Thyroid hormone reduces PCSK9 and stimulates bile acid

1 Thyroid hormone reduces PCSK9 and stimulates bile acid synthesis in humans Running title: Thyroid hormone and human lipid metabolism Ylva Bonde (y...
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Thyroid hormone reduces PCSK9 and stimulates bile acid synthesis in humans Running title: Thyroid hormone and human lipid metabolism

Ylva Bonde ([email protected])1,2, Olof Breuer ([email protected])3, Dieter Lütjohann ([email protected] )4, Stefan Sjöberg ([email protected])1, Bo Angelin ([email protected])1,2 and Mats Rudling ([email protected])1,2

1

Department of Medicine, and 2Department of Biosciences and Nutrition, Karolinska Institute at

Karolinska University Hospital Huddinge, S-14186 Stockholm, Sweden, 3Karo Bio AB, Novum, S-14186 Stockholm, Sweden, 4Institute of Clinical Chemistry and Clinical Pharmacology, University Clinics Bonn, D-53105 Bonn, Germany

Correspondence: Ylva Bonde, Karolinska Institutet, C2:84, Karolinska University Hospital Huddinge, S14186 Stockholm, Sweden; Phone: +46768721299; Fax: +4687110710; E-mail: [email protected]

Abbreviations and acronyms: 7α-hydroxy-4-cholestene-3-one; C4, chenodeoxycholic acid; CDCA, cholesterol 7alpha-hydroxylase; CYP7A1, cholic acid; CA, deoxycholic acid; DCA, euthyroid state; EU, farnesoid X receptor; FXR, fibroblast growth factor; FGF, free thyroxine; fT4, free triiodothyronine; fT3, hyperthyroid state; HY, lipoprotein(a); Lp(a), low density lipoprotein receptor; LDLR, off eprotirome; -E, on eprotirome; +E, proprotein convertase subtilisin/kexin type 9; PCSK9, sex hormone binding globulin; SHBG, sterol regulatory binding protein; SREBP, thyroid hormone; TH, thyroid stimulating hormone; TSH

2 ABSTRACT

Objectives. Reduced plasma LDL-cholesterol is a hallmark of hyperthyroidism and is caused by transcriptional stimulation of LDL receptors in the liver. Here, we investigated if thyroid hormone actions involve other mechanisms that may also account for the reduction in LDL-cholesterol, including effects on proprotein convertase subtilisin/kexin type 9 (PCSK9) and bile acid synthesis. Approach and Results. Twenty hyperthyroid patients were studied before and after clinical normalization, and the responses to hyperthyroidism were compared to those in fourteen healthy individuals after fourteen days of treatment with the liver-selective thyroid hormone analog eprotirome. Both hyperthyroidism and eprotirome treatment reduced circulating PCSK9, lipoprotein cholesterol, apoB and AI, and lipoprotein(a), while cholesterol synthesis was stable. Hyperthyroidism, but not eprotirome treatment, markedly increased bile acid synthesis and reduced fibroblast growth factor 19 (FGF19) and dietary cholesterol absorption. Eprotirome treatment, but not hyperthyroidism, reduced plasma triglycerides. Neither hyperthyroidism nor eprotirome treatment altered insulin, glucose or fibroblast growth factor 21 (FGF21) levels. Conclusions. Thyroid hormone reduces circulating PSCK9 thereby likely contributing to lower plasma LDL-cholesterol in hyperthyroidism. Thyroid hormone also stimulates bile acid synthesis although this response is not critical for its LDL lowering effect.

Keywords: Lipoproteins/Metabolism, Cholesterol 7alpha-hydroxylase, Cholesterol/Absorption, Bile acids and salts/Biosynthesis, fibroblast growth factor, FGF19, FGF21, proprotein convertase subtilisin/kexin type 9, eprotirome, Drug therapy/Hypolipidemic drugs

3 INTRODUCTION Thyroid hormone (TH) is a potent regulator of multiple metabolic pathways by interaction with TH nuclear receptors in various tissues (1-3). Lipoprotein metabolism is strongly influenced by TH and dyslipidemia is common in thyroid disorders (4). Reduced plasma LDL-cholesterol is a hallmark of hyperthyroidism and caused by increased transcription of LDL receptors (LDLRs) in the liver. In rodents, TH stimulates processes that contribute to elimination of cholesterol from the body, including the conversion of cholesterol into bile acids (5) and biliary secretion of bile acids and cholesterol (6). TH also diminishes intestinal absorption of dietary cholesterol (7) and stimulates cholesterol synthesis (5). The importance of these mechanisms for lowering LDL-cholesterol in humans is somewhat unclear, as is the possible involvement of novel regulators of lipid metabolism such as proprotein convertase subtilisin/kexin type 9 (PCSK9) (8) and fibroblast growth factor (FGF) 19 and 21 (9). The aim of this study was therefore to further characterize the effects of TH on cholesterol and lipoprotein metabolism in humans. For this purpose, two models of exposure to TH were used: (a) patients with hyperthyroidism before and after clinical normalization, and (b) healthy volunteers treated for fourteen days with a liverselective TH analog, eprotirome (10, 11).

4 MATERIALS AND METHODS

Subjects and study design. The first study (a) included 16 women and 4 men who had been referred to our outpatient unit due to hyperthyroidism. They were between 18 and 73 years old (mean±SD, 46±14 years) with serum levels of thyroid stimulating hormone (TSH) 6.5 pmol/L. Patients who were pregnant or had been diagnosed with malignancy were excluded. Diagnoses were based on serum levels of TSH and THs, presence of thyroid antibodies, and thyroid gland enlargement. Seventeen patients were diagnosed as having Grave’s disease; sixteen of these were treated with tiamazol (Thacapzol) and levothyroxine, and one received radioiodine treatment and levothyroxine. One patient was diagnosed as having toxic uninodular goiter and was treated with radioiodine. Two patients were diagnosed as having thyroiditis with transient nodular thyrotoxicosis; they became euthyroid without medical treatment. Blood samples were collected between 08:30-09:00 AM after overnight fast on two occasions: before start of treatment, and when serum fT3 was normalized (3.0-6.5 pmol/L). The interval between the samplings ranged between 4 and 25 weeks (mean±SD, 14±6 weeks). In the second study (b), samples were obtained from 14 healthy volunteers (7 women and 7 men) between 25 and 55 years old (mean±SD, 41±11 years), and with BMI between 22 and 29 kg/m2 (mean±SD, 26±3 kg/m2). They had been included in a study evaluating a potential drug interaction between eprotirome and warfarin using a double-blind cross-over design (KBT011; Eudra CT 2011-003029-92). Eprotirome is a liverselective TH receptor agonist that has been tested in human hypercholesterolemia (10-12). Despite promising results in early trials, the development program for eprotirome was discontinued in 2012 due to a toxicology study that revealed cartilage damage in dogs after long-term exposure. Samples taken after 14 days of treatment with 100 µg/d of eprotirome (Karo Bio AB, Sweden) were compared to samples obtained prior to treatment or after a wash-out period of 14 days after the last dose. Body composition. Body weight and composition were measured using a bioelectrical impedance scale (TBF-305, Umedico AB, Sweden).

5 THs, lipids and glucose. Serum levels of fT3, free thyroxine (fT4), TSH, insulin, and plasma levels of total cholesterol, triglycerides, and glucose were measured using a MODULAR ANALYTICS P170/P800 (Roche/Hitachi). Serum levels of cholesterol and triglycerides within VLDL, LDL, and HDL fractions, and glycerol, were measured by fast protein liquid chromatography (FPLC) (13). For all assays, kits from Roche Diagnostics GmbH (Mannheim, Germany) were used. In eprotirome-treated subjects, insulin levels were measured using ELISA kits (Mercodia AB, Uppsala, Sweden). Serum levels of sex hormone binding globulin (SHBG) was assayed using ELISA kits (SHBG, MX52011, IBL International GmbH, Hamburg, Germany) according to manufacturer’s instructions. Serum levels of free fatty acids (FFAs) were measured using kits from Kamiya Biomedical Company (Seattle, WA) and a Tecan Infinite M200. Apolipoproteins. Serum levels of apoAI (KAI-002), AII (KAI-003), B (KAI-004), CII (KAI-005), and CIII (KAI-006) were determined using immunoturbidimetric assays (Kamiya Biomedical Company). Serum levels of apoAIV were measured using ELISA kits from Millipore (EZHAP0A4-73K, Billerica, MA). All analyses were carried out in duplicate following the manufacturer’s instructions. Serum lipoprotein(a) [Lp(a)] levels were determined in duplicate samples with an immunoturbidimetric assay using kits from DiaSys Diagnostic Systems GmbH (Lp(a) 21 FS, Holzheim, Germany) and a Response 910 analyzer. PCSK9 and FGF19/21. ELISA kits were used to determine serum levels of PCSK9 (CY-8079, CycLex Co. Ldt., Nagano, Japan), FGF19 and FGF21 (DF1900 and DF2100, respectively; R&D Systems Europe Ltd., Abingdon, UK). All analyses were carried out following the manufacturer’s instructions. Bile acid synthesis. In patients, serum levels of 7α-hydroxy-4-cholestene-3-one (C4), a marker of bile acid synthesis (14-17), were assayed in duplicate samples as described (14) and normalized for plasma total cholesterol levels (18). In healthy volunteers, serum levels of the marker 7α-hydroxycholesterol (19) were assayed as described and normalized for plasma cholesterol (20). Cholesterol synthesis. Serum levels of the cholesterol synthesis marker lathosterol (21-24) were assayed in the hyperthyroid patients as described (21) and in eprotirome-treated subjects as described (25). Serum levels of lathosterol were normalized for plasma cholesterol.

6 Dietary cholesterol absorption. In patients, serum levels of campesterol and sitosterol were determined using GC-MS in duplicate samples as described (7) and in eprotirome-treated subjects as described (26). Serum levels of plant sterols were normalized for plasma cholesterol. Serum bile acids. Serum levels of chenodeoxycholic acid (CDCA), cholic acid (CA), deoxycholic acid (DCA), and their amino acid conjugates, were assayed using 250µL of serum in duplicate samples. Acetonitril was added to samples which were then centrifuged at 13000g for 15min. The upper phase was collected and dried under nitrogen before dissolved in methanol and analyzed by LC/MS/MS using D 4bile acids as internal standards. Bile acids in samples from subjects treated with eprotirome were analyzed as described (27). Statistics. Diagrams show individual data and horizontal bars represent mean levels. Two-tailed Wilcoxon matched-pairs test was used to test significance of differences. Correlations were tested by the Spearman rank correlation coefficient. Significance threshold was set at p

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