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Papers by Branislav Radovic

Autophagy

Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among ot... more Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among other autophagy-related (ATG) proteins, it is proposed that, like in yeast, both ATG5 and ATG7 are essential for LC3 conjugation. Using atg5-deficient (−/-) and atg7 −/macrophages, we provide evidence that loss of ATG5 but not of ATG7 resulted in LC3-II depletion. Accumulation of LC3-II in elicited atg7 −/macrophages in response to bafilomycin A 1 validated these data. Furthermore, complete loss of ATG3 in atg7 −/macrophages demonstrated that ATG7 and ATG3 are dispensable for LC3-PE conjugation. In contrast to thioglycolate-elicited macrophages, naïve peritoneal and bone marrow-derived atg7 −/macrophages exhibited no LC3-II, even under inflammatory stimuli in vitro. Hence, the macrophage metabolic status dictates the level of LC3-PE conjugation with a supportive but nonessential role of ATG7, disclosing the eukaryotic exception from the LC3 lipidation model based on yeast data.

Cells

Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of chol... more Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of cholesteryl esters and triglycerides at an acidic pH in lysosomes, resulting in the release of unesterified cholesterol and free fatty acids. However, the role of LAL in diet-induced adaptations is largely unexplored. In this study, we demonstrate that feeding a Western-type diet to Lal-deficient (LAL-KO) mice triggers metabolic reprogramming that modulates gut-liver cholesterol homeostasis. Induction of ileal fibroblast growth factor 15 (three-fold), absence of hepatic cholesterol 7α-hydroxylase expression, and activation of the ERK phosphorylation cascade results in altered bile acid composition, substantial changes in the gut microbiome, reduced nutrient absorption by 40%, and two-fold increased fecal lipid excretion in LAL-KO mice. These metabolic adaptations lead to impaired bile acid synthesis, lipoprotein uptake, and cholesterol absorption and ultimately to the resistance of LAL-KO mi...

Biochimica et biophysica acta, Jan 31, 2018

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and tri... more Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.

Oncotarget, Jan 16, 2017

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among var... more Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (-/-) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)-/- mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO...

Oncotarget, Jan 29, 2017

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipa... more Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH. We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils. Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus. Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes. The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis. To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/- mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2. Acute inhibition of ...

The Journal of biological chemistry, Aug 27, 2016

Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester (CE) ... more Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester (CE) and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of CEs and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell-line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum, but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with a RE gavage exhibited largely reduced ...

Diabetologia, 2016

Aims/hypothesis Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (T... more Aims/hypothesis Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal-/-) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). Methods We studied metabolic adaptations in Lal-/mice. Results Despite loss of adipose tissue, Lal-/mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [ 3 H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal-/mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal-/mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal-/mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal-/mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal-/mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. Conclusions/interpretation Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Electronic supplementary material The online version of this article

Atherosclerosis Supplements, 2010

Introduction: We previously showed both hepatic apoB100-and intestinal apoB48-containing lipoprot... more Introduction: We previously showed both hepatic apoB100-and intestinal apoB48-containing lipoprotein particles in human atherosclerotic plaques by immunohistochemistry, here we evaluated plaques by immuno-electronmicroscopy (IEM). Methods: Transmission electron microscopy was performed on human liver, intestine and carotid plaque after immuno-gold labeling; tissue was fixed overnight with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and embedded in LR-white-resin. Primary antibodies were incubated overnight diluted 1:100 in PBS-containing 0.1% acetylated BSA; secondary antibodies conjugated to immuno-gold-particles were incubated for 2 hrs. Primary antibodies for apoB (Abcam 20737) and apoB100 (Abcam 50069) were commercial, polyclonal rabbit antibodies recognizing the N-terminal and C-terminal of apoB, respectively. For apoB48, we used non-commercial, murine monoclonal antibody (4C8) recognizing the C-terminal of apoB48; this region is conformationally hidden in apoB100, preventing cross-reaction. Results: IEM confirmed the presence of ApoB in the rough endoplasmic reticulum (RER) of the hepatocytes and in the brush border of the enterocytes. ApoB100-particles were located in the RER of the hepatocytes and in the cytoplasm of the enterocytes and the Goblet cells. Interestingly, apoB48particles were localized in the glycogen storage vesicles of the hepatocyte and in the RER of the enterocytes. ApoB-, ApoB100-and ApoB48-particles were located throughout the atherosclerotic plaque. Conclusions: This is the first demonstration by IEM that the presence of both hepatic apoB100 and intestinal apoB48 contribute to human atherosclerosis. This may serve as clinical rationale to target both the hepatic and intestinal pathways.

Circulation, Nov 23, 2010

Atherosclerosis, 2015

Background and aims-Monoglyceride lipase (MGL) catalyzes the final step of lipolysis by degrading... more Background and aims-Monoglyceride lipase (MGL) catalyzes the final step of lipolysis by degrading monoglyceride (MG) to glycerol and fatty acid. MGL also hydrolyzes and thereby deactivates 2-arachidonoyl glycerol (2-AG), the most abundant endocannabinoid in the mammalian system. 2-AG acts as full agonist on cannabinoid receptor type 1 (CB1R) and CB2R, which are mainly expressed in brain and immune cells, respectively. Thus, we speculated that in the absence of MGL, increased 2-AG concentrations mediate CB2R signaling in immune cells to modulate inflammatory responses, thereby affecting the development of atherosclerosis. Methods and results-We generated apolipoprotein E (ApoE)/MGL double-knockout (DKO) mice and challenged them with Western-type diet for 9 weeks. Despite systemically increased 2-AG concentrations in DKO mice, CB2R-mediated signaling remains fully functional, arguing against CB2R desensitization. We found increased plaque formation in both en face aortae (1.3fold, p = 0.028) and aortic valve sections (1.5-fold, p = 0.0010) in DKO mice. Interestingly, DKO mice also presented reduced lipid (12%, p = 0.031) and macrophage content (18%, p = 0.061), elevated intraplaque smooth muscle staining (1.4-fold, p = 0.016) and thicker fibrous caps (1.8fold, p = 0.0032), together with a higher ratio of collagen to necrotic core area (2.5-fold, p = 0.0003) and expanded collagen content (1.6-fold, p = 0.0007), which suggest formation of less vulnerable atherosclerotic plaques. Treatment with a CB2R inverse agonist prevents these effects in DKO mice, demonstrating that the observed plaque phenotype in DKO mice originates from CB2R activation.

Atherosclerosis Supplements, 2011

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2015

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded ... more During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory

Atherosclerosis Supplements, 2010

Journal of lipid research, 2014

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator compar... more Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58(-/-) mice). CGI-58(-/-) macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL(-/-) macrophages. In contrast to ATGL(-/-) macrophages, however, CGI-58(-/-) macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58(-/-) macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-5...

Molecular Nutrition & Food Research, 2010

In the present study, we analyzed the influence of xanthohumol (XN) on thyroid hormone (TH) distr... more In the present study, we analyzed the influence of xanthohumol (XN) on thyroid hormone (TH) distribution and metabolism in rats. A potent and selective competition of XN for thyroxine (T4) binding to transthyretin (IC 50 5 1 mM at 1.7 nM [ 125 I]T4) was found in human and rat sera in vitro. Female rats treated orally with XN showed increased hepatic expression of T4-binding globulin and decreased transthyretin and albumin. Thyrotropin levels and hepatic type 1 deiodinase activity were moderately increased. Northern blot analysis revealed diminished expression of liver sulfotransferase (Sult1a1) and uridine-diphosphate glucuronosyltransferase (Ugt1a1) after XN treatment. The transcript levels of constitutive androstane receptor (CAR), known to be involved in regulation of enzymes metabolizing hormones, drugs and xenobiotics, was lower in rats treated with 410 mg XN/kg body weight per day. Immunoblot analysis indicates reduced amounts of CAR protein. The phenobarbitalinducible cytochrome P450 mRNA level was decreased in rats treated with 410 mg XN/kg/ day, in agreement with reduced CAR protein. Although only moderate changes in TH serum levels were observed, the XN-dependent altered expression of components involved in TH homeostasis might be important not only for hormone metabolism, but also for hepatic phase I and II elimination of drug metabolites and xenobiotics.

Molecular Nutrition & Food Research, 2013

Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits p... more Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits positive effects on lipid and glucose metabolism. Based on its favorable biological properties, we investigated whether XN attenuates atherosclerosis in western-type diet-fed apolipoprotein-E-deficient (ApoE⁻/⁻) mice. XN supplementation markedly reduced plasma cholesterol concentrations, decreased atherosclerotic lesion area, and attenuated plasma concentrations of the proinflammatory cytokine monocyte chemoattractant protein 1. Decreased hepatic triglyceride and cholesterol content, activation of AMP-activated protein kinase, phosphorylation and inactivation of acetyl-CoA carboxylase, and reduced expression levels of mature sterol regulatory element-binding protein (SREBP)-2 and SREBP-1c mRNA indicate reduced lipogenesis in the liver of XN-fed ApoE⁻/⁻ mice. Concomitant induction of hepatic mRNA expression of carnitine palmitoyltransferase-1a in ApoE⁻/⁻ mice-administered XN suggests increased fatty acid beta-oxidation. Fecal cholesterol concentrations were also markedly increased in XN-fed ApoE⁻/⁻ mice compared with mice fed western-type diet alone. The atheroprotective effects of XN might be attributed to combined beneficial effects on plasma cholesterol and monocyte chemoattractant protein 1 concentrations and hepatic lipid metabolism via activation of AMP-activated protein kinase.

Molecular Nutrition & Food Research, 2005

Sodium-iodide-symporter (NIS), an integral plasma membrane glycoprotein, mediates the sodium-depe... more Sodium-iodide-symporter (NIS), an integral plasma membrane glycoprotein, mediates the sodium-dependent active uptake of iodide (I(-)) into the thyroid gland, which is a fundamental step in thyroid hormone synthesis. In this work, we analyzed the influence of xanthohumol (XN), a prenylated chalcone from hops (Humulus lupulus L.), on the I(-) uptake in a cell culture model of normal, nontransformed rat thyrocytes (FRTL-5). Acute treatment with nanomolar concentrations of XN does not influence I(-) uptake, but after 2 and 3-days of XN stimulation an increase in I(-) uptake was observed; I(-) uptake was maximally increased by 50% compared to control after 3-days of XN stimulation at 1 nM. A clear time-dependent stimulation was observed which showed no marked concentration relationship, however. To investigate whether expression of NIS mRNA is also increased, we grew FRTL-5 cells for 3-days in a medium containing increasing concentrations of XN (0.1 nM-1 muM). Northern blot analysis showed no difference in NIS mRNA transcript levels between control cells and those treated with different concentrations of XN. This study revealed that nanomolar concentrations of XN, a unique compound with anticancer properties, exert stimulating effects on radioiodide uptake. In contrast to many other plant-derived phenolic secondary metabolites such as (iso-)flavonoids, which inhibit I(-) uptake, XN might be an interesting candidate for more efficient radioiodide therapy of thyroid and perhaps other cancer expressing NIS such as breast cancer.

The Journal of Lipid Research, 2008

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonis... more Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3b-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7a-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists.-Kratzer, A

Autophagy

Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among ot... more Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among other autophagy-related (ATG) proteins, it is proposed that, like in yeast, both ATG5 and ATG7 are essential for LC3 conjugation. Using atg5-deficient (−/-) and atg7 −/macrophages, we provide evidence that loss of ATG5 but not of ATG7 resulted in LC3-II depletion. Accumulation of LC3-II in elicited atg7 −/macrophages in response to bafilomycin A 1 validated these data. Furthermore, complete loss of ATG3 in atg7 −/macrophages demonstrated that ATG7 and ATG3 are dispensable for LC3-PE conjugation. In contrast to thioglycolate-elicited macrophages, naïve peritoneal and bone marrow-derived atg7 −/macrophages exhibited no LC3-II, even under inflammatory stimuli in vitro. Hence, the macrophage metabolic status dictates the level of LC3-PE conjugation with a supportive but nonessential role of ATG7, disclosing the eukaryotic exception from the LC3 lipidation model based on yeast data.

Cells

Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of chol... more Lysosomal acid lipase (LAL) is the sole enzyme known to be responsible for the hydrolysis of cholesteryl esters and triglycerides at an acidic pH in lysosomes, resulting in the release of unesterified cholesterol and free fatty acids. However, the role of LAL in diet-induced adaptations is largely unexplored. In this study, we demonstrate that feeding a Western-type diet to Lal-deficient (LAL-KO) mice triggers metabolic reprogramming that modulates gut-liver cholesterol homeostasis. Induction of ileal fibroblast growth factor 15 (three-fold), absence of hepatic cholesterol 7α-hydroxylase expression, and activation of the ERK phosphorylation cascade results in altered bile acid composition, substantial changes in the gut microbiome, reduced nutrient absorption by 40%, and two-fold increased fecal lipid excretion in LAL-KO mice. These metabolic adaptations lead to impaired bile acid synthesis, lipoprotein uptake, and cholesterol absorption and ultimately to the resistance of LAL-KO mi...

Biochimica et biophysica acta, Jan 31, 2018

Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and tri... more Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.

Oncotarget, Jan 16, 2017

Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among var... more Monoglyceride lipase (MGL) hydrolyzes monoglycerides (MGs) to glycerol and fatty acids. Among various MG species MGL also degrades 2-arachidonoylglycerol (2-AG), the most abundant endocannabinoid and potent activator of cannabinoid receptors (CBR) 1 and 2. MGL-knockout (-/-) mice exhibit pronounced 2-AG accumulation, but lack central cannabimimetic effects due to CB1R desensitization. We have previously shown that MGL affects plaque stability in apolipoprotein E (ApoE)-/- mice, an established animal model for dyslipidemia and atherosclerosis. In the current study, we investigated functional consequences of MGL deficiency on lipid and energy metabolism in ApoE/MGL double knockout (DKO) mice. MGL deficiency affected hepatic cholesterol metabolism by causing increased cholesterol elimination via the biliary pathway. Moreover, DKO mice exhibit lipid-triggered delay in gastric emptying without major effects on overall triglyceride and cholesterol absorption. The observed phenotype of DKO...

Oncotarget, Jan 29, 2017

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipa... more Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH. We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils. Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus. Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes. The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis. To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/- mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2. Acute inhibition of ...

The Journal of biological chemistry, Aug 27, 2016

Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester (CE) ... more Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester (CE) and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of CEs and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell-line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum, but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with a RE gavage exhibited largely reduced ...

Diabetologia, 2016

Aims/hypothesis Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (T... more Aims/hypothesis Lysosomal acid lipase (LAL) hydrolyses cholesteryl esters and triacylglycerols (TG) within lysosomes to mobilise NEFA and cholesterol. Since LAL-deficient (Lal-/-) mice suffer from progressive loss of adipose tissue and severe accumulation of lipids in hepatic lysosomes, we hypothesised that LAL deficiency triggers alternative energy pathway(s). Methods We studied metabolic adaptations in Lal-/mice. Results Despite loss of adipose tissue, Lal-/mice show enhanced glucose clearance during insulin and glucose tolerance tests and have increased uptake of [ 3 H]2-deoxy-D-glucose into skeletal muscle compared with wild-type mice. In agreement, fasted Lal-/mice exhibit reduced glucose and glycogen levels in skeletal muscle. We observed 84% decreased plasma leptin levels and significantly reduced hepatic ATP, glucose, glycogen and glutamine concentrations in fed Lal-/mice. Markedly reduced hepatic acyl-CoA concentrations decrease the expression of peroxisome proliferator-activated receptor α (PPARα) target genes. However, treatment of Lal-/mice with the PPARα agonist fenofibrate further decreased plasma TG (and hepatic glucose and glycogen) concentrations in Lal-/mice. Depletion of hepatic nuclear factor 4α and forkhead box protein a2 in fasted Lal-/mice might be responsible for reduced expression of microsomal TG transfer protein, defective VLDL synthesis and drastically reduced plasma TG levels. Conclusions/interpretation Our findings indicate that neither activation nor inactivation of PPARα per se but rather the availability of hepatic acyl-CoA concentrations regulates VLDL synthesis and subsequent metabolic adaptations in Electronic supplementary material The online version of this article

Atherosclerosis Supplements, 2010

Introduction: We previously showed both hepatic apoB100-and intestinal apoB48-containing lipoprot... more Introduction: We previously showed both hepatic apoB100-and intestinal apoB48-containing lipoprotein particles in human atherosclerotic plaques by immunohistochemistry, here we evaluated plaques by immuno-electronmicroscopy (IEM). Methods: Transmission electron microscopy was performed on human liver, intestine and carotid plaque after immuno-gold labeling; tissue was fixed overnight with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and embedded in LR-white-resin. Primary antibodies were incubated overnight diluted 1:100 in PBS-containing 0.1% acetylated BSA; secondary antibodies conjugated to immuno-gold-particles were incubated for 2 hrs. Primary antibodies for apoB (Abcam 20737) and apoB100 (Abcam 50069) were commercial, polyclonal rabbit antibodies recognizing the N-terminal and C-terminal of apoB, respectively. For apoB48, we used non-commercial, murine monoclonal antibody (4C8) recognizing the C-terminal of apoB48; this region is conformationally hidden in apoB100, preventing cross-reaction. Results: IEM confirmed the presence of ApoB in the rough endoplasmic reticulum (RER) of the hepatocytes and in the brush border of the enterocytes. ApoB100-particles were located in the RER of the hepatocytes and in the cytoplasm of the enterocytes and the Goblet cells. Interestingly, apoB48particles were localized in the glycogen storage vesicles of the hepatocyte and in the RER of the enterocytes. ApoB-, ApoB100-and ApoB48-particles were located throughout the atherosclerotic plaque. Conclusions: This is the first demonstration by IEM that the presence of both hepatic apoB100 and intestinal apoB48 contribute to human atherosclerosis. This may serve as clinical rationale to target both the hepatic and intestinal pathways.

Circulation, Nov 23, 2010

Atherosclerosis, 2015

Background and aims-Monoglyceride lipase (MGL) catalyzes the final step of lipolysis by degrading... more Background and aims-Monoglyceride lipase (MGL) catalyzes the final step of lipolysis by degrading monoglyceride (MG) to glycerol and fatty acid. MGL also hydrolyzes and thereby deactivates 2-arachidonoyl glycerol (2-AG), the most abundant endocannabinoid in the mammalian system. 2-AG acts as full agonist on cannabinoid receptor type 1 (CB1R) and CB2R, which are mainly expressed in brain and immune cells, respectively. Thus, we speculated that in the absence of MGL, increased 2-AG concentrations mediate CB2R signaling in immune cells to modulate inflammatory responses, thereby affecting the development of atherosclerosis. Methods and results-We generated apolipoprotein E (ApoE)/MGL double-knockout (DKO) mice and challenged them with Western-type diet for 9 weeks. Despite systemically increased 2-AG concentrations in DKO mice, CB2R-mediated signaling remains fully functional, arguing against CB2R desensitization. We found increased plaque formation in both en face aortae (1.3fold, p = 0.028) and aortic valve sections (1.5-fold, p = 0.0010) in DKO mice. Interestingly, DKO mice also presented reduced lipid (12%, p = 0.031) and macrophage content (18%, p = 0.061), elevated intraplaque smooth muscle staining (1.4-fold, p = 0.016) and thicker fibrous caps (1.8fold, p = 0.0032), together with a higher ratio of collagen to necrotic core area (2.5-fold, p = 0.0003) and expanded collagen content (1.6-fold, p = 0.0007), which suggest formation of less vulnerable atherosclerotic plaques. Treatment with a CB2R inverse agonist prevents these effects in DKO mice, demonstrating that the observed plaque phenotype in DKO mice originates from CB2R activation.

Atherosclerosis Supplements, 2011

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2015

During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded ... more During autophagy, autophagosomes fuse with lysosomes to degrade damaged organelles and misfolded proteins. Breakdown products are released into the cytosol and contribute to energy and metabolic building block supply, especially during starvation. Lipophagy has been defined as the autophagy-mediated degradation of lipid droplets (LDs) by lysosomal acid lipase. Adipose triglyceride lipase (ATGL) is the major enzyme catalyzing the initial step of lipolysis by hydrolyzing triglycerides (TGs) in cytosolic LDs. Consequently, most organs and cells, including macrophages, lacking ATGL accumulate TGs, resulting in reduced intracellular free fatty acid concentrations. Macrophages deficient in hormone-sensitive lipase (H0) lack TG accumulation albeit reduced in vitro TG hydrolase activity. We hypothesized that autophagy is activated in lipase-deficient macrophages to counteract their energy deficit. We therefore generated mice lacking both ATGL and HSL (A0H0). Macrophages from A0H0 mice showed 73% reduced neutral TG hydrolase activity, resulting in TG-rich LD accumulation. Increased expression of cathepsin B, accumulation of LC3-II, reduced expression of p62 and increased DQ-BSA dequenching suggest intact autophagy and functional lysosomes in A0H0 macrophages. Markedly decreased acid TG hydrolase activity and lipid flux independent of bafilomycin A1 treatment, however, argue against effective lysosomal degradation of LDs in A0H0 macrophages. We conclude that autophagy of proteins and cell organelles but not of LDs is active as a compensatory

Atherosclerosis Supplements, 2010

Journal of lipid research, 2014

Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator compar... more Cellular TG stores are efficiently hydrolyzed by adipose TG lipase (ATGL). Its coactivator comparative gene identification-58 (CGI-58) strongly increases ATGL-mediated TG catabolism in cell culture experiments. To investigate the consequences of CGI-58 deficiency in murine macrophages, we generated mice with a targeted deletion of CGI-58 in myeloid cells (macCGI-58(-/-) mice). CGI-58(-/-) macrophages accumulate intracellular TG-rich lipid droplets and have decreased phagocytic capacity, comparable to ATGL(-/-) macrophages. In contrast to ATGL(-/-) macrophages, however, CGI-58(-/-) macrophages have intact mitochondria and show no indications of mitochondrial apoptosis and endoplasmic reticulum stress, suggesting that TG accumulation per se lacks a significant role in processes leading to mitochondrial dysfunction. Another notable difference is the fact that CGI-58(-/-) macrophages adopt an M1-like phenotype in vitro. Finally, we investigated atherosclerosis susceptibility in macCGI-5...

Molecular Nutrition & Food Research, 2010

In the present study, we analyzed the influence of xanthohumol (XN) on thyroid hormone (TH) distr... more In the present study, we analyzed the influence of xanthohumol (XN) on thyroid hormone (TH) distribution and metabolism in rats. A potent and selective competition of XN for thyroxine (T4) binding to transthyretin (IC 50 5 1 mM at 1.7 nM [ 125 I]T4) was found in human and rat sera in vitro. Female rats treated orally with XN showed increased hepatic expression of T4-binding globulin and decreased transthyretin and albumin. Thyrotropin levels and hepatic type 1 deiodinase activity were moderately increased. Northern blot analysis revealed diminished expression of liver sulfotransferase (Sult1a1) and uridine-diphosphate glucuronosyltransferase (Ugt1a1) after XN treatment. The transcript levels of constitutive androstane receptor (CAR), known to be involved in regulation of enzymes metabolizing hormones, drugs and xenobiotics, was lower in rats treated with 410 mg XN/kg body weight per day. Immunoblot analysis indicates reduced amounts of CAR protein. The phenobarbitalinducible cytochrome P450 mRNA level was decreased in rats treated with 410 mg XN/kg/ day, in agreement with reduced CAR protein. Although only moderate changes in TH serum levels were observed, the XN-dependent altered expression of components involved in TH homeostasis might be important not only for hormone metabolism, but also for hepatic phase I and II elimination of drug metabolites and xenobiotics.

Molecular Nutrition & Food Research, 2013

Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits p... more Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits positive effects on lipid and glucose metabolism. Based on its favorable biological properties, we investigated whether XN attenuates atherosclerosis in western-type diet-fed apolipoprotein-E-deficient (ApoE⁻/⁻) mice. XN supplementation markedly reduced plasma cholesterol concentrations, decreased atherosclerotic lesion area, and attenuated plasma concentrations of the proinflammatory cytokine monocyte chemoattractant protein 1. Decreased hepatic triglyceride and cholesterol content, activation of AMP-activated protein kinase, phosphorylation and inactivation of acetyl-CoA carboxylase, and reduced expression levels of mature sterol regulatory element-binding protein (SREBP)-2 and SREBP-1c mRNA indicate reduced lipogenesis in the liver of XN-fed ApoE⁻/⁻ mice. Concomitant induction of hepatic mRNA expression of carnitine palmitoyltransferase-1a in ApoE⁻/⁻ mice-administered XN suggests increased fatty acid beta-oxidation. Fecal cholesterol concentrations were also markedly increased in XN-fed ApoE⁻/⁻ mice compared with mice fed western-type diet alone. The atheroprotective effects of XN might be attributed to combined beneficial effects on plasma cholesterol and monocyte chemoattractant protein 1 concentrations and hepatic lipid metabolism via activation of AMP-activated protein kinase.

Molecular Nutrition & Food Research, 2005

Sodium-iodide-symporter (NIS), an integral plasma membrane glycoprotein, mediates the sodium-depe... more Sodium-iodide-symporter (NIS), an integral plasma membrane glycoprotein, mediates the sodium-dependent active uptake of iodide (I(-)) into the thyroid gland, which is a fundamental step in thyroid hormone synthesis. In this work, we analyzed the influence of xanthohumol (XN), a prenylated chalcone from hops (Humulus lupulus L.), on the I(-) uptake in a cell culture model of normal, nontransformed rat thyrocytes (FRTL-5). Acute treatment with nanomolar concentrations of XN does not influence I(-) uptake, but after 2 and 3-days of XN stimulation an increase in I(-) uptake was observed; I(-) uptake was maximally increased by 50% compared to control after 3-days of XN stimulation at 1 nM. A clear time-dependent stimulation was observed which showed no marked concentration relationship, however. To investigate whether expression of NIS mRNA is also increased, we grew FRTL-5 cells for 3-days in a medium containing increasing concentrations of XN (0.1 nM-1 muM). Northern blot analysis showed no difference in NIS mRNA transcript levels between control cells and those treated with different concentrations of XN. This study revealed that nanomolar concentrations of XN, a unique compound with anticancer properties, exert stimulating effects on radioiodide uptake. In contrast to many other plant-derived phenolic secondary metabolites such as (iso-)flavonoids, which inhibit I(-) uptake, XN might be an interesting candidate for more efficient radioiodide therapy of thyroid and perhaps other cancer expressing NIS such as breast cancer.

The Journal of Lipid Research, 2008

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonis... more Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3b-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7a-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists.-Kratzer, A