SUMMARYThe basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. Currently, there is a debate regarding whether the specification of these lineages occurs early in the formation of the epicardium or later after the cells have entered the myocardium. Lineage tracing using a tamoxifen-inducible Cre expressed from the Tcf21 locus demonstrated that the majority of Tcf21-expressing epicardial cells are committed to the cardiac fibroblast lineage prior to initiation of epicardial epithelial-to-mesenchymal transition (EMT). Furthermore, Tcf21 null hearts fail to form cardiac fibroblasts, and lineage tracing of the null cells showed their inability to undergo EMT. This is the first report of a transcription factor essential for the development of cardiac fibroblasts. We demonstrate a unique role for Tcf21 in multipotent epicardial progenitors, prior to the process of EMT that is essential for cardiac fibroblast development.

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.

Tcf21 is a class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21iCre) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.

This article is available online at http://www.jlr.org an enzyme that catalyzes an early step in cholesterol biosynthesis, effectively lower plasma LDL-C levels and prevent CHD ( 2 ). Human genetic studies have provided several new targets for LDL-C lowering. Individuals who lack ApoB ( 3 ), or microsomal transfer protein (MTP), the enzyme that transfers TG to TG-rich lipoproteins in the liver and intestine ( 4 ), have very low LDL levels. Agents that suppress ApoB expression ( 5 ) or inhibit MTP activity ( 6 ) are now available for treatment of severe hypercholesterolemia in individuals with homozygous familial hypercholesterolemia. Loss-of-function mutations in PCSK9, a secreted proprotein convertase that promotes degradation of the LDL receptor (LDLR), cause a 30% reduction in LDL-C and substantial protection from CHD ( 7 ). Anti-PCSK9 antibodies lower circulating LDL-C levels ( 8 ) and clinical trials are now underway to determine whether there is an associated reduction in CHD ( 9 ).More recently, several families with inactivating mutations in angiopoietin-like protein 3 ( ANGPTL3 ) were identifi ed ( 10-12 ). Family members with two loss-of-function mutations in ANGPTL3 have striking pan-hypolipidemia; plasma levels of TGs, NEFAs, VLDL-cholesterol (VLDL-C), LDL-C, and HDL-cholesterol (HDL-C) are all markedly reduced. The mechanisms by which ANGPTL3 modulates TG metabolism have been extensively investigated ( 13 ). ANGPTL3 inhibits the activity of two intravascular lipases: LPL, which catalyzes hydrolysis of TGs in TG-rich lipoproteins, and endothelial lipase (EL), which hydrolyzes lipoprotein phospholipids ( 14-16 ). Thus, increased activity of LPL and EL may account for the low plasma levels of TG Abstract Humans and mice lacking angiopoietin-like protein 3 (ANGPTL3) have pan-hypolipidemia. ANGPTL3 inhibits two intravascular lipases, LPL and endothelial lipase, and the low plasma TG and HDL-cholesterol levels in ANG-PTL3 defi ciency refl ect increased activity of these enzymes. The mechanism responsible for the low LDL-cholesterol levels associated with ANGPTL3 defi ciency is not known. Here we used an anti-ANGPTL3 monoclonal antibody (REGN1500) to inactivate ANGPTL3 in mice with genetic defi ciencies in key proteins involved in clearance of ApoBcontaining lipoproteins. REGN1500 treatment consistently reduced plasma cholesterol levels in mice in which Apoe , Ldlr , Lrp1 , and Sdc1 were inactivated singly or in combination, but did not alter clearance of rabbit 125 I-␤ VLDL or mouse 125 I-LDL. Despite a 61% reduction in VLDL-TG production, VLDL-ApoB-100 production was unchanged in REGN1500-treated animals. Hepatic TG content, fatty acid synthesis, and fatty acid oxidation were similar in REGN1500 and control antibody-treated animals. Taken together, our fi ndings indicate that inactivation of ANGPTL3 does not affect the number of ApoB-containing lipoproteins secreted by the liver but alters the particles that are made such that they are cleared more rapidly from the circulation via a noncanonical pathway(...

Angiopoietin-like protein 3 (ANGPTL3) is a circulating inhibitor of lipoprotein and endothelial lipase whose physiological function has remained obscure. Here we show that ANGPTL3 plays a major role in promoting uptake of circulating very low density lipoprotein-triglycerides (VLDL-TGs) into white adipose tissue (WAT) rather than oxidative tissues (skeletal muscle, heart brown adipose tissue) in the fed state. This conclusion emerged from studies of Angptl3(-/-) mice. Whereas feeding increased VLDL-TG uptake into WAT eightfold in wild-type mice, no increase occurred in fed Angptl3(-/-) animals. Despite the reduction in delivery to and retention of TG in WAT, fat mass was largely preserved by a compensatory increase in de novo lipogenesis in Angptl3(-/-) mice. Glucose uptake into WAT was increased 10-fold in KO mice, and tracer studies revealed increased conversion of glucose to fatty acids in WAT but not liver. It is likely that the increased uptake of glucose into WAT explains the increased insulin sensitivity associated with inactivation of ANGPTL3. The beneficial effects of ANGPTL3 deficiency on both glucose and lipoprotein metabolism make it an attractive therapeutic target.

lipolysis in WAT (23, 24). Thus, A4 expression in WAT promotes flux of fatty acids toward oxidative tissues to supply them with energy substrate during fasting. A3 and A8 are functionally interdependent in vivo (11) and in vitro (25). Expression of A8 fails to suppress intravascular LPL of mice lacking A3 (11). The 2 family members form a complex in which the LPL binding domain of A8 (and not A3) is required for LPL inhibition (25). After feeding, A3 and A8 act together in the systemic circulation to inhibit LPL activity. Under these conditions, more circulating TGs bypass oxidative tissues and are hydrolyzed in WAT (7, 21). Feeding also increases expression of A8 in adipose tissue, but its role in WAT has not been defined (11, 19, 20). To begin to understand the relative roles of A8 in liver and in adipose tissue, we developed mice in which A8 is selectively inactivated in those 2 tissues. Here we show that A8 has distinct physiological functions in the 2 tissues. All detectable circulating A8 originates in the liver, and only this form of A8 complexes with A3 to inhibit intravascular lipolysis. A8 from adipose tissue makes no detectable contribution to circulating A8, but rather has local effects on substrate homeostasis. A8 expression in adipose tissue attenuates the LPL inhibitory actions of A4, thus ensuring a rapid replenishment of energy stores with feeding after a fast. Results Tissue-specific inactivation of A8. We developed C57BL/6N mice in which expression of A8 was ablated either in hepatocytes (liver-specific-A8-/mice; Ls-A8-/mice) or in adipocytes (adipose-specific-A8-/mice; As-A8-/mice). The mice were established using the Cre-lox system to remove exons 1 and 2 of A8 (Figure 1A). In the targeting construct, the neomycin gene is flanked by 2 rox sites that recombine to remove the Neo cassette, thus producing an allele with loxP sites flanking the first 2 exons of A8. Mice homozygous for the fl allele were used as controls for all experiments described in this paper, unless otherwise stated, and are referred to as WT. A8 was inactivated by crossing mice homozygous for the fl allele either with mice expressing Cre under control of the albumin promoter (B6N.Cg-Speer6-ps1 Tg(Alb-cre)21Mgn /J; Alb-Cre) (26) or with mice expressing Cre driven by the adiponectin promoter (Adipo-Cre) (27) to produce Ls-A8-/mice and As-A8-/mice, respectively. Both sexes transmitted the inactivated A8 allele, and the genotypes of the offspring conformed to the expected Mendelian ratios (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.138777DS1). Litter sizes were similar in KO and WT mice (Supplemental Table 2). Because A8 is not expressed in the fasting state, all experiments were performed in refed conditions unless otherwise stated. The mean body weights of the C57BL/6N A8-/mice were similar to those of WT controls at 9-10 weeks of age (Figure 1B). The mean total fat mass was significantly lower in the total A8-/mice (3.0± 0.2 vs. 1.7± 0.1 g) but not in th...

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.

Dietary triglyceride (TG) is the most efficient energy substrate. It is processed and stored at substantially lower metabolic cost than is protein or carbohydrate. In fed animals, circulating TGs are preferentially routed for storage to white adipose tissue (WAT) by angiopoietin-like proteins 3 (A3) and 8 (A8). Here, we show that mice lacking A3 and A8 (A3 −/− A8 −/− mice) have decreased fat mass and a striking increase in temperature (+1°C) in the fed (but not fasted) state, without alterations in food intake or physical activity. Subcutaneous WAT (WAT-SQ) from these animals had morphologic and metabolic changes characteristic of beiging. O 2 consumption rates (OCRs) and expression of genes involved in both fatty acid synthesis and fatty acid oxidation were increased in WAT-SQ of A3 , but not WT, mice. Antibodymediated inactivation of both circulating A3 and A8 induced hyperthermia in WT mice. Together, these data indicate that A3 and A8 are essential for efficient storage of dietary TG and that disruption of these genes increases feeding-induced thermogenesis and energy utilization.irculating triglycerides (TGs) are partitioned between oxidative tissues and white adipose tissue (WAT) by lipoprotein lipase (LPL), an enzyme located on capillary endothelia. LPL releases fatty acids (FAs) from TGs in circulating lipoproteins for uptake by underlying tissues. The activity of LPL in different tissue beds is regulated in response to nutritional cues. In fasted animals, LPL activity is high in oxidative tissues and low in WAT, thus favoring the uptake of circulating TG by oxidative tissues (1, 2). Feeding decreases LPL activity in oxidative tissues and increases the activity of the enzyme in WAT, thus replenishing WAT energy stores by increasing FA uptake.The changes in LPL activity associated with fasting and refeeding are coordinated by secreted proteins of the angiopoietin-like protein (ANGPTL) family: ANGPTL3 (A3), ANGPTL4 (A4), and ANGPTL8 (A8). All three proteins inhibit LPL activity, but A4 is increased by fasting and acts locally in WAT (3), whereas A8 is stimulated by feeding (4-6) and acts together with A3 to inhibit LPL via the systemic circulation (6). Consequently, in fasted animals, A4 powerfully suppresses LPL activity in WAT, directly suppressing uptake of FAs from circulating lipoproteins (2). The very low expression of A8 during fasting limits systemic inhibition of LPL activity by the A3/A8 complex, resulting in high LPL activity in oxidative tissues (6-8). Upon refeeding, A4 expression is decreased in WAT, leading to increased LPL activity and TG-FA uptake in that organ (2). Conversely, feeding strongly stimulates A8 expression, promoting the formation of A3/A8 complexes in the circulation and thus systemic inhibition of LPL activity and TG-FA uptake in oxidative tissues (6, 8-10).In addition to the striking effects of A3 and A8 on circulating lipoprotein metabolism, both proteins appear to be required for normal energy substrate selection. Mice lacking A3 (A3 −/− mice) or A8 (A8 −/− mice) fail to ...