Role Of Angiopoietin-Like 3 And 4 in Triacylglycerol Metabolism : From population studies to molecular mechanisms of action

Excessive accumulation of triacylglycerols in circulation (hypertriglyceridemia) and/or in adipose tissue (obesity) are major risk factors for type 2 diabetes and cardiovascular diseases, two major morbidities worldwide. This thesis provides insights into the function of angiopoietin-like 3 (Angptl3) and Angptl4 in human triacylglycerol metabolism. Immunological assays have been developed and validated to measure human Angptl3 and Angptl4. Studies performed using several population samples showed that circulating levels of Angptl3 and Angptl4 have no or minor effects on plasma triacylglycerol levels. In the case of Angptl3 only complete absence of the protein in plasma, due to a non-sense mutation, generated dramatic changes not only in triacylglycerols but also in all major plasma lipoproteins and lipids, a phenotype referred to as familial combined hypolipidemia. A role for Angptl4 in human obesity was suggested by the study of monozygotic twins discordant for obesity. Serum Angptl4 and adipose tissue ANGPTL4 mRNA levels are significantly decreased in obese twins as compared with their non-obese co-twins. In vitro experiments using human adipocytes revealed that Angptl4 significantly increases the breakdown of triacylglycerol stores, a mechanism that might explain the observations in human subjects. Data obtained using animal models and human genetics clearly show that Angptl4 inhibits LPL activity and may modulate plasma triacylglycerol levels. Because the circulating levels of Angptl4 apparently do not influence LPL activity and plasma triacylglycerols a question emerged whether Angptl4 can have more subtle roles at tissue level. In this thesis, studies using skeletal muscle cells in culture provided evidence that Angptl4 is part of a negative feedback mechanism that is likely to prevent the overload of cells with triacylglycerols. The data showed that free fatty acids, the products of LPL activity, act through peroxisome proliferator-activated receptor δ/retinoic X receptor to upregulate Angptl4 synthesis coupled to inhibition of LPL activity. Furthermore, Angptl4 appears to be regulated by insulin in a tissue specific manner suggesting a mechanism for insulin-mediated fuel partitioning in the body. Taken together this data establish Angptl3 and Angptl4 as important regulators of triacylglycerol metabolism in humans.Triglyseridit ovat kolmiarvoisia rasvoja ja niiden varastoiminen soluihin on tärkeää rasva-aineenvaihdunnan kannalta erityisesti niiden sisältämän suuren energiamäärän vuoksi. Mikäli triglyseridien määrä verenkierrossa kasvaa suureksi aiheuttaen hypertriglyseridemian ja toisaalta mikäli sitä kertyy suurina määrinä rasvakudokseen, niin tämä lisää huomattavasti aikuistyypin diabeteksen sekä sydän- ja verisuonitautien riskiä. Tässä väitöskirjatyössä on selvitetty kahden keskeisesti rasva-aineenvaihduntaan vaikuttavan proteiinin, angiopoetiinin kaltainen proteiini 3:n ja 4:n (Angptl3 ja 4) molekulaarisia mekanismeja erityisesti niiden yhteyttä triglyseridimetaboliaan. Työssä on tutkittu sekä ihmisaineistoja että ko. proteiinien toimintaa ihmislähtöisissä soluviljelmissä. Molemmat proteiinit voidaan määrittää kehitetyillä ELISA-menetelmillä seerumi/plasmanäytteistä sekä erilaisissa soluviljelyolosuhteissa. Työssä osoitettiin, että Angptl3 säätelee erityisesti verenkierron triglyseriditasoja kun taas toinen näistä proteiineista Angptl4 osallistuu rasvasolujen triglyseridi-kertymien säätelyyn edesauttaen niiden hajottamista vapaiksi rasvahapoiksi. Angptl3:n täydellinen puuttuminen viallisen geenin vuoksi aiheuttaa ihmisellä merkittävän hypolipideemisen tilan, jota kutsutaan nimellä familiaalinen kombinoitu hypolipidemia, jossa sekä kolesteroli- että triglyseriditasot verenkierrossa ovat erittäin alhaiset. Ihmisperäisissä soluviljelyolosuhteissa osoitettiin, että Angptl4 tehokkaasti estää triglyseridien hajotukseen osallistuvan avainentsyymin lipoproteiinilipaasin (LPL) aktiivisuuden, mikä johtaa näiden kolmiarvoisten rasvojen merkittävään nousuun. Angptl3 ja 4 proteiinien toiminnan tarkempi ymmärtäminen triglyseridimetaboliassa edesauttaa uusien terapiakeinojen/lääkeaineiden kehitystyössä, jota tarvitaan metaboolisen oireyhtymän, diabeteksen ja sydän- ja verisuonitautien taltuttamiseksi

Quantitation of serum angiopoietin-like proteins 3 and 4 in a Finnish population sample

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Human apoA-I increases macrophage foam cell derived PLTP activity without affecting the PLTP mass

Background: phospholipid transfer protein (PLTP) plays important roles in lipoprotein metabolism and atherosclerosis and is expressed by macrophages and macrophage foam cells (MFCs). The aim of the present study was to determine whether the major protein from HDL, apoA-I, affects PLTP derived from MFCs. Results: as cell model we used human THP-1 monocytes incubated with acetylated LDL, to generate MFC. The addition of apoA-I to the cell media increased apoE secretion from the cells, in a concentration dependent fashion, without affecting cellular apoE levels. In contrast, apoA-I had no effect on PLTP synthesis and secretion, but strongly induced the PLTP activity in the media. ApoA-I also increased phospholipid transfer activity of PLTP isolated from human plasma. This effect was dependent on apoA-I concentration but independent on apoA-I lipidation status. ApoE, ApoA-II and apoA-IV, but not immunoglobulins or bovine serum albumin, also increased PLTP activity. We also report that apoA-I protects PLTP from heat inactivation. Conclusion: apoA-I enhances the phospholipid transfer activity of PLTP secreted from macrophage foam cells without affecting the PLTP mass.Peer reviewe

VEGF-C is required for intestinal lymphatic vessel maintenance and lipid absorption

Vascular endothelial growth factor C (VEGF-C) binding to its tyrosine kinase receptor VEGFR-3 drives lymphatic vessel growth during development and in pathological processes. Although the VEGF-C/VEGFR-3 pathway provides a target for treatment of cancer and lymphedema, the physiological functions of VEGF-C in adult vasculature are unknown. We show here that VEGF-C is necessary for perinatal lymphangiogenesis, but required for adult lymphatic vessel maintenance only in the intestine. Following Vegfc gene deletion in adult mice, the intestinal lymphatic vessels, including the lacteal vessels, underwent gradual atrophy, which was aggravated when also Vegfd was deleted. VEGF-C was expressed by a subset of smooth muscle cells adjacent to the lacteals in the villus and in the intestinal wall. TheVegfc-deleted mice showed defective lipid absorption and increased fecal excretion of dietary cholesterol and fatty acids. When fed a high-fat diet, the Vegfc-deficient mice were resistant to obesity and had improved glucose metabolism. Our findings indicate that the lymphangiogenic growth factors provide trophic and dynamic regulation of the intestinal lymphatic vasculature, which could be especially important in the dietary regulation of adiposity and cholesterol metabolism.Peer reviewe

OSBP-Related Proteins (ORPs) in Human Adipose Depots and Cultured Adipocytes: Evidence for Impacts on the Adipocyte Phenotype

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Angiopoietin-Like 4 Mediates PPAR Delta Effect on Lipoprotein Lipase-Dependent Fatty Acid Uptake but Not on Beta-Oxidation in Myotubes

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Serum angiopoietin-like 4 protein levels and expression in adipose tissue are inversely correlated with obesity in monozygotic twins

Animal studies have suggested that angiopoietin-like 4 (Angptl4) regulates adiposity through central and peripheral mechanisms. The aim of this study was to investigate whether serum concentration and adipose tissue expression of Angptl4 are associated with obesity-related parameters in humans. Altogether, 75 dizygotic (DZ) and 46 monozygotic (MZ) twin pairs were studied, from the FinnTwin12 and FinnTwin16 cohorts. Among the MZ pairs, 21 were discordant for body mass index (BMI) (intra-pair BMI-difference >2.5 kg/m², age 23-33 years). Serum Angptl4 (s-Angptl4) levels were measured by ELISA, and adipose tissue gene expression was analyzed by genome-wide transcript profiling. In MZ twin pairs discordant for BMI, s-Angptl4 and adipose tissue ANGPTL4 mRNA (at-ANGPTL4) levels were significantly decreased (P = 0.04 and P = 0.03, respectively) in obese twins as compared with their nonobese cotwins. In all twins, intra-pair differences in s-Angptl4 levels were inversely correlated with intra-pair differences in BMI (r = -0.27, P = 0.003). In individual MZ twins, at-ANGPTL4 expression was inversely correlated with BMI (r = -0.44, P = 0.001) and positively correlated with at-LIPE (r = 0.24, P = 0.01) and at-ABHD5 (r = 0.41, P = 0.005) expression. Our results demonstrated that variation in Angptl4 concentration was only modestly accounted for by genetic factors and suggest a role for Angptl4 in acquired obesity in humans.Peer reviewe

Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling

Background: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure. Methods: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes. Results: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart. Conclusions: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.Peer reviewe

Blockade of VEGF-C and VEGF-D modulates adipose tissue inflammation and improves metabolic parameters under high-fat diet

Objective: Elevated serum levels of the lymphangiogenic factors VEGF-C and -D have been observed in obese individuals but their relevance for the metabolic syndrome has remained unknown. Methods: K14-VEGFR-3-Ig (sR3) mice that constitutively express soluble-VEGFR-3eIg in the skin, scavenging VEGF-C and -D, and wildtype (WT) mice were fed either chow or high-fat diet for 20 weeks. To assess the effect of VEGFR-3 blockage on adipose tissue growth and insulin sensitivity, we evaluated weight gain, adipocyte size and hepatic lipid accumulation. These results were complemented with insulin tolerance tests, FACS analysis of adipose tissue macrophages, in vitro 3T3-L1 differentiation assays and in vivo blocking antibody treatment experiments. Results: We show here that sR3 mice are protected from obesity-induced insulin resistance and hepatic lipid accumulation. This protection is associated with enhanced subcutaneous adipose tissue hyperplasia and an increased number of alternatively-activated (M2) macrophages in adipose tissue. We also show that VEGF-C and -D are chemotactic for murine macrophages and that this effect is mediated by VEGFR-3, which is upregulated on M1 polarized macrophages. Systemic antibody blockage of VEGFR-3 in db/db mice reduces adipose tissue macrophage infiltration and hepatic lipid accumulation, and improves insulin sensitivity. Conclusions: These results reveal an unanticipated role of the lymphangiogenic factors VEGF-C and -D in the mediation of metabolic syndrome-associated adipose tissue inflammation. Blockage of these lymphangiogenic factors might constitute a new therapeutic strategy for the prevention of obesity-associated insulin resistance. (C) 2014 The Authors. Published by Elsevier GmbH.Peer reviewe

VEGF-B-induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart

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