Margrit Schwarz

A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels ≈2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR−/− mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.

Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis.

The in vivo role of the nuclear receptor SHP in feedback regulation of bile acid synthesis was examined. Loss of SHP in mice caused abnormal accumulation and increased synthesis of bile acids due to derepression of rate-limiting CYP7A1 and CYP8B1 hydroxylase enzymes in the biosynthetic pathway. Dietary bile acids induced liver damage and restored feedback regulation. A synthetic agonist of the nuclear receptor FXR was not hepatotoxic and had no regulatory effects. Reduction of the bile acid pool with cholestyramine enhanced CYP7A1 and CYP8B1 expression. We conclude that input from three negative regulatory pathways controls bile acid synthesis. One is mediated by SHP, and two are SHP independent and invoked by liver damage and changes in bile acid pool size.

Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease.

We describe a metabolic defect in bile acid synthesis involving a deficiency in 7alpha-hydroxylation due to a mutation in the gene for the microsomal oxysterol 7alpha-hydroxylase enzyme, active in the acidic pathway for bile acid synthesis. The defect, identified in a 10-wk-old boy presenting with severe cholestasis, cirrhosis, and liver synthetic failure, was established by fast atom bombardment ionization-mass spectrometry, which revealed elevated urinary bile acid excretion, a mass spectrum with intense ions at m/z 453 and m/z 510 corresponding to sulfate and glycosulfate conjugates of unsaturated monohydroxy-cholenoic acids, and an absence of primary bile acids. Gas chromatography-mass spectrometric analysis confirmed the major products of hepatic synthesis to be 3beta-hydroxy-5-cholenoic and 3beta-hydroxy-5-cholestenoic acids, which accounted for 96% of the total serum bile acids. Levels of 27-hydroxycholesterol were > 4,500 times normal. The biochemical findings were consistent with a deficiency in 7alpha-hydroxylation, leading to the accumulation of hepatotoxic unsaturated monohydroxy bile acids. Hepatic microsomal oxysterol 7alpha-hydroxylase activity was undetectable in the patient. Gene analysis revealed a cytosine to thymidine transition mutation in exon 5 that converts an arginine codon at position 388 to a stop codon. The truncated protein was inactive when expressed in 293 cells. These findings indicate the quantitative importance of the acidic pathway in early life in humans and define a further inborn error in bile acid synthesis as a metabolic cause of severe cholestatic liver disease.

Disruption of Cholesterol 7α-Hydroxylase Gene in Mice II. BILE ACID DEFICIENCY IS OVERCOME BY INDUCTION OF OXYSTEROL 7α-HYDROXYLASE

Past experiments and current paradigms of cholesterol homeostasis suggest that cholesterol 7α-hydroxylase plays a crucial role in sterol metabolism by controlling the conversion of cholesterol into bile acids. Consistent with this conclusion, we show in the accompanying paper that mice deficient in cholesterol 7α-hydroxylase (Cyp7−/− mice) exhibit a complex phenotype consisting of abnormal lipid excretion, skin pathologies, and behavioral irregularities (Ishibashi, S., Schwarz, M., Frykman, P. K., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 261, 18017-18023). Aspects of lipid metabolism in the Cyp7−/− mice are characterized here to deduce the physiological basis of this phenotype. Serum lipid, cholesterol, and lipoprotein contents are indistinguishable between wild-type and Cyp7−/− mice. Vitamin D3 and E levels are low to undetectable in knockout animals. Stool fat content is significantly elevated in newborn Cyp7−/− mice and gradually declines to wild-type levels at 28 days of age. Several species of 7α-hydroxylated bile acids are detected in the bile and stool of adult Cyp7−/− animals. A hepatic oxysterol 7α-hydroxylase enzyme activity that may account for the 7α-hydroxylated bile acids is induced between days 21 and 30 in both wild-type and deficient mice. An anomalous oily coat in the Cyp7−/− animals is due to the presence of excess monoglyceride esters in the fur. These data show that 7α-hydroxylase and the pathway of bile acid synthesis initiated by this enzyme are essential for proper absorption of dietary lipids and fat-soluble vitamins in newborn mice, but not for the maintenance of serum cholesterol and lipid levels. In older animals, an alternate pathway of bile acid synthesis involving an inducible oxysterol 7α-hydroxylase plays a crucial role in lipid and bile acid metabolism.

Disruption of Cholesterol 7α-Hydroxylase Gene in Mice I. POSTNATAL LETHALITY REVERSED BY BILE ACID AND VITAMIN SUPPLEMENTATION

Mice deficient in cholesterol 7α-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis, were constructed by targeted disruption of the Cyp7 gene. The introduced mutation removed exons 3-5 of the gene and gave rise to a null allele that encoded no immunoreactive or enzymatically active protein. Heterozygous carriers of the disrupted gene (Cyp7+/−) were phenotypically normal. Homozygous animals (Cyp7−/−) appeared normal at birth, but died within the first 18 days of life. Approximately 40% of the animals died between postnatal days 1 and 4 and 45% between days 11 and 18. The addition of vitamins to the water of nursing mothers prevented deaths in the early period, whereas the addition of cholic acid to chow prevented deaths in the later period. Newborn Cyp7−/− mice whose mothers were maintained on unsupplemented chow failed to gain weight at a normal rate and developed oily coats, hyperkeratosis, and apparent vision defects. These symptoms waned at 3 weeks of life, and their disappearance was accompanied by a marked increase in survival. In the accompanying study, the induction of an alternate pathway of bile acid biosynthesis is shown to underlie this unusual time course (Schwarz, M., Lund, E. G., Setchell, K. D. R., Kayden, H. J., Zerwekh, J. E., Björkhem, I., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18024-18031). We conclude that cholesterol 7α-hydroxylase is an essential enzyme for normal postnatal development.

Two 7 alpha-hydroxylase enzymes in bile acid biosynthesis

The addition of a 7‐hydroxyl group is an early and often rate‐limiting step in the synthesis of bile acids. This reaction is catalysed by two cytochrome P450 enzymes known as cholesterol 7α‐hydroxylase and oxysterol 7α‐hydroxylase. cDNAs encoding these proteins have been isolated and used to define

Effects of peroxisome proliferator-activated receptor alpha/delta agonists on HDL-cholesterol in vervet monkeys.

The objective of this study was to demonstrate the efficacy of a novel peroxisome proliferator-activated receptor (PPAR) agonist and known PPARα and PPARδ agonists to increase HDL-cholesterol (HDL-C) in the St. Kitts vervet, a nonhuman primate model of atherosclerosis. Four groups (n = 6) were studied and each group was assigned one of the following “treatments”: a) vehicle only (vehicle); b) the PPARδ selective agonist GW501516 (GW); c) the PPARα/δ agonist T913659 (T659); and d) the PPARα agonist TriCor® (fenofibrate). No statistically significant changes were seen in body weight, total plasma cholesterol, plasma triglycerides, VLDL-C, LDL-C, or apolipoprotein B (apoB) concentrations. Each of the PPARα and PPARδ agonists investigated in this study increased plasma HDL-C, apoA-I, and apoA-II concentrations and increased HDL particle size in St. Kitts vervets. The maximum percentage increase in HDL-C from baseline for each group was as follows: vehicle, 5%; GW, 43%; T659, 43%; and fenofibrate, 20%. Treatment with GW and T659 resulted in an increase in medium-sized HDL particles, whereas fenofibrate showed increases in large HDL particles. These data provide additional evidence that PPARα and PPARδ agonists (both mixed and selective) have beneficial effects on HDL-C in these experimental primates.

The bile acid synthetic gene 3β-hydroxy-Δ5-C27-steroid oxidoreductase is mutated in progressive intrahepatic cholestasis

We used expression cloning to isolate cDNAs encoding a microsomal 3β-hydroxy-Δ5-C27-steroid oxidoreductase (C27 3β-HSD) that is expressed predominantly in the liver. The predicted product shares 34% sequence identity with the C19 and C21 3β-HSD enzymes, which participate in steroid hormone metabolism. When transfected into cultured cells, the cloned C27 3β-HSD cDNA encodes an enzyme that is active against four 7α-hydroxylated sterols, indicating that a single C27 3β-HSD enzyme can participate in all known pathways of bile acid synthesis. The expressed enzyme did not metabolize several different C19/21 steroids as substrates. The levels of hepatic C27 3β-HSD mRNA in the mouse are not sexually dimorphic and do not change in response to dietary cholesterol or to changes in bile acid pool size. The corresponding human gene on chromosome 16p11.2-12 contains six exons and spans 3 kb of DNA, and we identified a 2-bp deletion in the C27 3β-HSD gene of a patient with neonatal progressive intrahepatic cholestasis. This mutation eliminates the activity of the enzyme in transfected cells. These findings establish the central role of C27 3β-HSD in the biosynthesis of bile acids and provide molecular tools for the diagnosis of a third type of neonatal progressive intrahepatic cholestasis associated with impaired bile acid synthesis.

Activation of FFA1 mediates GLP-1 secretion in mice. Evidence for allosterism at FFA1.

FFA1 (GPR40) and GPR120 are G-protein-coupled receptors activated by long-chain fatty acids. FFA1 is expressed in pancreatic β-cells, where it regulates glucose-dependent insulin secretion, and GPR120 has been implicated in mediating GLP-1 secretion. We show here that FFA1 co-localizes with GLP-1 in enteroendocrine cells and plays a critical role in glucose management by mediating GLP-1 secretion in vivo. Corn oil induces GLP-1 secretion in wild type mice and in GPR120-/- mice, but not in FFA1-/- mice. α-Linolenic acid, an endogenous ligand of FFA1, induces GLP-1 secretion in GLUTag cells and in primary fetal mouse intestinal cells. Synthetic partial FFA1 agonists do not stimulate GLP-1 secretion in mice, but partial and full agonists combined function cooperatively to enhance receptor activation and GLP-1 secretion both in vitro and in vivo. We conclude that allosterism at FFA1 can contribute to postprandial glucose management by stimulating insulin secretion via an extrapancreatic mechanism of action, and that GPR120 in GLP-1 secretion requires further investigation.

Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine

Scavenger receptor class B, type I (SR-BI) is expressed in the intestines of rodents and has been suggested to be involved in the absorption of dietary cholesterol. The aim of this study was to determine whether intestinal SR-BI expression is affected in animal models with altered bile delivery to the intestine and impaired cholesterol absorption. SR-BI protein and mRNA levels were determined in proximal and distal small intestine from control, bile-duct-ligated and bile-diverted rats and from control and bile-duct-ligated mice. Two genetically altered mouse models were studied: multidrug resistance-2 P-glycoprotein-deficient [Mdr2((-/-))] mice that produce phospholipid/cholesterol-free bile, and cholesterol 7alpha-hydroxylase-deficient [Cyp7a((-/-))] mice, which exhibit qualitative and quantitative changes in the bile-salt pool. Cholesterol-absorption efficiency was quantified using a dual-isotope ratio method. SR-BI was present at the apical membrane of enterocytes in control rats and mice and was more abundant in proximal than in distal segments of the intestine. In bile-duct-ligated animals, levels of SR-BI protein were virtually absent and mRNA levels were decreased by approximately 50%. Bile-diverted rats, Mdr2((-/-)) mice and Cyp7a((-/-)) mice showed decreased levels of intestinal SR-BI protein while mRNA levels were unaffected. Cholesterol absorption was reduced by >90% in bile-duct-ligated and bile-diverted animals and in Cyp7a((-/-)) mice, whereas Mdr2((-/-)) mice showed an approximately 50% reduction. This study shows that SR-BI is expressed at the apical membrane of enterocytes of rats and mice, mainly in the upper intestine where cholesterol absorption is greatest, and indicates that bile components play a role in post-transcriptional regulation of SR-BI expression. Factors associated with cholestasis appear to be involved in transcriptional control of intestinal SR-BI expression. The role of SR-BI in the cholesterol-absorption process remains to be defined.