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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
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SUMMARY
Cardiovascular diseases (CVD), which are mainly caused by the development of atherosclerosis, are the leading cause of morbidity and mortality in Western Society.
Two main risk factors for the development of atherosclerosis are hyperlipidemia and inflammation, that cause accumulation of lipids and immune cells, respectively, in the arterial wall. While activation of brown adipose tissue (BAT) is a promising strategy to alleviate hyperlipidemia, reduction of inflammation is also thought to reduce atherosclerosis progression. In this thesis, we aimed to address two key objectives: 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 modulation of the immune system and evaluate their effects on atherosclerosis development. Chapter 1 serves as a general introduction in which hyperlipidemia and inflammation are introduced as the two main causes for atherosclerosis development. More specifically, firstly the physiology and role of BAT in lipoprotein metabolism and atherosclerosis development are explained.
Secondly, the contribution of pro- and anti-inflammatory cytokines as well as specific receptors on immune cells in propelling immune responses are explained in the context of atherosclerosis development.
Activated BAT takes up large amounts of triglyceride (TG)-derived fatty acids (FA) from the circulation in order to generate heat. However, the mechanism by which BAT takes up these TG-derived FA remained elusive. In chapter 2, we studied whether BAT takes up TG-derived FA after lipolysis from TG-rich lipoproteins (TRL), via uptake of whole TRL or via a combination of both. Glycerol tri[3H]oleate and [14C]cholesteryl oleate double- labeled TRL-mimicking particles of various sizes were injected into mice that were housed at 28°C (thermoneutrality; inactive BAT), 21°C (moderate BAT activity) or 7°C (high BAT activity). Subsequently, we determined the uptake of these radiolabels by several metabolic organs including BAT. We demonstrated that BAT takes up TG-derived FA mainly after lipoprotein lipase (LPL)-mediated lipolysis, rather than via whole particle uptake. This was the case for TRL-mimicking particles of all sizes and was independent of the activity level of BAT, although the total amount of TG-derived FA taken up by BAT was much larger when BAT was activated by an environmental temperature of 7°C.
We have also shown that, as a consequence of the above demonstrated increased uptake
of TG-derived FA by activated BAT, the formation and hepatic uptake of cholesterol- enriched TRL remnants is also accelerated. Via this mechanism, BAT activation alleviates hypercholesterolemia and atherosclerosis development in APOE*3-Leiden.CETP (E3L.
CETP) mice. Since statins increase the hepatic uptake of TRL remnants, we hypothesized in chapter 3 that statin treatment increases the lipid-lowering and anti-atherogenic effects of BAT activation by accelerating the clearance of TRL remnants generated by activated BAT. We tested this hypothesis by combining statin treatment with a β3-adrenergic receptor (β3-AR) agonist to activate BAT in hyperlipidemic E3L.CETP mice. Indeed, statin treatment on top of BAT activation further increased the hepatic uptake of cholesterol- enriched TRL remnants as compared to β3-AR agonism alone. As a consequence, concomitant treatment with the statin further increased the cholesterol-lowering and atheroprotective effects of BAT activation. From this, we concluded that combining statin treatment, which is the first choice in treatment of hypercholesterolemia, with BAT activation has therapeutic potential to reduce CVD further than can be reached by statin therapy alone.
In addition to increasing the hepatic uptake of cholesterol-enriched TRL remnants, BAT activation also increases the hepatic uptake of high-density lipoprotein (HDL)- cholesterol. BAT activation thus not only induces a flux of cholesterol to the liver via increasing the hepatic uptake of TRL remnants but also by enhancing the hepatic clearance of HDL-derived cholesterol. Since hepatic cholesterol is mainly used for the synthesis of bile acids (BAs) we studied the effect of BAT activation on hepatic cholesterol and BA metabolism in chapter 4. Prolonged BAT activation, by means of β3-AR agonism, reduced fecal BA excretion, increased BA levels in plasma, and induced hepatic cholesterol accumulation in E3L.CETP mice. Concomitant treatment with colesevelam, which is a BA sequestrant that blocks BA reabsorption from the gut, increased fecal BA excretion, normalized plasma BA levels, and reversed the hepatic cholesterol accumulation caused by β3-AR agonism. Moreover, plasma total cholesterol levels were further reduced by this combination compared to β3-AR agonism alone. We concluded that BAT activation combined with BA sequestration improves BA metabolism, prevents the accumulation of hepatic cholesterol, and further reduces plasma cholesterol as compared to BAT activation alone. Therefore, combining BAT activation with BA sequestration additively improves cholesterol metabolism and may further reduce atherosclerosis development.
The above-described studies increased our knowledge of the mechanistic effects of
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BAT activation on lipid and lipoprotein metabolism in mice. However, the effect of BAT activation on human lipid and lipoprotein metabolism has not been extensively studied yet. Therefore, in chapter 5, we studied the effect of BAT activation, by means of short- term cooling, on the serum lipoprotein profile and HDL functionality in men. Before and after exposure to a personalized cooling protocol for 2 hours, serum samples were collected from young, lean men. Short-term cooling increased serum levels of free FA, TG, and cholesterol. This was accompanied by an increased concentration of large very- low-density lipoprotein (VLDL) particles upon cooling. In addition, cooling increased the concentration of small HDL particles as well as their cholesterol content. Small HDL particles are efficient acceptors of cholesterol via adenosine triphosphate-binding cassette A1 (ABCA1). Indeed, the increase in small HDL particles was accompanied by increased ABCA1-dependent cholesterol efflux in vitro. Short-term cooling thus increases the concentration of large VLDL particles and increases the generation of small HDL particles. In addition, short-term cooling increases HDL functionality, which may confer atheroprotective properties.
In chapter 6 we shifted our focus from the anti-atherogenic effects of BAT activation to the role of inflammation in atherosclerosis development. We reasoned that immune modulators that reduce the pro-inflammatory environment may protect from atherosclerosis development. As the human anti-inflammatory cytokine interleukin (IL)-37 may be such an immune modulator, we investigated the effect of hematopoietic expression of IL-37 on atherosclerosis development under low-grade inflammatory conditions. To this end, low-density lipoprotein receptor-deficient (Ldlr-/-) mice were lethally irradiated and transplanted with bone marrow from IL-37-transgenic or control wild-type mice and fed a Western-type diet. While hematopoietic IL-37 expression did not influence body weight, food intake and plasma cholesterol levels during the study, IL-37 expression did reduce the inflammatory state. Hematopoietic IL-37 expression did not influence the atherosclerotic lesion area and only marginally influenced plaque composition. Although the smooth muscle content within the lesion was decreased, macrophage and collagen content were not different. We therefore concluded that, under low-grade inflammatory conditions, hematopoietic IL-37 expression does not influence atherosclerosis development, at least in hyperlipidemic Ldlr-/- mice.
The production of pro- and anti-inflammatory cytokines by immune cells is determined by activation of immune receptors on their cell membrane. Reducing immune receptor
activation may therefore also reduce the pro-inflammatory environment. In chapter 7, we evaluated the role of the C-type lectin receptor (CLR) family in the pathogenesis of atherosclerosis. We studied whether hematopoietic deletion of the CLR Dectin-2 or the CLR adaptor molecule CARD9 reduces inflammation and atherosclerosis development.
To do so, Ldlr-/- mice were transplanted with bone marrow from control wild-type (WT), Dectin-2-/- or Card9-/- mice and fed a Western-type diet. Deletion of hematopoietic Dectin-2 did not influence the systemic inflammatory state or atherosclerosis development.
Although deletion of hematopoietic CARD9 also did not influence the inflammatory state, it unexpectedly increased the atherosclerotic lesion size. From these data we concluded that deletion of hematopoietic Dectin-2 does not influence atherosclerosis development, while deletion of hematopoietic CARD9 increases atherosclerotic lesion size, suggesting that the presence of CARD9 may have a protective role in atherosclerosis development.
CLRs are classically involved in the recognition of sugars and sugar-like structures present on fungi. In chapter 8, we hypothesized that under hyperglycemic conditions glycosylated protein (sugar-like) structures activate CLRs leading to immune cell activation and increased atherosclerosis development. Absence of CLRs during hyperglycemia may thus protect from atherosclerosis. We therefore next evaluated the effect of deletion of hematopoietic Dectin-2 or CARD9 on inflammation and atherosclerosis development under hyperglycemic conditions. Ldlr-/- mice were lethally irradiated and transplanted with bone marrow from control WT mice, Dectin-2-/- or Card9-/- mice. To induce hyperglycemia, insulin production by the pancreas was eliminated by injecting streptozotocin 6 weeks after BMT. Two weeks later, mice were fed a Western-type diet for 10 weeks. Under hyperglycemic conditions, deletion of hematopoietic Dectin-2 reduced the number of circulating pro-inflammatory Ly6Chi monocytes, while the pro-inflammatory cytokine production by LPS- and Pam3Cys-stimulated macrophages was increased. Deletion of hematopoietic Dectin-2 did not influence atherosclerosis development. While deletion of hematopoietic CARD9 did not influence the inflammatory state or lesion size, it tended to reduce the macrophage and collagen content in atherosclerotic lesions. From these data we concluded that under hyperglycemic conditions, deletion of hematopoietic Dectin-2 or CARD9 does not influence atherosclerosis development.
In chapter 9, we evaluated the results of this thesis and discussed the translational value of our research regarding the role of BAT and inflammation in the protection
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against cardiometabolic diseases in humans. In addition, the future directions that are required to implement BAT activation and immune modulation in clinical practice to combat cardiometabolic diseases are discussed. Taken together, the studies described in this thesis increased our knowledge on the potential of BAT activation and immune modulation to combat CVD, and suggest that especially BAT activation is a promising strategy to further pursue.
