Divergent role of Mitochondrial Amidoxime Reducing Component 1 (MARC1) in human and mouse.

Recent human genome-wide association studies have identified common missense variants in MARC1, p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.

Angiopoietin-like protein 3 governs LDL-cholesterol levels through endothelial lipase-dependent VLDL clearance.

Angiopoietin-like protein (ANGPTL)3 regulates plasma lipids by inhibiting LPL and endothelial lipase (EL). ANGPTL3 inactivation lowers LDL-C independently of the classical LDLR-mediated pathway and represents a promising therapeutic approach for individuals with homozygous familial hypercholesterolemia due to LDLR mutations. Yet, how ANGPTL3 regulates LDL-C levels is unknown. Here, we demonstrate in hyperlipidemic humans and mice that ANGPTL3 controls VLDL catabolism upstream of LDL. Using kinetic, lipidomic, and biophysical studies, we show that ANGPTL3 inhibition reduces VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation. This suggests that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production. Mechanistically, we discovered that EL is a key mediator of ANGPTL3's novel pathway. Our experiments revealed that, although dispensable in the presence of LDLR, EL-mediated processing of VLDL becomes critical for LDLR-independent particle clearance. In the absence of EL and LDLR, ANGPTL3 inhibition perturbed VLDL catabolism, promoted accumulation of atypical remnants, and failed to reduce LDL-C. Taken together, we uncover ANGPTL3 at the helm of a novel EL-dependent pathway that lowers LDL-C in the absence of LDLR.

ANGPTL8 requires ANGPTL3 to inhibit lipoprotein lipase and plasma triglyceride clearance.

Angiopoietin-like (ANGPTL)3 and ANGPTL8 are secreted proteins and inhibitors of LPL-mediated plasma triglyceride (TG) clearance. It is unclear how these two ANGPTL proteins interact to regulate LPL activity. ANGPTL3 inhibits LPL activity and increases serum TG independent of ANGPTL8. These effects are reversed with an ANGPTL3 blocking antibody. Here, we show that ANGPTL8, although it possesses a functional inhibitory motif, is inactive by itself and requires ANGPTL3 expression to inhibit LPL and increase plasma TG. Using a mutated form of ANGPTL3 that lacks LPL inhibitory activity, we demonstrate that ANGPTL3 activity is not required for its ability to activate ANGPTL8. Moreover, coexpression of ANGPTL3 and ANGPTL8 leads to a far more efficacious increase in TG in mice than ANGPTL3 alone, suggesting the major inhibitory activity of this complex derives from ANGPTL8. An antibody to the C terminus of ANGPTL8 reversed LPL inhibition by ANGPTL8 in the presence of ANGPTL3. The antibody did not disrupt the ANGPTL8:ANGPTL3 complex, but came in close proximity to the LPL inhibitory motif in the N terminus of ANGPTL8. Collectively, these data show that ANGPTL8 has a functional LPL inhibitory motif, but only inhibits LPL and increases plasma TG levels in mice in the presence of ANGPTL3.

ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys.

Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia.

Genetic and Metabolic Determinants of Plasma Levels of ANGPTL8.

CONTEXT: ANGPTL8 (A8) plays a key role in determining the tissue fate of circulating triglycerides (TGs). Plasma A8 levels are associated with several parameters of glucose and TG metabolism, but the causality of these relationships and the contribution of genetic variants to differences in A8 levels have not been explored. OBJECTIVE: To characterize the frequency distribution of plasma A8 levels in a diverse population using a newly-developed enzyme-linked immunosorbent assay (ELISA) and to identify genetic factors contributing to differences in plasma A8 levels. METHODS: We studied a population-based sample of Dallas County, comprising individuals in the Dallas Heart Study (DHS-1, n = 3538; DHS-2, n = 3283), including 2131 individuals with repeated measurements 7 to 9 years apart (age 18-85 years; >55% female; 52% Black; 29% White; 17% Hispanic; and 2% other). The main outcome measures were associations of A8 levels with body mass index (BMI), plasma levels of glucose, insulin, lipids, and hepatic TGs, as well as DNA variants identified by exome-wide sequencing. RESULTS: A8 levels varied over a 150-fold range (2.1-318 ng/mL; median, 13.3 ng/mL) and differed between racial/ethnic groups (Blacks > Hispanics > Whites). A8 levels correlated with BMI, fasting glucose, insulin, and TG levels. A variant in A8, R59W, accounted for 17% of the interindividual variation in A8 levels but was not associated with the metabolic parameters correlated with plasma A8 concentrations. CONCLUSIONS: A8 levels were strongly associated with indices of glucose and TG metabolism, but the lack of association of genetic variants at the A8 locus that impact A8 levels with these parameters indicates that differences in A8 levels are not causally related to the associated metabolic phenotypes.

ANGPTL8 Blockade With a Monoclonal Antibody Promotes Triglyceride Clearance, Energy Expenditure, and Weight Loss in Mice.

Angiopoietin-like protein (ANGPTL)8 is a negative regulator of lipoprotein lipase-mediated plasma triglyceride (TG) clearance. In this study, we describe a fully human monoclonal antibody (REGN3776) that binds monkey and human ANGPTL8 with high affinity. Inhibition of ANGPTL8 with REGN3776 in humanized ANGPTL8 mice decreased plasma TGs and increased lipoprotein lipase activity. Additionally, REGN3776 reduced body weight and fat content. The reduction in body weight was secondary to increased energy expenditure. Finally, single administration of REGN3776 normalized plasma TGs in dyslipidemic cynomolgus monkeys. In conclusion, we show that blockade of ANGPTL8 with monoclonal antibody strongly reduced plasma TGs in mice and monkeys. These data suggest that inhibition of ANGPTL8 may provide a new therapeutic avenue for the treatment of dyslipidemia with beneficial effects on body weight.