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The following handle holds various files of this Leiden University dissertation:
http://hdl.handle.net/1887/61829
Author: Hoeke, G.
Title: A fatty battle: towards identification of novel genetic targets to comBAT cardiometabolic diseases
Issue Date: 2018-05-03
A FATTY BATTLE
Towards identification of novel genetic targets to comBAT cardiometabolic diseases
Geraldine Hoeke
A Fatty Battle
Towards identification of novel genetic targets to comBAT cardiometabolic diseases
©2018, Geerte Hoeke
Layout & printing: Proefschriftenprinten.nl ISBN: 978-94-92679-34-5
All rights are reserved. No part of this publication may be transformed, reproduced or transmitted in any form and by any means without prior permission of the author.
A FATTY BATTLE
Towards identification of novel genetic targets to comBAT cardiometabolic diseases
Proefschrift
Ter verkrijging van
de graad van Doctor aan de Universiteit leiden, op gezag van Rector Magnificus prof. Mr. C.J.J.M. Stolker
volgens besluit van het College voor Promoties te verdedigen op donderdag 3 mei 2018
klokke 16.15 uur
door
Geraldine Hoeke
Geboren te Soest In 1990
Promotor Prof. Dr. P.C.N. Rensen
Copromotoren Dr. J.F.P. Berbée Dr. M.R. Boon
Leden promotiecommissie Prof. dr. J.W. Jukema Prof. dr. M. van Eck
Prof. dr. A.K. Groen (AMC, Amsterdam) Dr. J.A. van Diepen (Radboud UMC, Nijmegen)
The work described in this thesis was performed at the Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Leiden, The Netherlands, and at Einthoven Laboratory for Experimental Vascular Medicine, Leiden, The Netherlands.
Geerte Hoeke was supported by a GENIUS project “Generating the best evidence-based pharmaceutical targets for atherosclerosis” (CVON2011–9) to P.C.N. Rensen.
TABLE OF CONTENTS
Chapter 1 General introduction and outline
Chapter 2 Brown adipose tissue takes up plasma triglycerides mostly
after lipolysis
Chapter 3 Atorvastatin accelerates clearance of lipoprotein remnants generated by activated brown fat to further reduce hypercholesterolemia and atherosclerosis
Chapter 4 The bile acid sequestrant colesevelam enhances the beneficial effects of brown adipose tissue activation on cholesterol metabolism in APOE*3-Leiden.CETP mice
Chapter 5 Short-term cooling increases serum triglycerides and small high-density lipoprotein levels in humans
Chapter 6 The effects of selective hematopoietic expression of human IL-37 on systemic inflammation and atherosclerosis in LDLr-
deficient mice
Chapter 7 Deletion of hematopoietic Dectin-2 or CARD9 does not protect against atherosclerotic plaque formation in hyperlipidemic mice
Chapter 8 Deletion of hematopoietic Dectin-2 or CARD9 does not protect against atherosclerosis development under
hyperglycemic conditions
Chapter 9 General discussion and future perspectives
Chapter 10
Summary
Nederlandse Samenvatting List of publications
Curriculum Vitae
Dankwoord
7
29
51
77
103
123
141
163
185
211 213 219 227 229 231
Chapter
General introduction and outline
Modified from:
Relevance of lipid metabolism for brown fat visualization and quantification Curr Opin Lipidol 2016; 27(3): 242-8.
Role of brown fat in lipoprotein metabolism and atherosclerosis development Circ Res 2016; 118(1): 173-82.
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1. LIPOPROTEIN METABOLISM AND ATHEROSCLEROSIS 1
Lipids, such as triglycerides (TG) and cholesterol, are hydrophobic molecules that are present in our diet. TG are esters of glycerol and three fatty acids (FA), and are the main source of energy in the body by providing these FA to metabolic organs. Cholesterol, either free or esterified with FA as cholesteryl esters (CE), is derived from animal source foods only, and does not provide energy to metabolic organs. Rather, cholesterol is used as precursor for cell membranes, steroid hormones, vitamin D, and bile acids (1). Because TG, cholesterol and CE are poorly water soluble, they are transported in the blood by so-called lipoproteins. These lipoproteins carry hydrophobic lipids in their core (i.e. TG and CE), while the outer shell is comprised of phospholipids (PL), free cholesterol and (apolipo)proteins (2). Lipoproteins can be divided into apolipoprotein (apo)B-containing lipoproteins and high-density lipoproteins (HDL), which contain apoAI instead of apoB as main apolipoprotein. ApoB-containing lipoproteins can be further divided in subclasses based on size, composition, and site of synthesis. The synthesis and catabolism of lipoproteins is discussed below.
1.1. ApoB-containing lipoproteins
ApoB-containing lipoproteins can be divided into TG-rich lipoproteins (TRLs) and low- density lipoproteins (LDL). TRLs include intestine-derived chylomicrons and liver-derived very-low-density lipoproteins (VLDL), both of which contain relatively high amounts of TG compared to cholesterol/CE. LDL is virtually devoid of TG and, therefore, carry mainly CE in their core.
TRLs can be synthesized within two pathways, the exogenous and endogenous pathway. The exogenous pathway is initiated by feeding and results in the formation of chylomicrons. In the process of chylomicron formation, dietary TG are hydrolyzed into 2-monoacylglycerol and FA, and CE are hydrolyzed into free cholesterol and FA.
These constituents are then taken up by the enterocytes, re-esterified into TG and CE, and form a lipid droplet surrounded by PL and cholesterol that is merged with apoB (apoB48 in humans, apoB100 in rodents) by microsomal triglyceride transfer protein (MTP). The formed chylomicrons are released into the lymphatic system, subsequently reach the circulation via the choroid plexus, and are transported towards metabolically active tissues. In the endogenous pathway, the liver synthesizes VLDL. Albeit that the liver continuously produces VLDL, it is especially important during fasting for FA delivery
towards metabolically active tissues such as the heart. VLDL is generated intracellularly in a similar way to chylomicrons, except that apoB100 is the main apolipoprotein in both humans and rodents (1) and that nascent chylomicrons do not contain apoE while this apolipoprotein is involved in the hepatic assembly of VLDL (3).
In the circulation, TRLs acquire additional apolipoproteins such as apoAI, apoAV, apoCI, apoCII, apoCIII and apoE, which are not only important structural components, but also serve to modulate enzymatic reactions (e.g. as cofactor) or interaction with cell-surface receptors. These apolipoproteins ensure the transport of TRLs towards metabolic tissues which use TLR-derived FA for their function e.g. skeletal muscle and heart for ATP production, white adipose tissue (WAT) for storage, or brown adipose tissue (BAT) for thermogenesis. Uptake of FA from TLRs by metabolic tissues involves an elaborate cascade of events. On the vascular bed of these metabolic tissues heparan sulfate proteoglycans (HSPGs) and glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) capture TRLs (4). Subsequently, FA are released from TRL-TG and taken up by the organs in a coordinated fashion involving lipoprotein lipase (LPL) for lipolysis and the FA transporter cluster of differentiation 36 (CD36) for subsequent uptake of the FA (5, 6). As a consequence of LPL-mediated lipolysis, TRL remnants are formed that acquire more apoE in the circulation, which enables the uptake of the TRL remnants via hepatic apoE-binding receptors on mainly hepatocytes.
While the low-density lipoprotein receptor (LDLR) is the most important receptor, other binding sites include the LDLR-related protein 1 (LRP1) and HSPGs (7-9). When VLDL remnants are not taken up by the liver, they remain in the circulation where they become further lipolysed leading to the formation of LDL particles as lipolytic end product. As a consequence of TG depletion, these particles are very rich in CE. In addition, as these particles lose virtually all exchangeable apolipoproteins during lipolysis, they contain apoB100 as sole apolipoprotein. LDL particles can also be taken up by the LDLR via interaction with apoB100, which however has lower affinity for LDLR compared to apoE (10) (Fig. 1).
TRL remnants and LDL (i.e. apoB-containing particles) are pro-atherogenic since these particles can accumulate in the arterial wall, leading to the development of early atherosclerosis (as explained in the section 1.3).
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Figure 1: Lipoprotein metabolism. See section 1.1 and 1.2 for explanation. B48, apolipoprotein B48; B100, apolipoprotein B100; BAT, brown adipose tissue; CE, cholesteryl ester; E, apolipoprotein E; FA, fatty acid;
HDL, high-density lipoprotein; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; LPL, lipoprotein lipase; LRP, low-density lipoprotein receptor-related protein; SR-B1, scavenger receptor class B type 1; TG, triglyceride; WAT, white adipose tissue.
1.2. High-density lipoproteins
ApoAI, the main apolipoprotein of HDL, is synthesized in the liver and small intestine.
ApoAI is secreted into the circulation with PL as a discoidal particle, which can take up additional PL from the surface remnants that are formed as a consequence of lipolysis of TRLs. These discoidal HDL particles are potent acceptors of cholesterol, which is effluxed from peripheral cells (e.g. macrophages) towards via the receptor ATP binding cassette transporter (ABCA1). Via this process, larger, lipidated HDL are formed that accept cholesterol that is effluxed via ATP binding cassette subfamily G, member (ABCG1).
The cholesterol that is acquired by HDL particles initially intercalates in the particles’
shell where it is esterified with a FA of phosphatidylcholine by lecithin-cholesterol- acyltransferase (LCAT) into CE that enters the core of the particle allowing the HDL
particle to become larger. HDL can selectively deliver CE to hepatocytes via scavenger receptor class B type 1 (SR-B1), after which the delipidated particles can accept additional cholesterol from peripheral cells (Fig. 1). The cholesterol that is delivered to hepatocytes can be used for VLDL synthesis, can be secreted into the bile as neutral sterols, or can be used for the synthesis of hydrophilic bile acids that are secreted into the bile as well.
If the bile acids are not reabsorbed by the intestine by enterohepatic circulation, this export route allows (derivatized) cholesterol to be excreted from the body via the feces.
This entire process of cholesterol uptake from the periphery, uptake of cholesterol by hepatocytes and subsequent excretion of cholesterol via the feces is referred to as the reverse cholesterol transport (often abbreviated as ‘RCT’) which is regarded as an anti- atherogenic process (11).
1.3. Role of lipoproteins in atherosclerosis
Cardiovascular diseases are predominantly caused by atherosclerosis, for which both hyperlipidemia and inflammation are the main risk factors. The development of atherosclerosis starts with damage of the arterial wall due to high shear stress in the vessel, mainly around bifurcation of vessels. Subsequently, apoB-containing lipoproteins including LDL and TRL remnants (12), can infiltrate and accumulate in the arterial wall.
These accumulated lipids become modified leading to the formation of oxidized LDL (oxLDL) that activate endothelial cells. In addition, pro-inflammatory cytokines and chemokines, such as tumour necrosis factor α (TNFα) and monocyte chemoattractant protein-1 (MCP-1), are locally produced that attract monocytes towards the lesion as where they maturate into macrophages. These macrophages scavenge oxLDL as well as aggregated LDL (13), resulting in the formation of lipid-laden foam cells and a fatty streak in the arterial wall. This fatty streak is the hallmark of atherosclerosis development (14). Activated endothelial cells and foam cells also produce pro-inflammatory cytokines and chemokines, leading to proliferation and migration of collagen-producing vascular smooth muscle cells (VMCs) to form a collagen cap, which stabilizes the growing lesion, and attraction of more immune cells towards the lesion. Due to cell death within the lesion, a necrotic core is formed, which contains dead cell debris and cholesterol crystals that are released from foam cells. The balance between collagen production and degradation (e.g. by MMPs produced by macrophages) determines the composition and stability of the lesion. A stable cap is defined by a thick collagen cap and a small necrotic core. In contrast, an unstable plaque is characterized by a thin cap, a large necrotic core and a high number of macrophages within the lesion. As a consequence of plaque
instability, a plaque can rupture causing blot clotting and the formation of a thrombus
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that will stop the blood flow. Occlusion of the coronary artery may cause a myocardial infarction, while occlusion of an artery providing blood to the brain may cause ischemic stroke (15).
2. SHADES AND FUNCTIONS OF ADIPOSE TISSUES
Adipose tissue comprises metabolically active organs, i.e. white adipose tissue (WAT) and brown adipose tissue (BAT), both of which largely contribute to the metabolism of lipids as well as glucose. The handling of lipids and glucose is very much dependent of the color of adipose tissue as detailed below. Moreover, adipose tissue dysfunction, for example due to obesity, can result in hyperlipidemia and inflammation (16). The three main types of adipose tissue, i.e. brown, white and beige adipose tissue, will be discussed below.
2.1. White adipose tissue
The most abundant adipose tissue in the body is WAT, which can be found in different locations (e.g. subcutaneous, visceral). White adipocytes are monolocular cells containing a large lipid droplet and only few mitochondria, and their main function is to store lipids and glucose in the form of TG during periods of excess nutrient availability. Because of their low mitochondrial content white adipocytes hardly combust FA. Rather, in periods of fasting a low concentration of insulin combined with a high concentration of glucagon induces the release of FA from WAT to provide other metabolic organs with energy (17).
Likewise, at an ambient temperature below thermoneutrality, sympathetic activation of WAT via norepinephrine triggers WAT to release FA that become available to BAT for a process called thermogenesis (18), which will be explained below.
2.2. Brown adipose tissue
While white adipocytes are monolocular cells with few mitochondria, brown adipocytes are multilocular cells with small lipid droplets and a high content of mitochondria, which contain the uncoupling protein-1 (UCP-1) within their inner membrane. UCP-1 provides BAT with the ability to generate heat from stored FA in a process called thermogenesis (19). This is a crucial process for newborns and small mammals to maintain their core body temperature. Until recently it was assumed that humans would gradually lose BAT
upon ageing. Intriguingly, almost a decade ago BAT returned into the spotlights when it became evident that BAT is not only present but also still active in adults (20). More specifically, the use of positron emission tomography-computed tomography (PET-CT) scans in combination with the glucose tracer 18F-fluorodeoxyglucose ([18F]FDG) revealed that upon cold induction symmetrical uptake of [18F]FDG was found in regions that corresponded to BAT, i.e. in the supraclavicular area and along the spine (21-24). Indeed, biopsies of these regions showed high expression of UCP-1 (20).
The most important physiological activator of BAT is cold, which is sensed through nerve terminals expressing certain transient receptor potential (TRP) channels (25). Activation of these TRP channels initiates a signal towards the hypothalamic temperature center and subsequently enhances sympathetic outflow towards BAT.
The importance of the sympathetic nervous system (SNS) for BAT function is reflected by the high number of nerve endings in the tissue. Each brown adipocyte is in close proximity to a nerve ending that releases norepinephrine upon sympathetic stimulation.
Norepinephrine subsequently binds to the β3-adrenergic receptor (β3-AR) on the membrane of the brown adipocyte. This results in activation of adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP) that activates protein kinase A (PKA) to phosphorylate the lipolytic enzymes adipose TG lipase (ATGL), hormone-sensitive lipase (HSL) and monoglycerol lipase (MGL), leading to increased intracellular lipolysis (26). FA that are released subsequently enter the mitochondria where they are broken down by β-oxidation into substrates for the citric acid cycle, leading to activation of the electron transport chain and uncoupled respiration through UCP-1. This sequence of events results in the generation of heat instead of ATP (19) (Fig. 2). In addition to being oxidized, FA can allosterically activate UCP-1 by causing a conformational change in UCP-1, thereby enhancing uncoupled respiration (27).
2.3. Beige adipose tissue
In addition to brown adipocytes, white adipocytes can turn into so-called recruitable brown adipocytes, or ‘beige’ or ‘brite’ cells, which also have thermogenic capacity (19, 28).
Sympathetic innervation of WAT via cold exposure (29) and several other stimuli, such as β3-AR agonism (30, 31)and peroxisome proliferator-activated receptor (PPAR)-γ and -α agonism (32, 33) orchestrate ‘browning’ of WAT depots. This process is characterized by appearance of multilocular adipocytes that express several BAT-related markers such as UCP-1. Although basal UCP-1 gene and protein expression are relatively low in beige
adipocytes, both can be markedly increased upon stimulation, reaching similar levels of
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UCP-1 expression as in brown adipocytes (28). The contribution of beige adipose tissue versus BAT to total body thermogenesis is still under active debate.
Figure 2: BAT physiology. See section 2.2 for explanation. ATGL, adipose triglyceride lipase; cAMP, cyclic adenosine monophosphate; CD36, differentiation 36; CREB, cAMP response element-binding protein; DAG, diacylglycerol; FA, fatty acids; FATP, fatty acid transport protein; GLUT1/4 glucose transporter 1 and 4; HSL, hormone sensitive lipase; LPL, lipoprotein lipase; MAG, monoacylglycerol; MGL, monoglyceride lipase; NE, norepinephrine; PGC1α, peroxisome proliferator-activated receptor-gamma co-activator; PKA, protein kinase A; TG, triglyceride; UCP1, uncoupling protein 1. Adapted from (34).
3. SUBSTRATE UTILIZATION BY ADIPOCYTES
3.1. Substrate utilization by white adipocytes
Following a meal, insulin stimulates translocation of LPL in WAT towards the vascular bed, which leads to a high flux of TRL-derived FA towards adipocytes for uptake via CD36.
Intracellularly, FA react with glycerol-3-phosphate (G3P) to form TG that are stored in the intracellular lipid droplet. White adipocytes can also take up high amounts of glucose via the insulin-stimulated glucose transporter-4 (GLUT4), not only to produce G3P for TG
assembly, but also to generate precursors for FA synthesis by de novo lipogenesis (35). As such, high consumption of sugars can cause obesity.
3.2. Glucose uptake and utilization by brown and beige adipocytes
PET-CT using the glucose tracer [18F]FDG is the golden standard to visualize and quantify (cold-) activated BAT in humans (21-23), and this technique has also been applied to visualize [18F]FDG uptake in mice (36, 37). Glucose is indeed taken up and used by brown adipocytes, although it was demonstrated that BAT does not oxidize glucose directly (38). Following its uptake by the glucose transporter-1 (GLUT1) and -4 (GLUT4) (26, 39), glucose is used for both de novo lipogenesis and ATP generation that supports general adipocyte function (38, 40). This is supported by the observation that BAT activation increases the expression of genes involved in the glycolysis and the pentose phosphate pathway, pathways that provide ATP and reducing equivalents for de novo lipogenesis, respectively (39, 41).
3.3. FA uptake and utilization by brown and beige adipocytes
Upon BAT activation, intracellular lipolysis causes depletion of intracellular TG stores that subsequently need to be replenished via de novo lipogenesis and via the uptake of FA from the circulation. The uptake of free FA by BAT is relatively low (39), but likely of seeming importance as cold exposure and subsequent sympathetic outflow stimulates lipolysis and release of FA by WAT, which correlates with BAT activity (42). Also in humans, activated BAT takes up free FA (43). However, the most important source of FA are probably TRL-derived FA. In fact, activation of BAT in hypertriglyceridemic mice by cold strongly reduces plasma TG, illustrating the importance of BAT in lipoprotein-TG metabolism (44). Due to the lack of suitable tracers, the relevance of lipoprotein-TG as source of human BAT is unknown as yet.
A current matter of debate is the mode of action via which BAT takes up TRL-TG-derived FA from plasma: either after LPL-mediated lipolysis of TG, via holoparticle uptake of TRL, or through a combination of both. Cold exposure of mice increases the expression of Lpl and Cd36 in BAT (44). LPL and CD36 are both classically involved in lipolysis-mediated uptake of FA by heart, skeletal muscle, WAT (45). Mice that lack the FA transporter CD36 have impaired uptake of FA by tissues (46),and also exhibit defective thermogenesis (44, 47). Collectively, it seems likely that BAT takes up TG-derived FA via selective delipidation of TRL in a similar manner as heart, skeletal muscle and WAT (5, 6), although this had not been demonstrated as yet.
Following uptake of FA by BAT, FA are incorporated into TG and stored in intracellular lipid
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droplets from where they are released upon BAT activation. This requires an adequate intracellular handling mechanism of FA. FA that are taken up by the brown adipocyte directly stimulate lipogenesis through activation of peroxisome proliferator-activated receptors (PPARs) (48). High expression of glycerol-3-phosphate acyltransferase-4 (GPAT4) in brown adipocytes, and thus fast conversion of FA into TG, limits the direct oxidation of exogenous FA (49). By comparing tracers for FA uptake ([18F]FTHA) and oxidative activity ([11C]acetate) the notion that internalized FA are first esterified into TG upon which FA can be liberated for mitochondrial β-oxidation was further supported (39). This mode of instant storage implies that BAT thermogenesis is primarily regulated through modulation of lipolysis. Indeed, thermogenesis is almost completely abolished upon inhibition of ATGL or HSL in mice (50) as well as upon suppression of intracellular TG lipolysis in humans (51).
4. ROLE OF BAT IN LIPOPROTEIN METABOLISM AND ATHEROSCLEROSIS
Although it was previously established that BAT takes up FA from plasma to be combusted towards heat (19), it was only recently shown that FA uptake by BAT can reach such extent that BAT activation, by means of cold exposure, largely reduces plasma TG levels of hypertriglyceridemic Apoa5-/- mice (44). Furthermore, plasma TG levels in mice are reduced upon activation of BAT using e.g. β3-AR agonism (31) metformin (52), salsalate (53), rimonabant (54), and VEGF-A overexpression (55). Although it can not be excluded that also alternative pathways contribute to the observed TG lowering, these studies thus indicate that BAT activation is a promising target to combat hypertriglyceridemia.
Several studies suggest that, also in humans, BAT contributes to TG metabolism.
BAT-positive subjects have lower plasma TG and higher HDL-cholesterol levels (56).
Furthermore, LPL expression in perivascular adipose tissue, a BAT depot surrounding the aorta, is negatively correlated with plasma TG levels (57). Studies using the FA tracer [18F]
FTHA have shown increased uptake of FA by BAT after acute cold stimulation (43, 58).
Short-term cold exposure, however, does not seem to be accompanied by decreased plasma TG levels (58, 59). Interestingly, acute cold activation increases hepatic VLDL-TG secretion in rats (60). Therefore, it is possible that acute cold exposure of humans causes
a rapid increase in hepatic VLDL-TG production that can mask a potential BAT-mediated decrease in plasma TG. This hypothesis still needs to be experimentally addressed.
Only recently, it has been shown that BAT activation not only alleviates hypertriglyceridemia, but also hypercholesterolemia and even atherosclerosis development. This atheroprotective effect was shown in hyperlipidemic APOE*3-Leiden.
CETP (E3L.CETP) mice, that express a natural occurring variant of human apoE3 with attenuated binding affinity for the LDLR. In contrast to E3L.CETP mice, β3-AR agonism did not reduce plasma cholesterol levels and atherosclerosis development in Ldlr-/- and Apoe-
/- mice (31, 61), indicating that an intact apoE-LDLR pathway is crucial for the cholesterol- lowering effect of BAT activation. BAT activation might thus be a promising tool to combat hypercholesterolemia and atherosclerosis development. It remains unknown whether BAT activation may be beneficial to further reduce hypercholesterolemia and atherosclerosis development in addition to classic lipid-lowering strategies by e.g. statins and bile acid sequestrants.
The effect of BAT activation on hypercholesterolemia and atherosclerosis development in humans is still largely unknown. Thus far, only few studies investigated the effect of BAT activation on plasma cholesterol levels. One study reported that subjects with detectable BAT have lower plasma total cholesterol and LDL-cholesterol levels as compared to subjects without detectable BAT (62). Moreover, prolonged daily cold exposure of 20 min for 90 days reduced total cholesterol, LDL-cholesterol and body mass in hypercholesterolemic individuals, without differences in physical activity and food intake (63). These data suggest that BAT activation may alleviate hypercholesterolemia in humans, thus decreasing atherosclerosis. In line, a recent study demonstrated that high BAT activity is associated with a reduced risk of CVD events (64). Interestingly, South Asians, a population with increased risk for developing type 2 diabetes, metabolic syndrome and cardiovascular diseases have lower energy expenditure, non-shivering thermogenesis and BAT volume compared to white Caucasians, which may contribute to their increased cardiometabolic risk (59). However, whether decreased BAT activity is causally related to the development of cardiovascular diseases in humans and whether BAT activation might prevent atherosclerosis development remains to be elucidated.
5. ROLE OF IMMUNE SYSTEM IN METABOLISM AND 1
ATHEROSCLEROSIS
5.1. Immune cells and cytokine production
In addition to lipids, also the immune system largely contributes to the development of atherosclerosis, i.e. by inducing a pro-inflammatory state. Immune cells originate from hematopoietic stem cells and can be divided into cells from the innate immune system and the adaptive immune system. The innate immune system is also known as the non- specific immune system as it recognizes pathogens in a generic way, and consists of several cell types such as monocytes and neutrophils. Monocytes can differentiate into macrophages or dendritic cells, phagocytic cells that engulf pathogens. After ingestion of pathogenic material, antigens are presented on the outer surface of these cells via major histocompatibility complex (MHC) molecules (65).
T-cells can be subdivided into T helper cells and cytotoxic T-cells and are part of the adaptive immune system. B-cells also belong to the adaptive immune system, but will be not further introduced in this thesis. The adaptive immune system is referred to as the specific immune system, since adaptive immune cells create immunological memory after initial exposure to pathogens (66). T helper cells do not have phagocytic or cytotoxic capacities, but mediate immune responses by recognizing antigens presented on MHC complexes, which leads to their activation. Upon T helper cell activation, cytokines are produced, resulting in recruitment of monocytes and activation of cytotoxic T cells.
Cytotoxic T cells are so-called killer cells and are responsible for cell death by producing cytotoxins that induce apoptosis of infected cells (67).
Cells from both the innate and adaptive immune system play an important role in the development and progression of atherosclerosis by producing cytokines and chemokines. These cytokines and chemokines serve as important signal molecules and can be either pro-inflammatory or anti-inflammatory. Pro-inflammatory cytokines and chemokines include TNFα and MCP1 that attract monocyte-derived macrophages to sites of inflammation, monocyte-colony stimulating factor (M-CSF) that stimulates differentiation of monocytes into macrophages, and interferon-γ that activates macrophages. In addition, a number of interleukins (i.e. group of cytokines; IL), such as IL1β, IL6 and IL12 are pro-inflammatory. In contrast, IL10 and IL37 are regarded as anti- inflammatory (68). While the anti-atherogenic role of IL-10 has been demonstrated (69),
the role of IL-37 in atherosclerosis progression is still unclear.
The cytokines that are produced largely control immune responses. For example, the cytokine environment dictates whether macrophages are classically activated, leading to the pro-inflammatory M1 phenotype, or alternatively activated, leading to the anti- inflammatory M2 phenotype (70). Since inflammatory diseases are characterized by a shift towards more pro-inflammatory cytokine production, modulating the balance towards the anti-inflammatory side may hold therapeutic potential in the development of for example atherosclerosis. Indeed, the recent CANTOS trial showed that anti- inflammatory therapy targeting the IL-1β innate immune pathway lowered recurrent cardiovascular events, even independent of lipid lowering (71).
5.2. Pattern recognition receptors
Cells from the innate immune cells recognize pathogens after binding to pattern recognition receptors (PRRs) that are expressed on the membrane of immune cells and are involved in translating danger signals to cellular responses. These danger signals can be either microbe-specific molecular signatures known as pathogen-associated molecular patterns (PAMPs) or self-derived molecules derived from damaged cells, referred to as damage-associated molecular patterns (DAMPs). Since there are many different pathogens, also various classes of PRRs are presented on the membrane of immune cells, such as toll-like receptors (TLR), retinoic acid-inducible gene I (RIG-I)- like receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, and C-type lectin receptors (CLRs). These PRRs cooperate in transducing pro-inflammatory responses via co-activation. For example, products produced by TRLs can also activate NOD-like receptors and some PRRs share the same downstream signaling molecule (72).
The TRLs and CLRs are the most studied PRRs and are responsible for the recognition of PAMPs. The TLR family mainly binds FA-like structures on pathogens. Activation of TLRs leads to downstream signaling, eventually leading to activation of nuclear factor-kappa B (NF-κB) and subsequent secretion of pro-inflammatory cytokines (73). In contrast, the CLR family is characterized by a carbohydrate-binding domain which can bind carbohydrates of e.g. fungi, mainly β-glucans. Well-known members of the CLR family are Dectin-1, Dectin-2 and Mincle, that signal through the PKCδ-CARD9-Bcl-10-MALT1 axis, which also leads to activation of NF-κB and secretion of pro-inflammatory cytokines. The caspase recruitment domain-containing protein (CARD) 9 is part of this axis and a master regulator in signal transduction of all CLRs (74, 75).
Although the classical function of PRR is to recognize pathogens, these receptors
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may also become activated after binding of endogenous ligands. TLR2 and TLR4 can be activated by endogenous saturated FA, thereby promoting the development of metabolic diseases (76). CLRs can also become activated by endogenous ligands in disease models of atherosclerosis (77) and cancer (78), although it is not always fully understood which endogenous structures serve as ligand. Although the aggravating effects of TLRs on atherosclerosis have been documented (79, 80), the effects of CLR family members and downstream signaling by CARD9 on atherosclerosis have as yet not been determined.
6. OUTLINE OF THIS THESIS
As outlined in this chapter (chapter 1), both hyperlipidemia and inflammation contribute to atherosclerosis. Since activated BAT largely alleviates hypertriglyceridemia and hypercholesterolemia, BAT activation is a promising therapeutic strategy to protect from atherosclerosis development. On the other hand, given the recent success of the CANTOS trial, anti-inflammatory strategies are also expected to be reduce atherosclerosis.
Therefore, we aimed to address two key objectives in this thesis: 1) to identify genetic targets in mice and men that are involved in BAT activity and evaluate their effects on lipoprotein metabolism and atherosclerosis development, and 2) to identify genetic targets in mice that are involved in immune system modulation and evaluate their effects on atherosclerosis development.
To address key objective 1, we first set out to investigate by which mechanism activated BAT alleviates hypertriglyceridemia. While activated BAT has been shown to take up lipids, whether BAT takes up FA by means of whole particle uptake or after liberation by LPL-mediated lipolysis of TG from TRLs remained to be elucidated. The aim of chapter 2 was, therefore, to evaluate how BAT takes up TRL-derived FA by using radiolabeled TRL- like particles of various sizes (i.e. small VLDL up to small chylomicrons). Since statins increase the hepatic uptake of cholesterol-enriched lipoprotein remnants, the aim of chapter 3 was to evaluate whether statin treatment enhances the lipid-lowering and anti-atherogenic effects of BAT activation. Hepatic cholesterol can be used to produce bile acids or is directly excreted via bile acids. In the intestines, bile acids are either reabsorbed and transported to the liver, or excreted from the body via the feces. Bile acid
metabolism is thus an important determinant of hepatic and plasma cholesterol levels.
In chapter 4, we investigated the effects of β3-AR agonism on bile acid metabolism and studied whether the addition of the bile acid sequestrant colesevelam (i.e. an inhibitor of bile acid reabsorption) on top of β3-AR agonism further lowers plasma lipid levels.
Next, we made a first attempt to investigate whether the effect of BAT activation on lipoprotein metabolism could also be translated to humans. Therefore, in chapter 5, the purpose was to evaluate the effects of BAT activation, by means of cold exposure, on lipoprotein metabolism in young, healthy men.
To meet our second key objective, we evaluated the effect of various immune modulators on atherosclerosis development. In chapter 6, we first studied the effect of hematopoietic IL-37 expression on atherosclerosis development. Next, we switched to the PRRs and specifically studied the involvement of the CLR family, and downstream signaling, in atherosclerosis development. In chapter 7 we investigated whether deletion of hematopoietic Dectin-2 or CARD9, responsible for binding of carbohydrate structures of pathogens, protects from atherosclerosis development. Since hyperglycemia causes glycosylation of proteins in the circulation, these receptors may also play a role in the progression of atherosclerosis in diabetic patients. Therefore, we next investigated the effect of deletion of hematopoietic Dectin-2 or CARD9 on atherosclerosis development under hyperglycemic conditions in chapter 8.
Finally, in chapter 9 the major findings of these studies and their implications for the development of therapeutic strategies are discussed.
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Chapter
Brown adipose tissue takes up plasma triglycerides mostly after lipolysis
P. Padmini S.J. Khedoe*, Geerte Hoeke*,Sander Kooijman, Wieneke Dijk, Jeroen T. Buijs,Sander Kersten,Louis M. Havekes, Pieter S. Hiemstra,Jimmy F.P. Berbée,Mariëtte R. Boon, Patrick C.N. Rensen
*Contributed equally
J Lipid Res 2015; 56: 51-9
2
2
ABSTRACT
Brown adipose tissue (BAT) produces heat by burning TG that are stored within intracellular lipid droplets and need to be replenished by the uptake of TG-derived FA from plasma. It is currently unclear whether BAT takes up FA via uptake of TG-rich lipoproteins (TRLs), after lipolysis-mediated liberation of FA, or via a combination of both.
Therefore, we generated glycerol tri[3H]oleate and [14C]cholesteryl oleate double-labeled TRL-mimicking particles with an average diameter of 45, 80 and 150 nm (representing small VLDL to chylomicrons) and injected these intravenously into male C57Bl/6J mice.
At room temperature (21°C), the uptake of 3H-activity by BAT, expressed per gram tissue, was much higher than the uptake of 14C-activity, irrespective of particle size, indicating lipolysis-mediated uptake of TG-derived FA rather than whole particle uptake. Cold exposure (7°C) increased the uptake of FA derived from the differently sized particles by BAT, while retaining the selectivity for uptake of FA over CE. At thermoneutrality (28°C), total FA uptake by BAT was attenuated, but the specificity of uptake of FA over CE was again largely retained. Altogether, we conclude that, in our model, BAT takes up plasma TG preferentially by means of lipolysis-mediated uptake of FA.
INTRODUCTION
Brown adipose tissue (BAT) is an important player in energy homeostasis due to its ability to combust energy towards heat by virtue of the presence of uncoupling protein 1 (UCP1), a process called non-shivering thermogenesis (1). The most well-known trigger for activation of BAT is cold, which increases sympathetic outflow from the hypothalamic temperature center towards BAT. Here, nerve endings release noradrenalin that binds to adrenergic receptors on the brown adipocyte membrane (2). Activation of an intracellular signaling cascade subsequently leads to a rapid induction of intracellular lipolysis, mediated by adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL) and monoglyceride lipase (MGL), resulting in release of FA from TG-filled lipid droplets (3). FA are directed to the mitochondria where they either allosterically activate UCP1 present on the inner membrane of the mitochondria or undergo β-oxidation within the mitochondrial matrix (2). Upon activation, UCP1 dissipates the proton gradient across the inner mitochondrial membrane that is generated by the respiratory chain, resulting in production of heat. Of note, FA used for activation of UCP1 and β-oxidation appear to be mainly derived from intracellular TG stores, rather than from directly internalized FA, as mice that lack ATGL exhibit defective thermogenesis (4). Therefore, replenishment of intracellular TG stores within the brown adipocyte is essential for non-shivering thermogenesis in BAT.
Replenishment of intracellular TG stores is mediated via three mechanisms: uptake of glucose followed by de novo lipogenesis, uptake of albumin-bound FA, and uptake of TG- rich lipoprotein (TRL)-derived FA from the plasma followed by incorporation of FA within TG (2, 3, 5). Circulating TRLs, i.e. VLDL (particle size 40-80 nm) and chylomicrons (particle size 100-500 nm), are the main source for FA stored as TG in BAT (3).
Only recently, BAT appeared as a major player in plasma TG clearance. Bartelt et al. (5) showed that 24 h of cold exposure markedly enhanced clearance of glycerol tri[3H]
oleate-labeled TRLs which was specifically mediated by BAT (5, 6). The authors suggested that upon cold exposure, BAT internalized TG from chylomicron-sized TRL-like particles (approx. 250 nm) via whole particle uptake. However, they also demonstrated that BAT activation by cold was accompanied by enhanced expression of Lpl and Cd36, and the presence of both appeared critical for the uptake of TG (5, 6). Interestingly, the critical involvement of LPL and CD36 suggests lipolysis-mediated uptake of TRL-derived FAs
2
rather than whole particle as would also occur in skeletal muscle, heart and white adipose tissue (WAT) (7). The formed remnant particles, depleted of TG, are subsequently taken up by the liver through an interaction of apoE with the low-density lipoprotein receptor (LDLR) (8).
The aim of the present study was to further investigate how BAT takes up lipoprotein- derived FA from the circulation, examining the importance of selective delipidation of circulating TRL by LPL and whole particle uptake of TRL. To this end, we assessed the uptake of FA into BAT by injecting glycerol tri[3H]oleate and cholesteryl [14C]oleate double-labeled TRL-mimicking particles with diameters ranging from small VLDL to chylomicrons (45-150 nm) in mice, while modulating the activity of BAT using various ambient temperatures (7°C, 21°C and 28°C).
MATERIALS AND METHODS
Animals and diet
For all studies 8-10 week old male C57Bl/6J mice (Jackson Laboratory, Bar Harbor, ME) were used. Mice were housed in conventional cages with a 12:12-h light-dark cycle and had free access to chow food and water. All mouse experiments were performed in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and have received approval from the Animal Ethical Committee (Leiden University Medical Center, Leiden, The Netherlands).
Acclimation to ambient temperature
Mice were single-housed one week prior to the experiment at an environmental temperature of 21°C. Subsequently they were randomized based on fasting plasma TG levels, total cholesterol (TC) levels and body weight in two groups that were exposed to an ambient temperature of 7°C or 21°C for 24 h. During the last 4 h, mice were fasted before performing a terminal kinetic experiment with TRL-mimicking particles (see below). For the first experiment, mice in each temperature group were divided into three groups that received glycerol tri[3H]oleate and cholesteryl[14C]oleate-labeled TRL-mimicking particles of different size (average 45, 80 or 150 nm, n=6 per group).
An additional set of mice received (non-degradable) [3H]cholesteryl oleoyl ether ([3H]
COE)-labeled TRL-mimicking particles of 150 nm. To investigate TG kinetics under
thermoneutral conditions, mice were randomized into two groups that were exposed for 4 h to an ambient temperature of 21°C or 28°C, while being fasted, prior to the kinetic experiment. For this experiment, mice in each temperature group were divided into three groups that received double-labeled TRL-mimicking particles of different size (average 45, 80 or 150 nm, n=6 per group).
Plasma parameters
At randomization and prior to the clearance experiment, a blood sample was collected from the tail vein of 4 h fasted mice into capillaries. Plasma was assayed for TG and TC using enzymatic kits from Roche Diagnostics (Mannheim, Germany).
Preparation of radiolabeled TRL-mimicking emulsion particles
Radiolabeled TRL-mimicking emulsion particles were prepared from 100 mg of total lipid including triolein (70 mg), egg yolk phosphatidylcholine (22.7 mg), lysophosphatidylcholine (2.3 mg), cholesteryl oleate (3.0 mg) and cholesterol (2.0 mg), with addition of glycerol tri[3H]oleate ([3H]TO) (100 μCi) and [14C]cholesteryl oleate ([14C]CO) (10 μCi) (9, 10). In addition, TRL-mimicking particles were prepared with the non-degradable label [3H]cholesteryl oleoyl ether ([3H]COE) (40 μCi). Sonification was performed using a Soniprep 150 (MSE Scientific Instruments, UK) that is equipped with a water bath for temperature (54°C) maintenance, at 10 μm output (9). The emulsion was fractionated by consecutive density gradient ultracentrifugation steps in a Beckman SW 40 Ti rotor. After centrifugation for 27 min at 20,000 rpm at 20°C, an emulsion fraction containing chylomicron-like particles (average size 150 nm) was removed from the top of the tube by aspiration and replaced by NaCl buffer (1.006 g/mL). After a subsequent centrifugation step for 27 min at 40,000 rpm large VLDL-like particles (average size 80 nm) were obtained in a similar manner. A third centrifugation step for 3 h at 40,000 rpm yielded small VLDL-like particles (average size 45 nm). The average size of the particles has previously been validated in numerous studies by means of photon correlation spectroscopy, as initially described (10). Characterization of emulsion fractions was done by determination of TG concentration (as described under Plasma parameters) and radioactivity. Emulsions were stored at 4°C under argon and used for in vivo kinetic experiments within 5 days following preparation.
In vivo clearance of radiolabeled TRL-mimicking emulsion particles
To study the in vivo clearance of radiolabeled TRL-mimicking emulsion particles, mice
