mutations in the ileal apical sodium-bile acid cotransporter gene (ASBT; gene symbol, SLC10A2) and in the genes for several of the nuclear receptors known to be important for ASBT expression: the farnesoid X receptor (FXR) and peroxisome proliferator activated receptor alpha (PPARalpha). The patients presented with a clinical history of idiopathic chronic watery diarrhea, which was responsive to cholestyramine treatment and consistent with IBAM. Bile acid absorption was determined using (75)Se-homocholic acid taurine (SeHCAT); bile acid synthesis was estimated by measuring the plasma levels of 7alpha-hydroxy-4-cholesten-3-one (C4). The ASBT, FXR, and PPARalpha genes in the affected and unaffected family members were analyzed using single stranded conformation polymorphism (SSCP), denaturing HPLC, and direct sequencing. No ASBT mutations were identified and the ASBT gene did not segregate with the bile acid malabsorption phenotype. Similarly, no mutations or polymorphisms were identified in the FXR or PPARalpha genes associated with the bile acid malabsorption phenotype. These studies indicate that the intestinal bile acid malabsorption in these patients cannot be attributed to defects in ASBT. In the absence of apparent ileal disease, alternative explanations such as accelerated transit through the small intestine may be responsible for the IBAM.

Normal or increased bile acid uptake in isolated mucosa from patients with bile acid malabsorption.

INTRODUCTION: Bile acid malabsorption as reflected by an abnormal Se-labelled homocholic acid-taurine (SeHCAT) test is associated with diarrhoea, but the mechanisms and cause-and-effect relations are unclear. OBJECTIVES: Primarily, to determine whether there is a reduced active bile acid uptake in the terminal ileum in patients with bile acid malabsorption. Secondarily, to study the linkage between bile acid malabsorption and hepatic bile acid synthesis. METHODS: Ileal biopsies were taken from patients with diarrhoea and from controls with normal bowel habits. Maximal active bile acid uptake was assessed in ileal biopsies using a previously validated technique based on uptake of C-labelled taurocholate. To monitor the hepatic synthesis, 7alpha-hydroxy-4-cholesten-3-one, a bile acid precursor, was assayed in blood. The SeHCAT-retention test was used to diagnose bile acid malabsorption. RESULTS: The taurocholate uptake in specimens from diarrhoea patients was higher compared with the controls [median, 7.7 (n=53) vs 6.1 micromol/g per min (n=17)] (P<0.01) but no difference was seen between those with bile acid malabsorption (n=18) versus diarrhoea with a normal SeHCAT test (n=23). The SeHCAT values and 7alpha-hydroxy-4-cholesten-3-one were inversely correlated. CONCLUSIONS: The data do not support bile acid malabsorption being due to a reduced active bile acid uptake capacity in the terminal ileum.

Bile acid synthesis in humans has a rapid diurnal variation that is asynchronous with cholesterol synthesis.

BACKGROUND & AIMS: The conversion of cholesterol to bile acids by the liver is an important regulator of body cholesterol homeostasis. In rodents, both cholesterol and bile acid synthesis have marked diurnal rhythms that peak synchronously at midnight. The aim of this study was to establish whether such diurnal rhythms are also present in healthy humans. METHODS: Serum levels of the markers 7alpha-hydroxy-4-cholesten-3-one (C4) monitoring bile acid biosynthesis and lathosterol reflecting cholesterol synthesis were determined at 90-minute intervals in 8 human volunteers during standardized dietary conditions. RESULTS: Serum C4 showed 2 distinct peaks (2- to 4-fold above baseline) during a 24-hour period, the first at 1:00 pm and the second at 9:00 pm. During the night, C4 levels declined, and they returned to baseline levels the next morning. In contrast, serum lathosterol levels peaked at night, between midnight and 4:00 am. The diurnal changes of C4 were not synchronous with serum lipid changes or with the postprandial increase in serum bile acids and were maintained in cholecystectomized subjects. CONCLUSIONS: Bile acid synthesis in humans has a diurnal rhythm, with 2 peaks during the daytime, that is opposite from the circadian rhythm of cholesterol synthesis. This is completely different from the pattern in rodents and indicates the presence of an important species variation in the regulation of cholesterol homeostasis.

Bile acid synthesis is increased in Chilean Hispanics with gallstones and in gallstone high-risk Mapuche Indians.

BACKGROUND & AIMS: Gallstone disease is an important, costly health-care problem in Western societies. It is still unclear whether hepatic lipid regulatory enzymes play primary or secondary roles in gallstone formation. In this study, the aim was to investigate whether the synthesis of bile acids and cholesterol is increased in gallstone disease and to test whether such a metabolic change, if present, might occur before gallstone formation. METHODS: A total of 125 Chilean Hispanic women (80 without gallstones and 45 with gallstones) matched for age and body mass index were investigated, along with 40 Chilean Mapuche Indian women (20 without gallstones and 20 with gallstones), a population group in which the prevalence for gallstone disease is very high. Fasting blood plasma samples were assayed for 7 alpha-hydroxy-4-cholesten-3-one and lathosterol, 2 strong indicators for hepatic bile acid and body cholesterol synthesis, respectively. RESULTS: Plasma 7 alpha-hydroxy-4-cholesten-3-one levels, corrected for plasma cholesterol, were significantly increased by 50% in Hispanic women with gallstones as compared with gallstone-free Hispanics (P < 0.006). As compared with Hispanic women without gallstones, plasma 7 alpha-hydroxy-4-cholesten-3-one levels were increased by > or =100% (P < 0.002) in Mapuche Indian women, independently of whether gallstones were present. Plasma lathosterol, corrected for plasma cholesterol, was significantly increased by 22% in Hispanic women with gallstones and in Mapuche Indian women compared with Hispanic women. CONCLUSIONS: The results indicate that the synthesis of bile acids and cholesterol is induced in gallstone disease and precedes gallstone development. These inductions presumably occur as a response to an increased intestinal loss of bile acids.

Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alpha-hydroxy-4-cholesten-3-one in peripheral blood.

We describe an accurate method for monitoring the enzymatic activity of hepatic cholesterol 7alpha-hydroxylase (C7alphaOH; CYP7A1), the rate-limiting and major regulatory enzyme in the synthesis of bile acids. Assay of 7alpha-hydroxy-4-cholesten-3-one (C4), an intermediate in bile acid synthesis, revealed that the level of C4 in peripheral blood serum or plasma showed a strong correlation to the enzymatic activity of hepatic C7alphaOH, both at steady-state conditions (r = 0.929) as well as during the rapid changes that occur during the diurnal phases. This assay should be of value in clarifying the regulation of bile acid synthesis in vivo in laboratory animals and humans since it allows for the monitoring of hepatic C7alphaOH activity using peripheral blood samples.

Pharmacological interference with intestinal bile acid transport reduces plasma cholesterol in LDL receptor/apoE deficiency.

Reduction of plasma cholesterol by statins is fundamental to prevent coronary heart disease. Such therapy is often sub-optimal, however, particularly in patients with reduced LDL receptors (familial hypercholesterolemia), and novel or adjuvant therapies are therefore warranted. Cholesterol elimination is profoundly influenced by the rate of its conversion to bile acids (BA), regulated by the enzyme Cyp7a1. Induced fecal loss of BA by resin treatment reduces plasma cholesterol, presumably through induction of hepatic LDL receptors (LDLR). We here describe the effect of PR835, a drug belonging to a new class of lipid-lowering agents that inhibit the Slc10a2 protein, the intestinal transporter responsible for active uptake of BA. Treatment reduced plasma cholesterol by 40% in mice devoid of both the LDLR and its ligand, apoE, while triglycerides and HDL cholesterol were unchanged. Cyp7a1 enzyme activity and mRNA were induced several-fold, and hepatic HMG CoA reductase mRNA increased, mirroring an induced synthesis of BA and cholesterol. The addition of a statin potentiated the effect, leading to reductions of plasma total and LDL cholesterol by 64% and 70%, respectively. These effects could not be attributed to induction of other known hepatic lipoprotein receptors and indicate the presence of new points of targeting in lipid-lowering therapy.

Prolonged stimulation of the adrenals by corticotropin suppresses hepatic low-density lipoprotein and high-density lipoprotein receptors and increases plasma cholesterol.

Pituitary ACTH has been shown to strongly stimulate adrenal receptors for low-density lipoprotein (LDL) and high-density lipoprotein (HDL) scavenger receptor class B type 1(SR-BI) to provide precursor cholesterol for glucocorticoid synthesis. The present study aimed to determine the effects of ACTH on hepatic cholesterol metabolism and plasma lipoproteins. Treatment of Sprague Dawley rats or normal C57BL/6J mice with ACTH for 3.5 d reduced hepatic SR-BI and LDL receptors. Simultaneously, cholesterol in plasma LDL and HDL was increased. None of these effects could be reproduced using glucocorticoids instead of ACTH, and they were abolished in adrenalectomized rats, indicating an obligate role of the adrenals for the effects of ACTH observed in the liver. When ACTH was given to LDL receptor-deficient mice, plasma LDL did not increase and the increase in HDL cholesterol remained, as did the suppression of hepatic SR-BI. Our data show that prolonged ACTH treatment suppresses hepatic SR-BI and LDL receptors in vivo in rodents, resulting in elevated plasma HDL and LDL. The adrenals are obligate for these effects, suggesting that ACTH releases some factor(s) that suppresses hepatic LDL and SR-BI receptors. Hypothetically, this novel mechanism would further promote channeling of cholesterol to the adrenals in situations of prolonged stress.