Scavenger receptor BI (SR-BI) has been suggested to modulate adipocyte function. To uncover the potential relevance of SR-BI for the development of obesity and associated metabolic complications,... Show moreScavenger receptor BI (SR-BI) has been suggested to modulate adipocyte function. To uncover the potential relevance of SR-BI for the development of obesity and associated metabolic complications, we compared the metabolic phenotype of wild-type and SR-BI deficient mice fed an obesogenic diet enriched in fat. Both male and female SR-BI knockout mice gained significantly more weight as compared to their wild-type counterparts in response to 12 weeks high fat diet feeding (1.5-fold; P < .01 for genotype). Plasma free cholesterol levels were ~2-fold higher (P < .001) in SR-BI knockout mice of both genders, whilst plasma cholesteryl ester and triglyceride concentrations were only significantly elevated in males. Strikingly, the exacerbated obesity in SR-BI knockout mice was paralleled by a better glucose handling. In contrast, only SR-BI knockout mice developed atherosclerotic lesions in the aortic root, with a higher predisposition in females. Biochemical and histological studies in male mice revealed that SR-BI deficiency was associated with a reduced hepatic steatosis degree as evident from the 29% lower (P < .05) liver triglyceride levels. Relative mRNA expression levels of the glucose uptake transporter GLUT4 were increased (+47%; P < .05), whilst expression levels of the metabolic PPARgamma target genes CD36, HSL, ADIPOQ and ATGL were reduced 39%-58% (P < .01) in the context of unchanged PPARgamma expression levels in SR-BI knockout gonadal white adipose tissue. In conclusion, we have shown that SR-BI deficiency is associated with a decrease in adipocyte PPARgamma activity and a concomitant uncoupling of obesity development from hepatic steatosis and glucose intolerance development in high fat diet-fed mice. Show less
Hoekstra, M.; Ouweneel, A.B.; Nahon, J.E.; Geerling, J.J.; Eck, M. van 2019
Scavenger receptor BI (SR-BI) plays a quantitative role in triglyceride metabolism. However, the relevance of SR-BI in pa- thologies associated with disturbances in triglyceride ... Show moreScavenger receptor BI (SR-BI) plays a quantitative role in triglyceride metabolism. However, the relevance of SR-BI in pa- thologies associated with disturbances in triglyceride homeostasis, i.e. obesity and fatty liver disease, remains largely unknown. We therefore investigated the metabolic phenotype of SR-BI knockout mice and wild- type controls after a high-fat diet challenge.Mice were fed a high-fat diet for 12 weeks.SR-BI knockout mice gained significantly more weight than wild- type control mice after 12 weeks of high-fat diet feeding (13±1 grams versus 9±1 grams, respectively; P<0.05). SR-BI deficiency was associated with higher plasma levels of free cholesterol (+111%; P<0.001) and cho- lesteryl esters (+89%; P<0.001) as well as hypertriglyceridemia (+40%; P<0.01). In contrast, liver triglyceride levels were lower (-29%; P<0.05) in SR-BI knockout mice, despite relatively high hepatic expression levels of lipogenic genes ACC (+54%; P<0.05) and SCD1 (+199%; P<0.01). Fasting blood glucose levels were higher in SR-BI knockout mice as compared to wild-type controls (+20%; P<0.01). However, SR-BI knockout mice could handle an oral bolus of glucose much better than wild-type mice as judged from the 45% lower (P<0.05) area under the curve of the oral glucose tolerance test. Notably, atherosclerotic lesions could be detected in the aortic root of all SR-BI knockout mice, while none were present in wild- type mice after the high-fat diet challenge.SR-BI deficiency disassociates hypertriglyceridemia, obesity, and atherosclerosis development from hepatic steatosis and glucose intolerance in high-fat diet-fed mice. Our studies provide additional prooffor the biological relevance of SR-BI in total body triglyceride metabolism. Show less
Hoekstra, M.; Nahon, J.E.; Jong, L.M. de; Kröner, M.J.; Leeuw, L.R. de; Eck, M. van 2019
The nuclear receptor liver X receptor (LXR) impacts on cholesterol metabolism as well as hepatic lipogenesis via transcriptional regulation. It is proposed that inhibition of the protein arginine... Show moreThe nuclear receptor liver X receptor (LXR) impacts on cholesterol metabolism as well as hepatic lipogenesis via transcriptional regulation. It is proposed that inhibition of the protein arginine methyltransferase 3 (PRMT3) uncouples these two transcriptional pathways in vivo by acting as a specific lipogenic coactivator of LXR. Here we validated the hypothesis that treatment with the allosteric PRMT3 inhibitor SGC707 will diminish the hepatic steatosis extent, while leaving global cholesterol metabolism, important in cholesterol-driven pathologies like atherosclerosis, untouched. For this purpose, 12-week old hyperlipidemic apolipoprotein E knockout mice were fed a Western-type diet for six weeks to induce both hepatic steatosis and atherosclerosis. The mice received 3 intraperitoneal injections with SGC707 or solvent control per week. Mice chronically treated with SGC707 developed less severe hepatic steatosis as exemplified by the 51% reduced (P < 0.05) liver triglyceride levels. In contrast, the extent of in vivo macrophage foam cell formation and aortic root atherosclerosis was not affected by SGC707 treatment. Interestingly, SGC707-treated mice gained 94% less body weight (P < 0.05), which was paralleled by changes in white adipose tissue morphology, i.e. reduction in adipocyte size and browning. In conclusion, we have shown that through PRMT3 inhibitor treatment specific functions of LXR involved in respectively the development of fatty liver disease and atherosclerosis can be uncoupled, resulting in an overall diminished hepatic steatosis extent without a negative impact on atherosclerosis susceptibility. As such, our studies highlight that PRMT3 inhibition may constitute a novel therapeutic approach to limit the development of fatty liver disease in humans. Show less
Nahon, J.E.; Hoekstra, M.; Harmelen, V. van; Rensen, P.C.N.; Dijk, K.W. van; Kooijman, S.; Eck, M. van 2019
ObjectiveProteoglycan 4 (Prg4) has emerged from human association studies as a possible factor contributing to weight gain, dyslipidemia and insulin resistance. In the current study, we... Show moreObjectiveProteoglycan 4 (Prg4) has emerged from human association studies as a possible factor contributing to weight gain, dyslipidemia and insulin resistance. In the current study, we investigated the causal role of Prg4 in controlling lipid and glucose metabolism in mice.MethodsPrg4 knockout (KO) mice and wild-type (WT) littermates were challenged with an obesogenic high-fat diet (45% of total calories as fat) for 16 weeks. To further stimulate the development of metabolic alterations, 10% fructose water was provided starting from week 13.ResultsPrg4 deficiency only tended to reduce diet-induced body weight gain, but significantly improved glucose handling (AUC: −29%; p < 0.05), which was also reflected by a tendency towards a reduced HOMA-IR score (−49%; p = 0.06 as compared to WT mice). This coincided with lower hepatic expression of glycolysis (Gck: −30%; p < 0.05) and lipogenesis (Acc: −21%; p < 0.05 and Scd1: −38%; p < 0.001) genes, which translated in significantly lower hepatic triglyceride levels (−56%; p < 0.001) in Prg4 KO mice as compared to WT mice. Prg4 KO mice likely had lower glucose utilization by skeletal muscle as compared to WT mice, judged by a significant reduction in the genes Glut4 (−29%; p < 0.01), Pfkm (−21%; p < 0.05) and Hk2 (−39%; p < 0.001). Moreover, Prg4 KO mice showed a favorable white adipose tissue phenotype with lower uptake of triglyceride-derived fatty acids (−46%; p < 0.05) and lower gene expression of inflammatory markers Cd68, Mcp1 and Tnfα (−65%, −81% and −63%, respectively; p < 0.01) than WT mice.ConclusionPrg4 KO mice are protected from high-fat diet-induced glucose intolerance and fatty liver disease. Show less
Hoekstra, M.; Ouweneel, A.B.; Nahon, J.E.; Geest, R. van der; Kröner, M.J.; Sluis, R.J. van der; Eck, M. van 2019
ObjectiveSince cholesterol is the sole precursor for glucocorticoid synthesis, it is hypothesized that genetic defects in proteins that impact the cellular cholesterol pool may underlie... Show moreObjectiveSince cholesterol is the sole precursor for glucocorticoid synthesis, it is hypothesized that genetic defects in proteins that impact the cellular cholesterol pool may underlie glucocorticoid insufficiency in humans. In the current study, we specifically focused on the cholesterol efflux mediator ATP-binding cassette transporter G1 (ABCG1) as gene candidate.MethodsThe adrenal transcriptional response to fasting stress was measured in wild-type mice to identify putative novel gene candidates. Subsequently, the adrenal glucocorticoid function was compared between ABCG1 knockout mice and wild-type controls.ResultsOvernight food deprivation induced a change in relative mRNA expression levels of cholesterol metabolism-related proteins previously linked to steroidogenesis, i.e. scavenger receptor class B type I (+149%; P < 0.001), LDL receptor (−70%; P < 0.001) and apolipoprotein E (−41%; P < 0.01). Strikingly, ABCG1 transcript levels were also markedly decreased (−61%; P < 0.05). In contrast to our hypothesis that decreasing cholesterol efflux would increase the adrenal cholesterol pool and enhance glucocorticoid output, ABCG1 knockout mice as compared to wild-type mice exhibited a reduced ability to secrete corticosterone in response to an ACTH challenge (two-way ANOVA: P < 0.001 for genotype) or fasting stress. As a result, glucocorticoid target gene expression levels in liver and hypothalamus were reduced and blood lymphocyte concentrations and spleen weights increased in ABCG1 knockout mice under fasting stress conditions. This was paralleled by a 48% reduction in adrenal cholesteryl ester stores and stimulation of adrenal NPC intracellular cholesterol transporter 2 (+37%; P < 0.05) and apolipoprotein E (+59%; P < 0.01) mRNA expression.ConclusionABCG1 deficiency is associated with mild glucocorticoid insufficiency in mice. Show less
Nahon, J.E.; Hoekstra, M.; Havik, S.R.; Santbrink, P.J. van; Dallinga-Thie, G.M.; Kuivenhoven, J.A.; ... ; Eck, M. van 2018
Stabilin-1 (STAB1) is a scavenger receptor expressed on alternatively activated macrophages and sinusoidal endothelial cells. Its ligands include oxidized low-density lipoprotein (LDL) and the... Show moreStabilin-1 (STAB1) is a scavenger receptor expressed on alternatively activated macrophages and sinusoidal endothelial cells. Its ligands include oxidized low-density lipoprotein (LDL) and the extracellular matrix glycoprotein SPARC and it is present in both human and murine atherosclerotic lesions. We aimed to investigate the effect of specific deletion of STAB1 in bone marrow-derived cells, including macrophages on atherosclerotic lesion formation in mice.Lethally irradiated hypercholesterolemic LDL receptor knockout mice received either wildtype (WT) or STAB1 knockout (STAB1 KO) bone marrow. Bone marrow transplanted mice were fed a Western-type diet for 9 weeks to induce atherosclerotic lesion formation. Interestingly, LDL receptor knockout mice reconstituted with STAB1 KO bone marrow showed increased body weight gain (two-way ANOVA: p < 0.001) and larger white adipocyte cell sizes (43% increase in cell area; p < 0.05) as compared to WT bone marrow transplanted mice, which correlated positively (r = 0.82; p < 0.001). This was paralleled by a significant increase in white adipose tissue relative mRNA expression levels of the adipokine leptin (+94% p < 0.05). Despite these changes, no differences in serum lipid levels, the extent of in vivo macrophage foam cell formation or circulating leukocyte concentrations were observed. Moreover, the size and composition of atherosclerotic lesions was not different between the two experimental groups. Bone marrow-specific Stabilin-1 deletion does not affect the susceptibility for atherosclerosis in mice. However, the increased body weight gain and adipocyte cell size highlight a potential role for leukocyte STAB1 in the development of metabolic disorders. Show less
Proteoglycan 4 (Prg4) is a mediator in inflammatory processes, either directly through interactions with CD44 [1] or toll-like receptors [2], or indirectly as a growth factor for hematopoietic cell... Show moreProteoglycan 4 (Prg4) is a mediator in inflammatory processes, either directly through interactions with CD44 [1] or toll-like receptors [2], or indirectly as a growth factor for hematopoietic cell lineages [3]. In line with this, in our article “Proteoglycan 4 regulates macrophage function without altering atherosclerotic lesion formation in a murine bone marrow-specific deletion model” published in Atherosclerosis, we showed that Prg4 deficiency attenuated the LPS-induced pro-inflammatory response in macrophages [4]. Show less
Cardiovascular disease is the number one cause of death worldwide. The most important risk factor for developing this disease is high cholesterol levels in the blood. Other risk factors... Show moreCardiovascular disease is the number one cause of death worldwide. The most important risk factor for developing this disease is high cholesterol levels in the blood. Other risk factors contributing to cardiovascular disease can develop in individuals which are overweight. The clinical consequences of being overweight are clustered in the medical term: metabolic syndrome. Included in the metabolic syndrome are high blood pressure, dyslipidemia and glucose intolerance. At present, most cardiovascular disease patients are treated with statins which lower blood cholesterol levels. However, this treatment is not as effective in all patients and can cause some adverse drug reactions. Therefore, it is essential that novel therapeutic targets for the treatment of cardiovascular disease are identified. In this thesis, potential novel therapeutic targets in cardiovascular disease and metabolic syndrome are validated. In total, three potential targets were investigated: proteoglycan 4, protein arginine methyltransferase 3 and stabilin 1. Our studies showed the involvement of two of these targets in the development of cardiovascular disease and metabolic syndrome. Moreover, our results stress (1) that cardiovascular disease and metabolic syndrome are complex, multifactorial diseases with overlapping mechanisms and (2) that integration of research into both diseases can benefit therapeutic target identification and validation. Show less
Kritikou, E.; Duijn, J. van; Nahon, J.E.; Heijden, T. van der; Bouwman, M.; Groeneveldt, C.; ... ; Bot, I. 2018
The development of atherosclerosis is tightly regulated by the innate and adaptive immune system. Communication between these two compartments occurs, among others, upon presentation of lipid... Show moreThe development of atherosclerosis is tightly regulated by the innate and adaptive immune system. Communication between these two compartments occurs, among others, upon presentation of lipid antigens to the NKT cell population by CD1d-expressing antigen-presenting cells. Recent evidence states that also mast cells express CD1d and can directly communicate with NKT cells. However, no such relationship has been reported in atherosclerosis. Here, we aimed to elucidate in vivo the CD1d-mediated interaction between mast cells and NKT cells upon atherosclerosis progression.\n mice and subsequently placed the animals on a Western-type diet for 10 weeks.\n circulating T cells.\nThis study is the first to illustrate that disruption of the CD1d communication pathway between mast cells and NKT cells aggravates atherosclerosis, through a shift towards pro-inflammatory T cell responses. This ability of mast cell action during plaque progression sheds new light on their role in atherosclerosis. Show less
Nahon, J.E.; Groeneveldt, C.; Geerling, J.J.; Eck, M. van; Hoekstra, M. 2018
Agonists for the liver X receptor (LXR) are considered promising therapeutic moieties in cholesterol-driven diseases by promoting cellular cholesterol efflux pathways. However, current clinical... Show moreAgonists for the liver X receptor (LXR) are considered promising therapeutic moieties in cholesterol-driven diseases by promoting cellular cholesterol efflux pathways. However, current clinical application of these agents is hampered by concomitant LXR-induced activation of a lipogenic transcriptional network, leading to hepatic steatosis. Recent studies have suggested that protein arginine methyltransferase 3 (PRMT3) may act as a selective co-activator of LXR activity. Here, we verified the hypothesis that PRMT3 inhibition selectively disrupts the ability of LXR to stimulate lipogenesis while maintaining its capacity to modulate macrophage cholesterol homeostasis. A combination of the LXR agonist T0901317 and palm oil was administered to C57BL/6 mice to maximally stimulate LXR and PRMT3 activity. PRMT3 activity was inhibited using the allosteric inhibitor SGC707. Treatment with SGC707 did not negatively influence the T0901317/palm oil-induced up-regulation of the cholesterol efflux ATP-binding cassette transporter genes, ABCA1 and ABCG1, in peritoneal cells. In contrast, SGC707 treatment was associated with a significant decrease in the hepatic expression of the lipogenic gene fatty acid synthase (-64%). A similar trend was observed for stearoyl-coenzyme A desaturase and acetyl CoA carboxylase expression (-43%; -56%). This obstruction of lipogenic gene transcription coincided with a significant 2.3-fold decrease in liver triglyceride content as compared with the T0901317 and palm oil-treated control group. We showed that inhibition of PRMT3 activity by SGC707 treatment selectively impairs LXR-driven transcription of hepatic lipogenic genes, while the positive effect of LXR stimulation on macrophage cholesterol efflux pathways is maintained. Show less
Nahon, J.E.; Hoekstra, M.; Havik, S.R.; Santbrink, P.J. van.; Dallinga-Thie, G.M.; Kuivenhoven, J.A.; ... ; Eck, M. van 2018
Proteoglycan 4 (Prg4) has a high structural similarity with the established atherosclerosis-modulating proteoglycan versican, but its role in atherogenesis is still unknown. Therefore, the impact... Show moreProteoglycan 4 (Prg4) has a high structural similarity with the established atherosclerosis-modulating proteoglycan versican, but its role in atherogenesis is still unknown. Therefore, the impact of Prg4 deficiency on macrophage function in vitro and atherosclerosis susceptibility in vivo was investigated.The presence and localization of Prg4 was studied in atherosclerotic lesions. Furthermore, the effect of Prg4 deficiency on macrophage foam cell formation, cholesterol efflux and lipopolysaccharide (LPS) response was determined. Finally, susceptibility for atherosclerotic lesion formation was investigated in bone marrow-specific Prg4 knockout (KO) mice.Prg4 mRNA expression was induced 91-fold (p<0.001) in murine initial atherosclerotic lesions and Prg4 protein co-localized with human lesional macrophages. Murine Prg4 KO macrophages showed increased foam cell formation (+2.1-fold, p<0.01). In parallel, the expression of the cholesterol efflux genes ATP-binding cassette transporter A1 and scavenger receptor type B1 was lower (-35%, p<0.05;-40%, p<0.05) in Prg4 KO macrophages. This translated into an impaired cholesterol efflux to high-density lipoprotein (-13%, p<0.001) and apolipoprotein A1 (-8%, p<0.05). Furthermore, Prg4 KO macrophages showed an impaired LPS-induced rise in TNFα secretion as compared to wild-type controls (-31%, p<0.001), indicating a reduced inflammatory response. Combined, these pro- and anti-atherogenic effects did not translate into a significant difference in atherosclerotic lesion formation upon bone marrow-specific deletion of Prg4 in low-density lipoprotein receptor KO mice.Prg4 is present in macrophages in both murine and human atherosclerotic lesions and critically influences macrophage function, but deletion of Prg4 in bone marrow-derived cells does not affect atherosclerotic lesion development. Show less
Nahon, J.E.; Groeneveldt, C.; Eck, M. van; Hoekstra, M. 2018
Agonists for the liver X receptor (LXR) are considered promising therapeutic moieties in cholesterol-driven diseases by promoting cellular cholesterol efflux. However, current clinical application... Show moreAgonists for the liver X receptor (LXR) are considered promising therapeutic moieties in cholesterol-driven diseases by promoting cellular cholesterol efflux. However, current clinical application of these agents is hampered by the concomitant LXR-induced activation of a lipogenic transcriptional network, leading to hepatic steatosis. Recent studies have suggested that protein arginine methyltransferase 3 (PRMT3) may act as a selective co-activator of LXR activity. Here we verified the hypothesis that PRMT3 inhibition selectively disrupts the ability of LXR to stimulate lipogenesis, while maintaining the capacity of LXR to modulate cholesterol homeostasis.A combination of the LXR agonist T0901317 and palm oil was administered to C57BL/6 mice to maximally stimulate LXR and PRMT3 activity, in absence and presence of the allosteric PRMT3 inhibitor SGC707.Treatment with the PRMT3 inhibitor SGC707 did not negatively influence the T0901317/palm oil induced upregulation of the cholesterol efflux genes ABCA1 and ABCG1 in peritoneal cells. In contrast, SGC707 treatment was associated with a significant decrease in the hepatic expression of the lipogenic gene FAS (-64%; p<0.01). A similar trend was observed for ACC (-56%) and SCD1 expression (-43%). This obstruction of lipogenic gene transcription coincided with a significant 2.3-fold (p<0.01) decrease in liver triglyceride content as compared to the T0901317 and palm oil treated control group.Inhibition of PRMT3 activity by SGC707 treatment selectively impairs LXR-driven transcription of hepatic lipogenic genes, while the positive effect of LXR stimulation on cholesterol efflux pathways is maintained. Show less
Ouweneel, A.B.; Sluis, R.J. van der; Nahon, J.E.; Eck, M. van; Hoekstra, M. 2017
Statin treatment disrupts HMG-CoA reductase-mediated endogenous cholesterol synthesis and lowers plasma LDL-cholesterol levels. Although statin treatment can theoretically impair adrenal steroid... Show moreStatin treatment disrupts HMG-CoA reductase-mediated endogenous cholesterol synthesis and lowers plasma LDL-cholesterol levels. Although statin treatment can theoretically impair adrenal steroid hormone synthesis, thus far, no effect on glucocorticoid output has been described, as LDL-cholesterol levels usually remain within the physiological range. However, novel statin-based treatment regimens that dramatically decrease LDL-cholesterol levels are currently employed. Here, we assessed whether inhibition of cholesterol synthesis under these relatively hypocholesterolemic conditions may alter adrenal glucocorticoid output. Hypocholesterolemic apolipoprotein A1 (apoA1) knockout mice were administered high dose simvastatin twice daily for 3 days. Simvastatin treatment did not change plasma cholesterol levels or modify the adrenal expression levels of genes involved in cholesterol metabolism. However, simvastatin treatment lowered basal plasma levels of the primary glucocorticoid corticosterone (-62%; p < 0.05). Upon injection with adrenocorticotropic hormone, control-treated apoA1 knockout mice already showed only a mild increase in plasma corticosterone levels, indicative of relative glucocorticoid insufficiency. Importantly, simvastatin treatment further diminished the adrenal glucocorticoid response to adrenocorticotropic hormone exposure (two-way ANOVA p < 0.05 for treatment). Peak corticosterone levels were 49% lower (p < 0.01) upon simvastatin treatment. We have shown that simvastatin treatment aggravates the glucocorticoid insufficiency associated with hypocholesterolemia in mice. Our data suggest that (1) HMG-CoA reductase activity controls the adrenal steroidogenic capacity under hypocholesterolemic conditions and (2) imply that it might be important to monitor adrenal function in humans subjected to statin-based treatments aimed at achieving sub-physiological LDL-cholesterol levels, as these may potentially execute a negative impact on the glucocorticoid function in humans. Show less
