surface of the HDL particle, LCAT maintains a chemical gradient that facilitates a unidirectional net fl ow of cholesterol from the cell surface of peripheral tissues to HDL particles in the blood ( 2 ). Mature HDL particles ultimately transport cholesterol (in the form of CE) back to the liver in pathways that involve cholesteryl ester transfer protein, LDL/VLDL, LDL receptor, and scavenger receptor class B type I. Hence, LCAT has been hypothesized to play a central role in driving HDL remodeling and reverse cholesterol transport (RCT) from the peripheral tissues to the liver.Loss-of-function mutations in the LCAT protein, along with their corresponding phenotypes, have been described ( 3 ). Familial LCAT defi ciency (FLD) is caused by a complete loss of LCAT activity, either through an absence of the LCAT protein itself or the presence of a mutant LCAT protein lacking any corresponding LCAT activity. In patients, FLD is characterized by HDL defi ciency along with corneal opacity, anemia, and loss of renal function. Fisheye disease (FED) is a partial LCAT defi ciency and is characterized by the presence of LCAT protein with decreased LCAT activity. Patients with FED manifest with low HDL and corneal opacity later in life. Interestingly, a defi nitive correlation between loss-of-function LCAT mutations and coronary heart disease remains elusive ( 4 ).Glomset ( 5, 6 ) fi rst described LCAT enzymatic activity in 1962 and subsequently purifi ed the protein from plasma. LCAT purifi ed from plasma has a mass of ‫ف‬ 65 kDa as assessed by SDS-PAGE. Later analysis showed that LCAT is a 416-amino-acid protein with a calculated molecular mass of ‫ف‬ 47 kDa, and that the extra mass of plasma LCAT is a result of N-linked carbohydrates at Asn20, Asn84, Asn272, and Asn384. Glycosylation of LCAT has been shown to be important for its activity and secretion ( 7,8 ). LCAT is predicted to have an ␣ / ␤ hydrolase fold with Ser181, LCAT (EC 2.3.1.43) is a plasma enzyme that catalyzes the conversion of cholesterol into long-chain cholesteryl esters (CEs). This reaction occurs on the surface of the HDL particle where, after activation by ApoA-I (the structural protein of HDL particles), LCAT hydrolyzes phosphatidylcholine (lecithin) at the sn -2 position and subsequently transfers the fatty acyl to cholesterol ( 1 ). The increased hydrophobicity of the resulting CE forces it into the interior core of the HDL particle and in the process transforms HDL from nascent discoidal particles into larger CEenriched spherical particles. By reducing cholesterol on the 11 June 2015. Published, JLR Papers in Press, July 20, 2015 DOI 10.1194 The high-resolution crystal structure of human LCAT Abbreviations: CE, cholesteryl ester; Endo H, endoglycosidase H; FED, fi sh-eye disease; FLD, familial LCAT defi ciency; mAb, monoclonal antibody; RCT, reverse cholesterol transport; RMSD, root-mean-square deviation . Abstract LCAT is intimately involved in HDL maturation All authors are or have been employees and shareholders of Amgen. X-ray diffraction da...

The intestinal absorption of cholesterol is mediated by a multipass membrane protein, Niemann-Pick C1-Like 1 (NPC1L1), the molecular target of a cholesterol lowering therapy ezetimibe. While ezetimibe gained Food and Drug Administration approval in 2002, its mechanism of action has remained unclear. Here, we present two cryo–electron microscopy structures of NPC1L1, one in its apo form and the other complexed with ezetimibe. The apo form represents an open state in which the N-terminal domain (NTD) interacts loosely with the rest of NPC1L1, leaving the NTD central cavity accessible for cholesterol loading. The ezetimibe-bound form signifies a closed state in which the NTD rotates ~60°, creating a continuous tunnel enabling cholesterol movement into the plasma membrane. Ezetimibe blocks cholesterol transport by occluding the tunnel instead of competing with cholesterol binding. These findings provide insight into the molecular mechanisms of NPC1L1-mediated cholesterol transport and ezetimibe inhibition, paving the way for more effective therapeutic development.

hormone ( 3 ). In lieu of heparan sulfate binding, FGF19 requires a protein cofactor, ␤ Klotho, to effectively interact with and activate FGF receptors ( 4-6 ). The requirement for a co-receptor is a unique feature common to the FGF19 subfamily and is further exemplifi ed by another subfamily member, FGF21, which also lacks heparan sulfate affi nity and uses ␤ Klotho as its co-receptor ( 4 ). Consistent with their shared ability to use the same co-receptor for signaling, there is extensive overlap in the reported pharmacological effects of FGF19 and FGF21. FGF19 and FGF21 transgenic mice, as well as chronic administration of recombinant FGF19 or FGF21 proteins, similarly lowered serum glucose, triglyceride (TG), and cholesterol levels and improved insulin sensitivity and reduced body weight in high-fat diet-induced obesity models ( 7-9 ). Chronic treatment with FGF19 or FGF21 similarly reduced blood glucose levels and improved glucose disposal in ob/ob (B6.V-Lep ob /J) leptin-defi cient mice; however, plasma TG and cholesterol levels after treatment with FGF19 have not been reported in this model ( 8,9 ).The common co-receptor for FGF19 and FGF21, ␤ Klotho, is a single-pass transmembrane protein with two homologous extracellular domains that share sequence homology to ␤ -glucosidases in bacteria and plants ( 10 ). ␤ Klotho has a short intracellular domain and is unlikely to signal by itself. Its primary role is believed to mediate interactions between these two FGF molecules and FGF receptors (FGFR) to activate FGFR tyrosine kinase activity ( 11 ). ␤ Klotho interacts with only four of the seven major FGFRs, the "c" isoforms of FGFR1, -2, -3. and -4 ( 11 ). FGF19 and FGF21 can activate FGFR1c, -2c, and -3c complexed with ␤ Klotho in vitro ( 4, 6, 11-13 ). Recent results using an engineered FGF19 variant with altered receptor specifi city Abstract Elevated triglyceride (TG) and cholesterol levels are risk factors for cardiovascular disease and are often associated with diabetes and metabolic syndrome. Recent reports suggest that fi broblast growth factor (FGF)19 and FGF21 can dramatically improve metabolic dysfunction, including hyperglycemia, hypertriglyceridemia, and hypercholesterolemia. Due to their similar receptor specifi cities and co-receptor requirements, FGF19 and FGF21 share many common properties and have been thought to be interchangeable in metabolic regulation. Here we directly compared how pharmacological administration of recombinant FGF19 or FGF21 proteins affect metabolism in B6.VLep ob /J leptin-defi cient mice. FGF19 and FGF21 equally improved glucose parameters; however, we observed increased serum TG and cholesterol levels after treatment with FGF19 but not with FGF21. Increases in serum TGs were also observed after a 4-day treatment with FGF19 in C57BL6/J mice on a high-fat diet. This is in contrast to many literature reports that showed signifi cant improvements in hyperlipidemia after chronic treatment with FGF19 or FGF21 in high-fat diet models. We propose that FGF19 has lipid-raising an...

The metabolic syndrome is a group of disorders including obesity, insulin resistance, atherogenic dyslipidemia, hyperglycemia, and hypertension. To date, few animal models have been described to recapitulate the phenotypes of the syndrome. In this study, we generated and characterized two lines of triple-knockout mice that are deficient in either apolipoprotein E (Apoe−/−) or low-density lipoprotein receptor (Ldlr−/−) and express no leptin (Lepob/ob) or apolipoprotein B-48 but exclusively apolipoprotein B-100 (Apob100/100). These two lines are referred to as Apoe triple-knockout-Apoe 3KO (Apoe−/−Apob100/100Lepob/ob) and Ldlr triple-knockout-Ldlr 3KO (Ldlr−/−Apob100/100Lepob/ob) mice. Both lines develop obesity, hyperinsulinemia, hyperlipidemia, hypertension, and atherosclerosis. However, only Apoe 3KO mice are hyperglycemic and glucose intolerant and are more obese than Ldlr 3KO mice. To evaluate the utility of these lines as pharmacological models, we treated both with leptin and found that leptin therapy ameliorated most metabolic derangements. Leptin was more effective in improving glucose tolerance in Ldlr 3KO than Apoe 3KO animals. The reduction of plasma cholesterol by leptin in Ldlr 3KO mice can be accounted for by its suppressive effect on food intake. However, in Apoe 3KO mice, leptin further reduced plasma cholesterol independently of its effect on food intake, and this improvement correlated with a smaller plaque lesion area. These effects suggest a direct role of leptin in modulating VLDL levels and, likewise, the lesion areas in VLDL-enriched animals. These two lines of mice represent new models with features of the metabolic syndrome and will be useful in testing therapies targeted for combating the human condition.

Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.