👤 Sander Kooijman

Dysregulated blood lipids are a major predictor of cardiovascular events. A recent genome-wide association study (GWAS) with five clinically relevant lipid traits in 1.65 million individuals implicated over 770 genomic regions in regulating blood lipid metabolism. To translate these associations into clinical applications, a functional understanding of their roles in lipoprotein metabolism, transport and remodeling (LPmtr) is required. Here, we report the deep molecular fine-mapping of 554 of these lipid risk loci using 168 lipoprotein-related traits and all possible ratios between them in over 273,000 participants of the UK Biobank. We identified new ratio-based markers of pathways shared by multiple LPmtr genes, such as the linoleic acid fraction of the polyunsaturated fatty acid pool to reveal potential causal genes at poorly characterized lipid risk loci, the percentage of esterified cholesterol moieties in LDL particles as a proxy for soluble LDL receptor levels, and the HDL fraction of total lipoprotein particle number as a predictor of incident myocardial infarction. We demonstrate how lipoprotein fine-mapping can generate new hypotheses for drug target development while uncovering new mechanisms relevant to hyperlipidemia. Ratio-driven clustering further implicated miR-148 in TG secretion, linking ER-stress responses at postprandial state to VLDL metabolism via mTORC1, shown through series of integrated cellular assays and mouse studies. Moreover, consistent with its regulatory influence on lipid flux we identify miR-148a a previously unrecognized determinat of Show less

The chimeric cytokine IC7Fc conveys the metabolic signaling properties of the glycoprotein 130 receptor cytokines interleukin-6 and ciliary neurotrophic factor via membrane-bound signaling. IC7Fc was previously shown to slow the progression of type 2 diabetes mellitus, and here, we demonstrate its effect on atherosclerotic development. In APOE*3-Leiden.CETP mice, an atherosclerosis-prone model with a humanized lipoprotein metabolism, IC7Fc markedly lowered plasma triglyceride and total cholesterol levels. This was mechanistically explained by an inhibition of de novo lipogenesis in the liver, increased synthesis of bile acids from cholesterol, and down-regulated apolipoprotein B synthesis, which resulted in decreased cholesterol secretion in very low-density lipoprotein particles. As a consequence, IC7Fc treatment considerably reduced atherosclerotic lesion formation and vascular inflammation compared with current antihyperlipidemic therapy. In conclusion, IC7Fc is a promising pharmacological treatment for cardiometabolic diseases targeting hyperlipidemia and inflammation. Show less

LIPC encodes hepatic lipase (HL), a liver-bound protein with both phospholipase and triglyceride lipase activity, and involved in the catabolism of circulating lipoproteins. We recently identified the gain-of-function variant HL-E97G, with selectively increased phospholipase activity, as a new genetic cause of familial combined hypocholesterolaemia in humans. The role of HL in the development of atherosclerosis remains controversial. In this context, the action of HL-E97G on the development of atherosclerosis remains unknown. To evaluate the lipid-lowering and anti-atherogenic properties of HL-E97G vs. wildtype HL (HL-WT) in hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism, and to assess dependence of these effects on the LDL receptor (LDLR) pathway in LDLR-deficient (Ldlr-/-) mice. APOE*3.Leiden.CETP mice or Ldlr-/- mice received an intravenous injection of AAV8 expressing either eGFP (control), HL-WT or HL-E97G (3 × 1011 GC/mouse) while being fed pro-atherogenic diets. Plasma cholesterol levels were measured monthly, and aortic atherosclerotic lesion sizes were assessed at termination. HL-E97G largely decreased plasma total cholesterol exposure in APOE*3-Leiden.CETP mice (-63% vs. control; -58% vs. HL-WT), resulting at least in part from increased uptake of (V)LDL by the liver, accompanied by a marked decrease in atherosclerotic lesion size (-98% vs. control; -97% vs. HL-WT) in the aortic root. Importantly, HL-E97G also strongly reduced plasma cholesterol exposure in Ldlr-/- mice (-80% vs. control; -77% vs. HL-WT), and decreased atherosclerotic lesion size in the aortic root (-54% vs. control; -41% vs. HL-WT) and the aortic arch (-73% vs. control; -70% vs. HL-WT). HL-E97G strongly reduces plasma cholesterol levels, by increasing the uptake of (V)LDL, to decrease atherosclerosis development in mice independently of the LDLR pathway. These data suggest that modulating HL function is a promising tool in patients with familial hypercholesterolaemia. Show less

[D APOE*3-Leiden.CETP mice were treated with saline, acyl-ExD3 or acyl-ExF1 via intraperitoneal injections for 6 weeks or intracerebroventricular infusion for 18 days. Body weight and composition were monitored at regular intervals, as were plasma glucose, triglyceride and cholesterol levels. At endpoint, mice were injected with very low-density lipoprotein (VLDL)-like particles containing glycerol tri[ Upon peripheral treatment, body weight gain was prevented and plasma glucose levels were reduced by acyl-ExF1, but circulating lipids were not affected by either acyl-ExF1 or acyl-ExD3. In contrast, central administration of either agonist strongly reduced plasma triglyceride and cholesterol levels, but did not affect glucose levels. Acyl-ExD3 and acyl-ExF1 increased [ The oppositely biased GLP-1 receptor agonists acyl-ExD3 and acyl-ExF1 do not differentially affect lipid metabolism in APOE*3-Leiden.CETP mice, while effects on glucose homeostasis and prevention of body weight gain are more pronounced upon peripheral acyl-ExF1 treatment. Show less

Elevated von Willebrand factor (VWF) levels correlate with higher risk of atherosclerosis-related arterial thrombosis (atherothrombosis). Silencing the This study aimed to investigate whether the LNP-siRNA strategy could achieve endothelium-specific Female transgenic mice expressing a variant of human The LNP-siRNA targeting Show less

Tirzepatide, a glucose-dependent insulinotropic polypeptide/glucagon-like peptide 1 receptor (GIPR/GLP-1R) agonist, has, in clinical trials, demonstrated greater reductions in glucose, body weight, and triglyceride levels compared with selective GLP-1R agonists in people with type 2 diabetes (T2D). However, cellular mechanisms by which GIPR agonism may contribute to these improved efficacy outcomes have not been fully defined. Using human adipocyte and mouse models, we investigated how long-acting GIPR agonists regulate fasted and fed adipocyte functions. In functional assays, GIPR agonism enhanced insulin signaling, augmented glucose uptake, and increased the conversion of glucose to glycerol in a cooperative manner with insulin; however, in the absence of insulin, GIPR agonists increased lipolysis. In diet-induced obese mice treated with a long-acting GIPR agonist, circulating triglyceride levels were reduced during oral lipid challenge, and lipoprotein-derived fatty acid uptake into adipose tissue was increased. Our findings support a model for long-acting GIPR agonists to modulate both fasted and fed adipose tissue function differentially by cooperating with insulin to augment glucose and lipid clearance in the fed state while enhancing lipid release when insulin levels are reduced in the fasted state. Show less

Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid (FA) uptake from triglyceride (TG)-rich lipoproteins such as very low density lipoproteins (VLDL). While pharmacological inhibition of ANGPTL3 is being evaluated as a lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aims to compare the therapeutic potential of liver-specific Angptl3 and Angptl4 silencing to attenuate hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established humanized model for lipoprotein metabolism. Mice were subcutaneously injected twice per week with saline or liver-targeted antisense oligonucleotides against Angptl3, Angptl4, both, or a scrambled oligonucleotide. Plasma lipid levels, VLDL clearance, and hepatic VLDL production were determined, and atherosclerosis development was assessed. For toxicological evaluation, cynomolgus monkeys were treated with three dosages of liver-targeted ANGPTL4-silencing oligonucleotides. Liver-targeted Angptl4 silencing reduced plasma TGs (-48%) and total cholesterol (-56%), explained by higher VLDL-derived FA uptake by brown adipose tissue and lower VLDL production by the liver. Accordingly, Angptl4 silencing reduced atherosclerotic lesion size (-86%) and improved lesion stability. Hepatic Angptl3 silencing similarly attenuated hyperlipidemia and atherosclerosis development. While Angptl3 and Angptl4 silencing lowered plasma TGs in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma TGs independent of the nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects. Liver-targeted Angptl4 silencing potently attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, and liver-targeted ANGPTL4 silencing is well tolerated in non-human primates. These data warrant further clinical development of liver-targeted ANGPTL4 silencing. Show less

Brown adipose tissue (BAT) combusts lipids and glucose to generate heat. Via this process of nonshivering thermogenesis, BAT plays a pivotal role in thermoregulation in cold environments, but its contribution to immune-induced fever is less clear. Male APOE∗3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism, and wild-type mice were given an intraperitoneal injection of Salmonella enterica serovar Typhimurium (S.tm). Energy expenditure and substrate utilization, plasma lipid levels, fatty acid (FA) uptake by adipose tissues, and lipid content and thermogenic markers in adipose tissues were examined. S.tm infection led to a set of characteristic symptoms, including elevated body temperature and decreased body weight. Whole-body energy expenditure was significantly decreased 72 h postinfection, but fat oxidation was increased and accompanied by a substantial reduction in plasma triglyceride (TG) levels as demonstrated in APOE∗3-Leiden.CETP mice. S.tm infection strongly increased uptake of FAs from TG-rich lipoproteins by BAT, which showed a positive correlation with body temperature in infected mice. Upon histological examination of BAT from wild-type or APOE∗3-Leiden.CETP mice, elevated levels of tyrosine hydroxylase were observed, indicative of stimulated sympathetic activity. In addition, the gene expression profile was consistent with more adrenergic stimulation, while lipid content was reduced. Furthermore, browning of white adipose tissue was observed, evidenced by a modest increase in TG-derived FA uptake, the presence of multilocular cells, and induction of uncoupling protein 1 expression. We proposed that BAT, or thermogenic adipose tissue in general, is involved in the maintenance of elevated body temperature upon invasive bacterial infection. Show less

The liver X receptor (LXR) is considered a therapeutic target for atherosclerosis treatment, but synthetic LXR agonists generally also cause hepatic steatosis and hypertriglyceridemia. Desmosterol, a final intermediate in cholesterol biosynthesis, has been identified as a selective LXR ligand that suppresses inflammation without inducing lipogenesis. Δ24-Dehydrocholesterol reductase (DHCR24) converts desmosterol into cholesterol, and we previously showed that the DHCR24 inhibitor SH42 increases desmosterol to activate LXR and attenuate experimental peritonitis and metabolic dysfunction-associated steatotic liver disease. Here, we aimed to evaluate the effect of SH42 on atherosclerosis development in APOE∗3-Leiden.CETP mice and low-density lipoproteins (LDL) receptor knockout mice, models for lipid- and inflammation-driven atherosclerosis, respectively. In both models, SH42 increased desmosterol without affecting plasma lipids. While reducing liver lipids in APOE∗3-Leiden.CETP mice, and regulating populations of circulating monocytes in LDL receptor knockout mice, SH42 did not attenuate atherosclerosis in either model. Show less

Cardiovascular disease, the primary cause of human mortality globally, is predominantly caused by a progressive disorder known as atherosclerosis. Atherosclerosis refers to the process of accumulation of cholesterol-enriched lipoproteins and the concomitant initiation of inflammatory processes in the arterial wall, including the recruitment of immune cells. This leads to the formation of atherosclerotic plaques, initially causing a thickening of the arterial wall and narrowing of arteries. However, as plaque formation progresses, atherosclerotic plaques may become unstable and rupture, leading to a blood clot that blocks the affected artery or travels through the blood to block blood flow elsewhere. In the early 1990s, emerging gene editing methods enabled the development of apolipoprotein E knockout (Apoe-/- ) and low-density lipoprotein receptor knockout (Ldlr-/- ) mice. These mice have been instrumental in unraveling the complex pathogenesis of atherosclerosis. Around the same time, human APOE*3-Leiden transgenic mice were generated, which were more recently cross-bred with human cholesteryl ester transfer protein (CETP) transgenic mice to generate APOE*3-Leiden.CETP mice. This model appears to closely mimic human lipoprotein metabolism and responds to classic lipid-lowering interventions due to an intact ApoE-LDLR pathway of lipoprotein remnant clearance. In this review, we describe the role of lipid metabolism and inflammation in atherosclerosis development and highlight the characteristics of the frequently used animal models to study atherosclerosis, with a focus on mouse models, discussing their advantages and limitations. Moreover, we present a detailed methodology to quantify atherosclerotic lesion area within the aortic root region of the murine heart, as well as details required for scoring atherosclerotic lesion severity based on guidelines of the American Heart Association adapted for mice. Show less

Combined glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP1R) agonism is superior to single GLP1R agonism with respect to glycemic control and weight loss in obese patients with or without type 2 diabetes. As insulin resistance and obesity are strong risk factors for nonalcoholic fatty liver disease (NAFLD), in the current study we investigated the effects of combined GIPR/GLP1R agonism on NAFLD development. Male APOE∗3-Leiden.CETP mice, a humanized model for diabetic dyslipidemia and NAFLD when fed a high-fat high-cholesterol diet, received subcutaneous injections with either vehicle, a GIPR agonist, a GLP1R agonist, or both agonists combined every other day. GIPR and GLP1R agonism reduced body weight and additively lowered fasting plasma levels of glucose, triglycerides and total cholesterol. Strikingly, we report an additive reduction in hepatic steatosis as evidenced by lower hepatic lipid content and NAFLD scores. Underlying the lipid-lowering effects were a reduced food intake and intestinal lipid absorption and an increased uptake of glucose and triglyceride-derived fatty acids by energy-combusting brown adipose tissue. Combined GIPR/GLP1R agonism also attenuated hepatic inflammation as evidenced by a decreased number of monocyte-derived Kupffer cells and a reduced expression of inflammatory markers. Together, the reduced hepatic steatosis and inflammation coincided with lowered markers of liver injury. We interpretate that GIPR and GLP1R agonism additively attenuate hepatic steatosis, lower hepatic inflammation, ameliorate liver injury, together preventing NAFLD development in humanized APOE∗3-Leiden.CETP mice. We anticipate that combined GIPR/GLP1R agonism is a promising strategy to attenuate NAFLD progression in humans. This work was supported by a grant from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences [CVON-GENIUS-II] to P.C.N.R., a Lilly Research Award Program [LRAP] Award to P.C.N.R. and S.K., a Dutch Heart Foundation [2017T016] grant to S.K., and an NWO-VENI grant [09150161910073] to M.R.B.; J.F.D.B. is supported by the Nutrition and Health initiative of the University of Groningen; Z.Y. is supported by a full-time PhD scholarship from the China Scholarship Council (201806850094 to Z.Y.). Show less

Combined agonism of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP1R) is superior to single GLP1R agonism in terms of glycemic control and lowering body weight in individuals with obesity and with or without type 2 diabetes mellitus. As both GIPR and GLP1R signaling have also been implicated in improving inflammatory responses and lipid handling, two crucial players in atherosclerosis development, here we aimed to investigate the effects of combined GIPR/GLP1R agonism in APOE*3-Leiden.CETP mice, a well-established mouse model for human-like lipoprotein metabolism and atherosclerosis development. Female APOE*3-Leiden.CETP mice were fed a Western-type diet (containing 16% fat and 0.15% cholesterol) to induce dyslipidemia, and received subcutaneous injections with either vehicle, a GIPR agonist (GIPFA-085), a GLP1R agonist (GLP-140) or both agonists. In the aortic root area, atherosclerosis development was assessed. Combined GIPR/GLP1R agonism attenuated the development of severe atherosclerotic lesions, while single treatments only showed non-significant improvements. Mechanistically, combined GIPR/GLP1R agonism decreased markers of systemic low-grade inflammation. In addition, combined GIPR/GLP1R agonism markedly lowered plasma triglyceride (TG) levels as explained by reduced hepatic very-low-density lipoprotein (VLDL)-TG production as well as increased TG-derived fatty acid uptake by brown and white adipose tissue which was coupled to enhanced hepatic uptake of core VLDL remnants. Combined GIPR/GLP1R agonism attenuates atherosclerosis severity by diminishing inflammation and increasing VLDL turnover. We anticipate that combined GIPR/GLP1R agonism is a promising strategy to lower cardiometabolic risk in humans. Show less

Circadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has been associated with elevated risk for atherosclerotic cardiovascular disease (asCVD) in humans, but evidence for the effectiveness of prevention strategies is lacking. Here, we applied time-restricted feeding (TRF) as a strategy to counteract atherosclerosis development during CD in female APOE∗3-Leiden.CETP mice, a well-established model for humanized lipoprotein metabolism. Control groups were subjected to a fixed 12:12 h light-dark cycle, while CD groups were subjected to 6-h phase advancement every 3 days. Groups had either ad libitum (AL) access to food or were subjected to TRF with restricted food access to the dark phase. TRF did not prevent the increase in the relative abundance of circulating inflammatory monocytes and elevation of (postprandial) plasma triglycerides during CD. Nonetheless, TRF reduced atherosclerotic lesion size and prevented an elevation in macrophage content of atherosclerotic lesions during CD, while it increased the relative abundance of anti-inflammatory monocytes, prevented activation of T cells, and lowered plasma total cholesterol levels and markers of hepatic cholesterol synthesis. These effects were independent of total food intake. We propose that time restricted eating could be a promising strategy for the primary prevention of asCVD risk in shift workers, which warrants future study in humans. This work was funded by the Novo Nordisk Foundation, the Netherlands Ministry of Social Affairs and Employment, Amsterdam Cardiovascular Sciences, and the Dutch Heart Foundation. Show less

Short-chain fatty acids, including butyrate, have multiple metabolic benefits in individuals who are lean but not in individuals with metabolic syndrome, with the underlying mechanisms still being unclear. We aimed to investigate the role of gut microbiota in the induction of metabolic benefits of dietary butyrate. We performed antibiotic-induced microbiota depletion of the gut and fecal microbiota transplantation (FMT) in APOE*3-Leiden.CETP mice, a well-established translational model for developing human-like metabolic syndrome, and revealed that dietary butyrate reduced appetite and ameliorated high-fat diet-induced (HFD-induced) weight gain dependent on the presence of gut microbiota. FMT from butyrate-treated lean donor mice, but not butyrate-treated obese donor mice, into gut microbiota-depleted recipient mice reduced food intake, attenuated HFD-induced weight gain, and improved insulin resistance. 16S rRNA and metagenomic sequencing on cecal bacterial DNA of recipient mice implied that these effects were accompanied by the selective proliferation of Lachnospiraceae bacterium 28-4 in the gut as induced by butyrate. Collectively, our findings reveal a crucial role of gut microbiota in the beneficial metabolic effects of dietary butyrate as strongly associated with the abundance of Lachnospiraceae bacterium 28-4. Show less

Activation of brown adipose tissue (BAT) with the β3-adrenergic receptor agonist CL316,243 protects mice from atherosclerosis development, and the presence of metabolically active BAT is associated with cardiometabolic health in humans. In contrast, exposure to cold or treatment with the clinically used β3-adrenergic receptor agonist mirabegron to activate BAT exacerbates atherosclerosis in apolipoprotein E (ApoE)- and low-density lipoprotein receptor (LDLR)-deficient mice, both lacking a functional ApoE-LDLR pathway crucial for lipoprotein remnant clearance. We, therefore, investigated the effects of mirabegron treatment on dyslipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a humanized lipoprotein metabolism model with a functional ApoE-LDLR clearance pathway. Mirabegron activated BAT and induced white adipose tissue (WAT) browning, accompanied by selectively increased fat oxidation and attenuated fat mass gain. Mirabegron increased the uptake of fatty acids derived from triglyceride (TG)-rich lipoproteins by BAT and WAT, which was coupled to increased hepatic uptake of the generated cholesterol-enriched core remnants. Mirabegron also promoted hepatic very low-density lipoprotein (VLDL) production, likely due to an increased flux of fatty acids from WAT to the liver, and resulted in transient elevation in plasma TG levels followed by a substantial decrease in plasma TGs. These effects led to a trend toward lower plasma cholesterol levels and reduced atherosclerosis. We conclude that BAT activation by mirabegron leads to substantial metabolic benefits in APOE*3-Leiden.CETP mice, and mirabegron treatment is certainly not atherogenic. These data underscore the importance of the choice of experimental models when investigating the effect of BAT activation on lipoprotein metabolism and atherosclerosis. Show less

The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1 h five times a week for 4 weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less

Brown adipose tissue (BAT) burns fatty acids (FAs) to produce heat, and shows diurnal oscillation in glucose and triglyceride (TG)-derived FA-uptake, peaking around wakening. Here we aimed to gain insight in the diurnal regulation of metabolic BAT activity. RNA-sequencing, chromatin immunoprecipitation (ChIP)-sequencing, and lipidomics analyses were performed on BAT samples of wild type C57BL/6J mice collected at 3-hour intervals throughout the day. Knockout and overexpression models were used to study causal relationships in diurnal lipid handling by BAT. We identified pronounced enrichment of oscillating genes involved in extracellular lipolysis in BAT, accompanied by oscillations of FA and monoacylglycerol content. This coincided with peak lipoprotein lipase (Lpl) expression, and was predicted to be driven by peroxisome proliferator-activated receptor gamma (PPARγ) activity. ChIP-sequencing for PPARγ confirmed oscillation in binding of PPARγ to Lpl. Of the known LPL-modulators, angiopoietin-like 4 (Angptl4) showed the largest diurnal amplitude opposite to Lpl, and both Angptl4 knockout and overexpression attenuated oscillations of LPL activity and TG-derived FA-uptake by BAT. Our findings highlight involvement of PPARγ and a crucial role of ANGPTL4 in mediating the diurnal oscillation of TG-derived FA-uptake by BAT, and imply that time of day is essential when targeting LPL activity in BAT to improve metabolic health. Show less

Brown adipose tissue (BAT) contributes to cardiometabolic health by taking up glucose and lipids for oxidation, a process that displays a strong diurnal rhythm. While aging has been shown to reduce thermogenic characteristics of BAT, it is as yet unknown whether this reduction is specific to the time of day. Therefore, we assessed whole-body and BAT energy metabolism in young and middle-aged male and female C57BL/6J mice and studied the consequences for lipid metabolism in humanized APOE*3-Leiden.CETP mice (also on a C57BL/6J background). We demonstrate that in middle-aged versus young mice body temperature is lower in both male and female mice, while uptake of triglyceride (TG)-derived fatty acids (FAs) by BAT, reflecting metabolic activity, is attenuated at its peak at the onset of the dark (wakeful) phase in female mice. This coincided with delayed plasma clearance of TG-rich lipoproteins and TG-depleted lipoprotein core remnants, and elevated plasma TGs at the same time point. Furthermore, middle-aged female mice showed increased adiposity, accompanied by lipid accumulation, increased expression of genes involved in lipogenesis, and reduced expression of genes involved in fat oxidation and the intracellular clock machinery in BAT. Peak abundance of lipoprotein lipase (LPL), a crucial regulator of FA uptake, was attenuated in BAT. Our findings suggest that LPL is a potential therapeutic target for restoring diurnal metabolic BAT activity, and that efficiency of strategies targeting BAT may be improved by including time of day as an important factor. Show less

Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, Using next-generation sequencing, we identified a novel dominant rare variant in the Family members carrying the We identified and characterized a novel rare variant in the Show less

Fibroblast growth factor (FGF) 21, a key regulator of energy metabolism, is currently evaluated in humans for treatment of type 2 diabetes and non-alcoholic steatohepatitis. However, the effects of FGF21 on cardiovascular benefit, particularly on lipoprotein metabolism in relation to atherogenesis, remain elusive. Here, the role of FGF21 in lipoprotein metabolism in relation to atherosclerosis development was investigated by pharmacological administration of a half-life extended recombinant FGF21 protein to hypercholesterolaemic APOE*3-Leiden.CETP mice, a well-established model mimicking atherosclerosis initiation and development in humans. FGF21 reduced plasma total cholesterol, explained by a reduction in non-HDL-cholesterol. Mechanistically, FGF21 promoted brown adipose tissue (BAT) activation and white adipose tissue (WAT) browning, thereby enhancing the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT and into browned WAT, consequently accelerating the clearance of the cholesterol-enriched remnants by the liver. In addition, FGF21 reduced body fat, ameliorated glucose tolerance and markedly reduced hepatic steatosis, related to up-regulated hepatic expression of genes involved in fatty acid oxidation and increased hepatic VLDL-triglyceride secretion. Ultimately, FGF21 largely decreased atherosclerotic lesion area, which was mainly explained by the reduction in non-HDL-cholesterol as shown by linear regression analysis, decreased lesion severity, and increased atherosclerotic plaque stability index. FGF21 improves hypercholesterolaemia by accelerating triglyceride-rich lipoprotein turnover as a result of activating BAT and browning of WAT, thereby reducing atherosclerotic lesion severity and increasing atherosclerotic lesion stability index. We have thus provided additional support for the clinical use of FGF21 in the treatment of atherosclerotic cardiovascular disease. Show less

We analyzed the effects of the common BMI-increasing melanocortin 4 receptor (MC4R) rs17782313-C allele with a minor allele frequency of 0.22-0.25 on (1) cardiovascular disease outcomes in two large population-based cohorts (Copenhagen City Heart Study and Copenhagen General Population Study, Show less

Pharmacological blockade of the cannabinoid type 1 receptor, a G protein-coupled receptor expressed in the central nervous system and various peripheral tissues, reverses diet-induced obesity and dyslipidemia through the reduction of food intake and altered nutrient partitioning. This strategy is being explored for a number of therapeutic applications; however, its potency for the treatment of atherosclerotic cardiovascular disease via improvements in lipid metabolism remains unclear. Therefore, here, we aimed to investigate whether inhibition of the endocannabinoid system can attenuate atherosclerosis development through improvement of dyslipidemia. Lean, dyslipidemic female APOE∗3-Leiden.CETP transgenic mice were fed a Western-type diet supplemented with or without the cannabinoid type 1 receptor inverse agonist rimonabant (20 mg·kg body weight Show less

Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition, by increasing hepatic low density lipoprotein (LDL) receptor (LDLR) levels, has emerged as a strategy to reduce atherosclerosis by lowering circulating very low density lipoprotein (VLDL)-cholesterol. We hypothesized that the therapeutic effectiveness of PCSK9 inhibition can be increased by accelerating the generation of VLDL remnants, which typically have a high affinity for the LDLR. Therefore, we aimed to investigate whether accelerating lipolytic processing of VLDL by brown fat activation can further lower (V)LDL and reduce atherosclerosis on top of PCSK9 inhibition. APOE*3-Leiden.CETP mice were fed a Western-type diet and treated with the anti-PCSK9 antibody alirocumab or saline. After 2 weeks, both groups of mice were randomized to receive either the selective β3-adrenergic receptor (AR) agonist CL316,243 to activate brown fat or saline for 3 additional weeks to evaluate VLDL clearance or 12 additional weeks to analyze atherosclerosis development. β3-AR agonism and alirocumab combined decreased (V)LDL-cholesterol compared to alirocumab alone, which was explained by an accelerated plasma clearance of VLDL-cholesteryl esters that were mainly taken up by the liver. In addition, the combination promoted the transfer of VLDL-phospholipids to HDL to a higher extent than alirocumab alone, accompanied by higher plasma HDL-cholesterol levels and increased cholesterol efflux capacity. Consequently, combination treatment largely reduced atherosclerotic lesion area compared to vehicle. Together, β3-AR agonism enhances the lipoprotein-modulating effects of alirocumab to further improve dyslipidemia and non-significantly further attenuate atherosclerosis development. Our findings demonstrate that brown fat activation may enhance the therapeutic effects of PCSK9 inhibition in dyslipidemia. Show less

Artificial light exposure is associated with dyslipidemia in humans, which is a major risk factor for the development of atherosclerotic cardiovascular disease. However, it remains unclear whether artificial light at night can exacerbate atherosclerosis. In this study, we exposed female APOE*3-Leiden.CETP mice, a well-established model for human-like lipid metabolism and atherosclerosis, to either a regular light-dark cycle or to constant bright light for 14 weeks. Mice exposed to constant light demonstrated a minor reduction in food intake, without any effect on body weight, body composition, or the weight of metabolic organs. Constant light increased the plasma levels of proatherogenic non-high-density lipoprotein (HDL) cholesterol but did not increase the size or severity of atherosclerotic lesions in the aortic root. Mice exposed to constant light did show lower immune cell counts, which could explain the absence of an effect of atherosclerosis despite increased non-HDL cholesterol levels. Behavioral analysis demonstrated variability in the response of mice to the light intervention. Constant light completely blunted behavioral rhythms in some mice, while others extended their behavioral period. However, rhythm strength was not an important determinant of atherosclerosis. Altogether, these results demonstrate that constant bright light does not affect atherosclerosis in APOE*3-Leiden.CETP mice. Whether artificial light exposure contributes to cardiovascular disease risk in humans remains to be investigated. Show less

Exercise has been shown to improve cardiometabolic health, yet neither the molecular connection nor the effects of exercise timing have been elucidated. The aim of this study was to investigate whether ad libitum or time-restricted mild exercise reduces atherosclerosis development in atherosclerosis-prone dyslipidemic APOE*3-Leiden.CETP mice and whether mild exercise training in men with obesity affects lipoprotein levels. Mice were group-housed and subjected to ad libitum or time-restricted (first or last 6 hours of the active phase) voluntary wheel running for 16 weeks while on a cholesterol-rich diet, after which atherosclerosis development was assessed in the aortic root. Furthermore, nine men with obesity followed a 12-week mild exercise training program. Lipoprotein levels were measured by nuclear magnetic resonance spectroscopy in plasma collected pre and post exercise training. Wheel running did not affect plasma lipid levels, uptake of triglyceride-derived fatty acids by tissues, and aortic atherosclerotic lesion size or severity. Markers of training status were unaltered. Exercise training in men with obesity did not alter lipoprotein levels. Mild exercise training does not reduce dyslipidemia or atherosclerosis development in APOE*3-Leiden.CETP mice or affect lipoprotein levels in humans. Future research on the effects of (time-restricted) exercise on atherosclerosis or lipid metabolism should consider more vigorous exercise protocols. Show less

Disruption of circadian rhythm by means of shift work has been associated with cardiovascular disease in humans. However, causality and underlying mechanisms have not yet been established. In this study, we exposed hyperlipidemic APOE*3-Leiden.CETP mice to either regular light-dark cycles, weekly 6 hours phase advances or delays, or weekly alternating light-dark cycles (12 hours shifts), as a well-established model for shift work. We found that mice exposed to 15 weeks of alternating light-dark cycles displayed a striking increase in atherosclerosis, with an approximately twofold increase in lesion size and severity, while mice exposed to phase advances and delays showed a milder circadian disruption and no significant effect on atherosclerosis development. We observed a higher lesion macrophage content in mice exposed to alternating light-dark cycles without obvious changes in plasma lipids, suggesting involvement of the immune system. Moreover, while no changes in the number or activation status of circulating monocytes and other immune cells were observed, we identified increased markers for inflammation, oxidative stress, and chemoattraction in the vessel wall. Altogether, this is the first study to show that circadian disruption by shifting light-dark cycles directly aggravates atherosclerosis development. Show less

Many favorable metabolic effects have been attributed to thermogenic activity of brown adipose tissue (BAT). Yet, time of day has rarely been considered in this field of research. Here, we show that a diurnal rhythm in BAT activity regulates plasma lipid metabolism. We observed a high-amplitude rhythm in fatty acid uptake by BAT that synchronized with the light/dark cycle. Highest uptake was found at the onset of the active period, which coincided with high lipoprotein lipase expression and low angiopoietin-like 4 expression by BAT. Diurnal rhythmicity in BAT activity determined the rate at which lipids were cleared from the circulation, thereby imposing the daily rhythm in plasma lipid concentrations. In mice as well as humans, postprandial lipid excursions were nearly absent at waking. We anticipate that diurnal BAT activity is an important factor to consider when studying the therapeutic potential of promoting BAT activity. Show less