Report of the 33rd annual conference of the european lipoprotein club, tutzing, 6–9 september 2010

ARTICLE IN PRESS
Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing,6–9 September 2010 Franco Bernini , Rachel Fisher , Dilys Freeman , Albert Groen , Athina Kalopissis , Ernst Malle ,Monique Mulders , Andreas Niemeier , Paolo Parini , Anne Tybjaerg Hansen ,Arnold von Eckardstein a Dipartimento di Scienze Farmacologiche, Biologiche e Chimiche Applicate, Universita‘di Parma, 43100 Parma, Italyb Atherosclerosis Research Unit, Department of Medicine, Karolinska University Hospital, SE-171 76, Stockholm, Swedenc Centre for Population and Health Sciences, University of Glasgow, Western Infirmary Glasgow Glasgow G11 6NT, United Kingdomd Department of Pediatrics (Room Y2-145), University Medical Centre Groningen, 9713ZG Groningen, The Netherlandse INSERM, U872, Equipe 4, Centre de Recherche des Cordeliers, F-75006 Paris, Francef Medical University of Graz, Center of Molecular Medicine, Institute of Molecular Biology and Biochemistry, A - 8010 GRAZ, Austriag Erasmus Medical Center, Department of Internal Medicine, Department of Pharmacology, Vascular and Metabolic Diseases, 3015 CE Rotterdam, The Netherlandsh University Medical Center Hamburg-Eppendorf, Department of Orthopaedics, D — 20246 Hamburg, Germanyi Division of Clinical Chemistry, Department of Laboratory Medicine, and Molecular Nutrition Unit, Department of Biosciences, Karolinska Institutet at Huddinge University Hospital,S-141 86 Stockholm, Swedenj Department Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen Ø, Denmarkk University Hospital Zurich, Institute of Clinical Chemistry, CH 8091 Zurich, Switzerland Article history:Received 3 December 2010Accepted 3 December 2010Available online xxx The 33rd meeting of the European Lipoprotein Club was held and its role in hepatic steatosis” which was then presented orally from 6 to 9 September 2010 at the Evangelische Akademie in Tutzing Germany and attended by 115 participants from all overEurope and the United States. It was the last co-organized by AthinaKalopissis (Paris, France) and Paolo Parini (Stockholm, Sweden).
They will be followed in 2011 by Philippe Costet (Nantes, France) 1. Key note lecture
and Ewa Ehrenborg (Stockholm, Sweden). In addition, Barbara Field-ing (Oxford, UK) will come onto the committee to overlap with the After the welcome by Arnold von Eckardstein the scientific part outgoing treasurer Dilys Freeman, who will leave in 2012.
of this year’s meeting was opened by a key note lecture from The program was composed of seven scientific sessions most Stephen O’Rahilly (Cambridge, UK) on “Common Metabolic Disor- of which were introduced by the lecture of an invited speaker.
ders: Lessons from Extreme Human Phenotypes”. Overweight and All other oral presentations as well as the poster presentations obesity cause 80% of all cases of type 2 diabetes, 35% of ischemic were selected by rating of abstracts through the ELC commit- heart diseases and 55% of diseases related to high blood pres- tee members. Two types of awards were presented to two young sure amongst adults in the European Union. Currently more than scientists. The young investigator award was presented to Max 200 million people worldwide suffer from diabetes but the World Nieuwdorp (Amsterdam, The Netherlands) for his presentation enti- Health Organisation estimates that the number of deaths as a result tled “Metabolic effects of transplantating gut microbiota from of diabetes will increase by more than 50% in the next 10 years.
lean donors to subjects with metabolic syndrome”. The poster Scientifically these findings raise several principle questions which award was presented to Marcela Aparicio-Vergara (Groningen, The Stephen O’Rahilly and his colleagues addressed by unravelling the Netherlands) for her poster entitled “Hyperstimulation of TNFR1 genetic basis of monogenic disorders of body weight control andinsulin action: 1. Why are some people fat and some people lean?2. Why does obesity frequently lead to insulin resistance? 3. How does impaired insulin-stimulated glucose metabolism lead to its Corresponding author. Tel.: +41 44 255 2260; fax: +41 44 255 4590.
E-mail address: (A. von Eckardstein).
0021-9150/$ – see front matter 2010 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Bernini F, et al. Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing, 6–9 September2010. Atherosclerosis (2011), ARTICLE IN PRESS
F. Bernini et al. / Atherosclerosis xxx (2010) xxx–xxx 1. After their discovery of the first mutations, namely in the heart triglyceride content secondary to diacylglycerol acyltrans- PCSK1 and leptin genes, which lead to obesity in humans, ferase 1 (DGAT1) overexpression in the heart was created. DGAT1 Stephen O’Rahilly and his colleague Sadaf Farooqi described the catalyzes the final step in mammalian triglyceride (TG) synthe- molecular basis of several other genetic obesity syndromes, sis. The DGAT1 transgenic mice displayed reduced levels of free including a deficiency in the Melanocortin 4 receptor (MC4R), fatty acids (FFAs), diacylglycerol (DAG) and ceramide in the hearts.
a disorder which occurs in more than 1 million patients. A com- Mice with a combined heart overexpression of DGAT1 and fatty prehensive analysis of these patients revealed that the most acyl CoA synthetase, an enzyme upstream of DGAT in TG synthe- important mechanism in the development of obesity amongst sis and whose overexpression leads to dilated cardiomyopathy, these patients was based on a deficiency in appetite control. After had increased fatty acid oxidation and improved survival. Reduced discovery of the underlying defects the principle that knowledge ceramide and DAG and increased FA oxidation were beneficial of mechanism could advance therapy of obesity was established in this model. Dr. Goldberg also presented data from a lipotox- by the dramatic effects of daily administration of recombinant icity model due to heart overexpression of PPAR␥ heart. When leptin in children with congenital leptin deficiency. Extensive these mice were crossed with PPAR␣ knockout mice TG accumula- genome analyses revealed that mutations in these and several tion was increased and large lipid droplets were found adjacent other genes play an important role for determining body mass to irregularly shaped mitochondria. Again, these animals were characterized by increased fatty acid oxidation and a concomi- 2. Patients with inherited forms of lipodystrophy because of muta- tantly reduced mortality. Gene expression data from the PPAR␥ tions in the genes encoding for AGPAT2, Seipin BSCL2, Caveolin × PPAR␣−/− mice suggest that these transcription factors com- 1, Lamin A/C, PPAR gamma, AKT2 or the recently identified pete and do not have overlapping functions. The study also gave CIDEC present with clinical signs of insulin resistance includ- further support to the notion that increased FA oxidation can ing hyperinsulinemia, decreased glucose tolerance, dyslipidemia correct lipotoxicity. With regard to the question of which sig- and acanthosis nigricans, that are otherwise predominantly seen naling pathways are involved in lipotoxic cardiomyopathy, he in overweight and obese patients. These at first sight paradoxi- demonstrated that ␤adrenergic receptor signaling was affected cal findings as well as the weight gain observed under treatment by lipotoxicity in cardiomyocytes. In addition, he showed that with the insulin sensitizing PPAR␥-agonists provided evidence mice overexpressing PPAR␥ in the heart develop ventricular fib- that insulin resistance is not necessarily a direct consequence rillation from which they eventually die. He also showed studies of fat-loaded adipocytes but rather caused by overspill of fat in progress attempting to corroborate the murine data by mea- into other tissues, especially muscle and liver. Stephen O’Rahilly surements of lipids in hearts from humans with severe heart now described a novel form of inherited insulin resistance, which manifests itself by acanthosis nigricans, hirsutism, and postpran- Alexander Bartelt (Hamburg, Germany) presented a study about dial hyperinsulinemia. The genetic basis is a missense mutation the biological significance of brown adipose tissue (BAT) for the in the gene encoding for AS160 which as a downstream tar- metabolism of triglyceride-rich lipoproteins. He demonstrated that get of Akt modulates the translocation of GLUT4 to the plasma acute cold exposure resulted in increased uptake of dietary lipids membrane. This defect can well explain the limitation of hyper- selectively into BAT. Using nanotechnology and confocal and mag- insulinaemia to the postprandial phase since translocation of netic resonance imaging, the increased uptake was visualized and GLUT 4 is necessary for post-prandial uptake of glucose into quantified in vivo. Finally, using hyperlipidemic Apoa5 KO and diet- skeletal muscle but not for insulin’s action to control hepatic induced obese mice, he demonstrated that increasing BAT activity glucose output in the fasting state.
might be a promising approach for treating hyperlipemia and obe- 3. Many individuals with insulin resistance present with hep- atosteatosis and hypertriglyceridemia. As yet it is not completely Janna van Diepen from Leiden (The Netherlands) reported on understood of whether the increased lipogenesis is due to hep- the importance of the NLRP3 inflammasome for the development atic insulin resistance or the resulting hyperinsulinemia or an of obesity and insulin resistance in mice. Using knockout mice for as yet unknown third factor. By comparing the hepatic liver the major components of the NLRP3 inflammasome (NLRP3−/−, content and plasma triglycerides of individuals with different ASC−/−, caspase 1−/−) in a model of high fat diet (HFD)-induced monogenic forms of insulin resistance O’Rahilly and colleagues obesity, she demonstrated that each of the knockout strains gained provided strong evidence that hyperinsulinemia is the etiologi- less total body weight upon HFD feeding for 16 weeks as com- cal factor: Defects at the level of the insulin receptor completely pared to wildtype controls. In addition, ASC deficiency was shown protected against the development of hepatic steatosis and dys- to protect against HFD-induced obesity, to display increased insulin lipidemia, despite the presence of severe insulin resistance. In sensitivity and glucose tolerance, but to have no impact on adipose contrast patients with similar degrees of insulin resistance due tissue inflammation. Similar to ASC−/− mice, caspase 1−/− mice to lipodystrophy or due to AKT2 mutations have severe dyslipi- were also protected against HFD-induced obesity and displayed an demia and fatty liver. Notably, this suggest that, in human liver, improved insulin sensitivity, furthermore, they displayed a reduced insulin’s activation of the AKT2 pathway may not be as closely level of adipose tissue inflammation. The inflammasome pathway linked to fat accumulation as it is to the control of carbohydrate was thus proposed as a potential target for the treatment of insulin Juliane Bogner-Strauss from Graz (Austria) presented work on the adipocyte plasma membrane-associated protein (APMAP).
2. Session I: metabolism and storage of triglycerides
Using different cell lines (3T3-L1, MEF, SGBS), she demonstrated (chaired and summarized by Andreas Niemeier (Hamburg,
that APMAP is upregulated during adipogenesis and can further Germany))
be rosiglitazone-induced. Silencing of APMAP strongly reduced thein vitro adipogenic differentiation potential of 3T3-L1 cells. Addi- Ira Goldberg (New York, USA) addressed several aspects of lipo- tionally, APMAP was shown to be a direct & functional PPAR␥ toxicity in cardiac muscle. He proposed that morbid obesity can target gene and to translocate from the ER to the plasma mem- lead to lipotoxic cardiomyopathy, and offered several basic ques- brane during adipogenesis. Ongoing work by the group currently tions related to this disease that need to be answered. One of these focuses on further elucidation of the function of APMAP in adipo- questions is which lipids are toxic. A mouse model of increased Please cite this article in press as: Bernini F, et al. Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing, 6–9 September2010. Atherosclerosis (2011), ARTICLE IN PRESS
F. Bernini et al. / Atherosclerosis xxx (2010) xxx–xxx 3. Session II: inflammation and hepatic steatosis (chaired
level. The mixed dyslipidemia and atherosclerosis in E2KI mice and summarized by Monique Mulder (Rotterdam, The
are responsive to PPAR␣ agonists, such as fenofibrate. Besides Netherlands))
inhibiting inflammatory responses, PPAR␣ agonists are known todecrease plasma triglyceride levels, to reduce levels of small dense Jeffrey Esko (San Diego, USA) as an invited speaker pre- LDL and increase levels of anti-inflammatory HDL. In E2KI mice, sented exciting data indicating proteoglycans as a driving force fenofibrate decreases liver steatosis, and atherosclerotic lesion size in lipoprotein clearance. Elevated plasma triglyceride levels are and macrophage content. The effect of PPAR␣ gene levels on dys- now also well recognized as risk factor for premature atheroscle- lipidemia, inflammation and atherosclerosis was investigated by rosis. Accumulation of triglyceride-rich lipoproteins in mice can crossing E2KI mice with PPAR␣-knockout mice. The absence of be caused by diminished sulfation of heparan sulfates in hepato- PPAR␣ does not affect plasma lipids, or atherosclerosis, but aggra- cytes, due to diminished hepatic clearance of lipoproteins. Evidence vates liver steatosis in E2KI mice. Although, the development of was provided indicating syndecan-1 as the specific proteogly- atherosclerosis is not affected by the PPAR␣ gene level, its regres- can involved in hepatic triglyceride-rich lipoprotein clearance in sion upon fenofibrate treatment is affected by the number of genes, mice. Syndecan-1, which is present specifically on the microvilli of with two genes giving the best response. For the anti-inflammatory hepatocyte basal membranes facing the space of Disse, exhibited response, however, two genes are required.
saturable binding of VLDL. The decreased clearance of triglyceride- Veerle Bieghs (Maastricht, The Netherlands) provided evidence rich lipoproteins in syndecan-1 knockout mice could be restored indicating CYP27, the enzyme converting cholesterol into its metabolite 27-hydroxycholesterol (27HC), as a novel player in diet- Both binding and uptake of triglyceride-rich lipoproteins by induced NASH. Recently, they reported an association between heparan sulfate proteoglycans depends on the degree of sulfation hepatic inflammation and lysosomal cholesterol accumulation in of their chains. Hepatocyte-specific inactivation of the enzyme, Kupffer cells. Because 27HC can mobilize cholesterol from the lyso- N-acetylglucosamine N-deacetylase-N-sulfotransferase 1 lead to somes to the cytoplasm of the cell in vitro, they hypothesized that accumulation of triglyceride-rich lipoproteins in plasma in mice.
CYP27 can redirect the intracellular cholesterol pool from the lyso- Using other mice containing loxP-franked conditional alleles of somes to the cytoplasm of the Kupffer cells (KCs) in vivo, thereby uronyl 2-O sulfotransferase and glucosaminyl 6-O sulfotransferase- reducing hepatic inflammation. To investigate the role of 27HC in 1 and the bacterial Cre recombinase expressed in hepatocytes, it NASH, Ldlr−/− mice were transplanted with bone marrow from was shown that the 2-O-sulfate group, specifically and not an over- mice lacking Cyp27. As expected, increased lysosomal cholesterol all charge of the chains, was involved in lipoprotein clearance.
accumulation in KCs, increased amount of cholesterol precipita- Esko also reported that a previously unrecognized phenotype of tions inside the lipid droplets and increased hepatic inflammation fasting hypertriglyceridemia in humans with Knobloch Syndrome was observed in Cyp27−/−-tp mice compared to Wt-tp mice after 3 caused by a null mutation in the vascular form of Type XVIII colla- months of HFC diet. Steatosis, however, was not affected at all. In gen (Col18). The presented data indicated that Col18 is involved conclusion, the present study showed for the first time the poten- in the presentation of Lipoprotein lipase (Lpl) on endothelia in tial of 27HC to modulate the intracellular cholesterol distribution extrahepatic tissue. Lpl acts on triglyceride-rich lipoproteins in inside KCs in vivo and to affect hepatic inflammation.
the peripheral circulation, liberating free fatty acids for energy Ryan Temel (Winston Salem, USA) has been searching for a good metabolism or storage. The enzyme can bind to heparan sul- pharmacological treatment for non-alcoholic fatty liver disease.
fate proteoglycans, but is generally assumed to be bound to the Fatty livers are characterized by the closely correlated accumu- endothelial cell surface primarily by a specific receptor GPIHBP1.
lation of triglycerides and cholesteryl esters. Fat accumulation in However, surprisingly, the absence of Col18 was found to result livers of mice, induced by a high carbohydrate, high cholesterol diet, in decreased lipolysis of triglyceride-rich lipoproteins and sub- can be prevented by knocking down the gene for the cellular choles- sequently, hypertriglyceridemia. This appeared to be due to a terol esterifying enzyme acyl-CoA:cholesterol O-acyltransferase 2 decreased presentation of the enzyme, since its production rate was (ACAT2), or by a administration of antisense oligonucleotides (ASO) normal and as well as the amounts released after heparin injec- against ACAT2. The presented data now show that ASO-mediated tion. Also accumulated lipoproteins were susceptible to lipolysis knockdown of ACAT2, not only can prevent, but also can regress diet induced hepatic lipid accumulation in mice. The absence of ACAT2 Atherosclerosis in humans and in rodents is aggravated by activity is suggested to induce the release of stored triglycerides by chronic inflammation that can be induced by lipopolysaccharide secretion in the form of very low density lipoproteins. They suggest (LPS) from Gram-negative bacteria via Toll-like receptors (TLRs).
that it may have similar effects in humans with non-alcoholic fatty Jimmy Berbée (Leiden, The Netherlands) previously reported that apoCI avidly binds to LPS and, thereby, enhances the LPS-inducedinflammatory response stimulating the development of atheroscle-rosis. Using N- and C-terminal apoCI-derived peptides, he shows 4. Session III: nutrition and metabolism (chaired and
that both the N- and the C-terminal helix of apoCI contain struc- summarized by Rachel Fisher (Stockholm, Sweden) and
tural elements that bind to LPS and prolong its retention time Ernst Malle (Graz, Austria))
in plasma. Both full-helical peptides enhance the LPS-inducedTNFalpha response, in vitro and in vivo via the CD14/TLR4 path- As the invited speaker, Fredrik Karpe, Oxford Centre for Diabetes, way. Conclusively, apoCI enhances the LPS-induced inflammatory Endocrinology and Metabolism (University of Oxford, UK), gave a response via a mechanism similar to LPS-binding protein (LBP).
talk entitled “Channelling of dietary fat into adipose tissue”. He A problem closely associated with obesity and insulin resis- began by questioning the dogma that fasting concentrations of cir- tance is non-alcoholic fatty liver (NAFL) or hepatic steatosis, which culating non-esterified fatty acids (NEFAs) increase with increasing is considered benign and likely reversible. However, when com- fat mass. To investigate the diurnal regulation of NEFA concen- bined with hepatic inflammation it can proceed into non-alcoholic trations in lean and overweight individuals, subjects were fed steatohepatitis (NASH) that may lead to further liver damage.
breakfast, lunch and dinner with each meal containing a different Bart Staels (Lille, France) now reports that in a model of NASH, tracer fatty acid (13C), and a simultaneous intravenous infusion of the apolipoprotein E2 knock-in (E2KI) mouse, lipid metabolism a 2H-labelled fatty acid (to label endogenous fatty acids) was per- and inflammation are differentially modulated by the PPAR␣ gene formed over the entire study period. While insulin responses to Please cite this article in press as: Bernini F, et al. Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing, 6–9 September2010. Atherosclerosis (2011), ARTICLE IN PRESS
F. Bernini et al. / Atherosclerosis xxx (2010) xxx–xxx the meals were greater in the overweight compared to the lean as well as the presence of SR-BI inhibitors or ligand competitors subjects, NEFA concentrations were remarkably similar over the specifically impaired this sensing process. These results suggest a 24-h period in the two groups. While the diurnal absolute rate new role for SR-BI in the intestine.
of NEFA appearance was greater in overweight than lean sub- In a double blind, randomized controlled trial, Max Nieuwdorp jects, this difference was abolished once adjustments were made (Amsterdam, The Netherlands) investigated metabolic effects of for lean body mass, and in fact the relationship was reversed if transplantating gut microbial from lean donors to subjects with adjustment for fat mass was performed, i.e. overweight individ- metabolic syndrome. Results from this study pointed out that lean uals released fewer fatty acids per unit of fat mass compared to donor faecal infusion improves both hepatic, peripheral insulin lean subjects. Collection of blood samples from a vein draining resistance and fasting lipids in metabolic syndrome underscoring the abdominal subcutaneous adipose tissue combined with mea- the potential role of gut microbial even as a potential target for surements of blood flow enabled calculation of rates of removal of chylomicron–triacylglycerol by adipose tissue (equivalent to theactivity of adipose tissue lipoprotein lipase). The overweight indi-viduals removed much less chylomicron–triacylglycerol than the 5. Session IV: functional genomics and genetics (chaired
lean subjects after each meal and the net postprandial uptake of and summarized by Albert Groen (Groningen, The
meal-derived fatty acids into adipose tissue was similarly reduced.
Netherlands) and Anne Tybjaerg-Hansen (Copenhagen,
In the lean subjects the proportion of the meal fat deposited in Denmark))
adipose tissue increased sequentially from breakfast to lunch todinner. However, there was no such increase in the overweight Metabolomics is an emerging field that is based on the quan- subjects and the proportion of meal-fat deposited in their adipose titative measurement of small organic molecules occurring in tissue was significantly less than in the lean subjects. Fredrik Karpe a biological sample on a very broad scale. Due to recent tech- concluded that the adipose tissue of overweight subjects appears to nical advances, metabolomics can now be used widely as an adapt to its enlarged size by down-regulating both fatty acid release analytical high-throughput technology in drug testing and epi- and uptake, thereby ensuring a relatively normal delivery of fatty demiological metabolome and genome wide association studies.
acids to the circulation. Indeed, the inefficient storage of dietary Using metabolite concentration ratios as proxies for enzymatic fatty acids in obese adipose tissue may be the basis for ectopic fat reaction rates, functionality of SNPs on enzyme activity can be analyzed. Karsten Suhre (Munich, Germany) demonstrated in a Leanne Hodson (Oxford, UK) presented data relating to hepatic very elegant presentation application of this novel approach in a fatty acid partitioning in the postprandial period. Data collected genome-wide association study (GWAS) in which 163 metabolic from the same study of lean and abdominally obese men that was traits covering amino acids, sugars and phospholipids were mea- presented by F. Karpe demonstrated that the contribution of meal- sured in human blood from 1809 participants from the KORA derived fatty acids to VLDL–triacylglycerol was similar in the two population. The results were replicated and validated in 422 par- groups. However, the overweight individuals had greater rates of ticipants of the TwinsUK cohort. For eight out of nine replicated ketogenesis and whole body oxidation, which may represent an loci (FADS1, ELOVL2, ACADS, ACADM, ACADL, SPTLC3, ETFDH adaptive response to prevent hepatic fat accumulation.
and SLC16A9), the genetic variant was located in or near genes Barbara Fielding (Oxford, UK) investigated fatty acid metabolism encoding enzymes or solute carriers whose functions match the in gluteofemoral and subcutaneous abdominal adipose tissue associating metabolic traits. In their study, the use of metabo- depots. Proportions of saturated fatty acids were greater, while lite concentration ratios as proxies for enzymatic reaction rates those of monounsaturated fatty acids were lower in abdominal reduced the variance and yielded robust statistical associations compared to gluteofemoral adipose tissue. The abdominal depot with very low P values ranging from 3 × 10(−24) to 6.5 × 10(−179).
had greater expression of genes involved in fatty acid synthesis These loci explained a considerable percentage (5.6–36.3%) of and elongation, but lower expression of stearoyl CoA desaturase the observed variance in metabolite concentrations. For several compared to the gluteofemoral depot. This provides evidence for loci, associations with clinically relevant parameters have been tissue-specific regulation of de novo lipogenesis and fatty acid reported previously showing the great promise of this novel Sander Kersten (Wageningen, The Netherlands) reported that Marcel Wolfs and co-workers (Groningen, The Netherlands) angiopoietin-like protein 4 (Angptl4, containing a lipoprotein exploited the fact that some morbidly obese subjects are metabol- lipase inhibiting domain in the N-terminal portion) acts as an ically healthy, while others show severe insulin resistance or efficient protector against proinflammatory effects of dietary satu- non-alcoholic steatohepatitis. Whole-transcriptome gene expres- rated fatty meals. In peritoneal macrophages incubated with chyle, sion levels in subcutaneous and visceral adipose tissue (SAT and Angptl4 dramatically reduced foam cell formation and inflamma- VAT) of 70 severely obese individuals (BMI > 35) was carried out tory gene expression, mimicking the lipoprotein lipase inhibitor using micro arrays. The gene expression data were organized orlistat. These observations reveal a novel mechanism in which in modules of coexpressed genes, based on the individual dif- induction of macrophage angptl4 by fatty acids serves to reduce ferences in expression patterns of these patients. In both SAT postprandial lipid uptake from fatty chyle into mesenteric lymph and VAT around 70 modules of highly co-expressed genes were node-resident macrophages by inhibiting triglyceride hydrolysis.
detected. Assessment of the correlation between each module and Veronique Carrière (Paris, France) investigated the intestinal the metabolic traits revealed that in VAT a module was significantly secretion of triglyceride-rich lipoproteins during postprandial correlated to glucose levels, and in SAT three modules were sig- period. In Caco-2/TC7 cells, a human enterocytes model, she nificantly negatively correlated to HDL levels. The genes within demonstrated the existence of a specific sensing of postprandial this module in VAT and within these three modules in SAT were lipid micelles at the apical pole of enterocytes and a role for the largely the same. Importantly, genes that constituted these mod- scavenger receptor, class B, type I (SR-BI) in this process. This sens- ules were highly enriched in immune-related genes (GO-terms).
ing is characterized by a rapid activation of p42/44 and p38 MAP Hence, increased expression of immune genes correlated with low kinases and the delocalization of apoB from the apical domain plasma HDL and high plasma glucose levels.
toward the secretory compartment and is accompanied by the clus- To identify potentially novel variants associated with HDL-C, tering of SR-BI at the apical membrane. Both, the absence of SR-BI Pirkka-Pekka Laurila (Helsinki, Finland) and co-workers genotyped Please cite this article in press as: Bernini F, et al. Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing, 6–9 September2010. Atherosclerosis (2011), ARTICLE IN PRESS
F. Bernini et al. / Atherosclerosis xxx (2010) xxx–xxx 450 Finns from the EUFAM population sample, either having Philippa Talmud (London, UK) presented 50K SNP chip genotype extremely high (>90th percentile) or low (<10th percentile) HDL- data from the Whitehall II study where the APOA5 − 1131T>C was C levels. Out of these, subcutaneous fat biopsies were obtained the SNP showing the strongest effect on plasma triglyceride lev- from 54 individuals, and their global expression profiles were ana- els. This provided confidence that this variant could be used in lyzed using Affymetrix microarrays. Interestingly, the 3rd and 10th Mendelian randomization to assess whether plasma triglycerides best hits (rs7766109, p = 10 − 5 and rs4959377, p = 3.45 × 10 − 5) were causal of coronary heart disease (CHD) In a large collabora- were located within coagulation factor XIII A1 subunit with no tive study the relationship between APOA5 − 1131T>C (rs662799), previously reported lipid associations. At the mRNA level, F13A1 triglyceride concentration, and risk of CHD was assessed. In meta- was over-expressed in the adipose tissue of individuals with analysis (n ∼300.000), the rare −1131 C-allele (8%) associated low HDL (p = 0.007), and a dose-dependent effect of rs7766109 with a 16% higher triglyceride level per allele. The corresponding genotype on both F13A1 expression (p = 0.02) and HDL-levels odds ratio for CHD per C-allele was 1.18 (95% confidence interval (p = 0.004) were observed. Laurila et al. also analyzed the adipose 1.11–1.26), concordant with the hazard ratio of 1.10(1.08–1.12) for tissue expression profiles of insulin sensitive and insulin resis- the same triglyceride increase in prospective studies. These results tant Finnish females before and after a euglycemic insulin clamp.
are consistent with a causal role for triglycerides in the develop- Again, F13A1 expression was 6-fold higher in IR compared to IS individuals (p = 0.003). Moreover, an insulin-induced decrease Stefan Coassin (Innsbruck, Austria) screened the ATGL gene in F13A1 expression was observed in IR (p = 0.047) but not in region (8 kb) for genetic variation in the SAPHIR Study (n = 1473), IS subjects. Altogether these data suggest that F13A1 controls and expressed all protein mutations identified in COS7 cells. The plasma HDL-C levels but the mechanism requires further inves- screening revealed numerous rare variants even in this healthy population, none of which had, however, serious impact on the fatty GALNT2 encoding polypeptide N-acetyl-galactosaminyl trans- acid levels, probably due to their heterozygous state. On the cellular ferase 2 (GalNAc-T2) was recently identified in genome-wide level, the same variants showed a large variability in their activity, association studies to be involved in high-density lipoprotein (HDL) and are therefore likely associated with a slight modification of metabolism. Subsequent studies into GALNT2 variation in cohort studies as well as overexpression and silencing approaches in mice Jorie Versmissen (Rotterdam, The Netherlands) compared famil- have provided supportive evidence that this enzyme, which cat- ial hypercholesterolemia (FH) patients with (n = 1383) and without alyzes O-linked glycosylation of Thr and Ser residues of target (n = 1013) LDLR mutations to assess the interaction between the proteins in the Golgi, indeed regulates HDL cholesterol levels. Jan APOE genotype and the presence of an LDLR mutation on risk of Albert Kuivenhoven and co-workers (Amsterdam, The Netherlands) coronary heart disease (CHD). The presence of an LDLR mutation studied the role of GalNAc-T2 in human lipid metabolism through had a protective effect on CHD risk amongst FH patients who were studies in individuals with high HDL cholesterol levels and their homozygous for apoE4 (hazard ratio (HR) 0.16, 95% confidence families. Mutations in the promoter (n = 1), coding (n = 3) and non- interval (CI) 0.05–0.58), and homozygosity for apoE4 was also pro- coding regions (n = 1) of GALNT2 were identified in 5 individuals tective amongst FH patients with an LDLR mutation (HR 0.26, CI with HDL cholesterol levels above the 95th percentile forage and 0.08–0.80). Because the latter patients had lower lipoprotein(a) lev- gender (all heterozygotes). Carriers presented with increased HDL els and a less atherogenic lipid profile than E3 homozygotes, this cholesterol levels (1.89 ± 0.26 vs. 1.37 ± 0.60 mmol/l, p < 0.01) and might explain why the LDLR was protective in this study. This might decreased triglyceride levels (0.90 (IQR 0.73–1.07) vs. 1.04 (IQR be caused by a more prominent role of other ApoE receptors when 0.74–1.32) mmol/l, p < 0.05). In addition, these individuals showed unchanged activities of lecithin:cholesterol acyltransferase (an Steve Humphries (London, UK) presented data from the prospec- O-linked glycosylated enzyme involved in HDL metabolism). Con- tive Whitehall II study (n = 5059) using 21 common LDL cholesterol centrations of other O-linked glycosylated proteins such as apoAII, variants. The data showed that the likelihood of lipid-modifying apoE and apoCIII were also similar in both groups. The role by which intervention and risk of coronary heart disease increased with the mutated GalNAc-T2 affects lipid metabolism is under investiga- number of LDL-raising alleles present, particularly for those in the top quartile of the allele distribution, with the likely mechanism GATA4, a zinc finger domain transcription factor is expressed being through the alleles influencing plasma LDL cholesterol levels.
in various tissues including the heart, adrenal glands and smallintestine not only during organogenesis but also in adulthood.
Its expression is conserved across several vertebrate and inver- 6. Session V: therapeutic approaches to dyslipidemia and
tebrate species. In 2006 intestinal GATA4 was shown to confer atherosclerosis (chaired and summarized by Franco Bernini
jejunal identity. Using intestine specific GATA4 knockouts gener- (Parma, Italy))
ated by Battle et al., Jay Patankar and co-workers (Graz, Austria)showed for the first time that intestinal GATA4 deficiency not only The session was opened with an invited lecture given by Eric J.
leads to decreased triglyceride uptake but also to a resistance to Niesor (Basel, Switzerland) on the HDL as a therapeutic target. He diet induced obesity. The intestinal specific GATA4−/− mice stayed reported results obtained from investigations of the mechanism by insulin sensitive after 20 weeks of high fat diet. In human associa- which cholesteryl ester transfer protein (CETP) activity affects HDL tion studies, Patankar et al. observed that SNPs in and around the metabolism, using agents that selectively target CETP (dalcetrapib, GATA4 gene were strongly associated with plasma TG levels. Inter- torcetrapib, anacetrapib). Results showed that, contrary to torce- estingly, one of the highly associated SNP in a conserved regulatory trapib and anacetrapib, dalcetrapib requires cysteine 13 (Cys13) to intron was observed to confer a gender specific effect on intestinal decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from Stefan Stender (Copenhagen, Denmark) presented data from the HDL3 to HDL2. Moreover, the study showed that only dalcetrapib Copenhagen City Heart Study which showed that homozygosity induced a conformational change in CETP, when added to human for two promoter variants in LXR alpha predicted risk of ischemic plasma in vitro. This effect was also observed in vivo and corre- heart disease, myocardial infarction (MI), ischemic cerebrovascular lated with CETP activity. No change in LCAT activity was observed.
disease, and ischemic stroke (IS) with hazards ratios of 1.6 and 1.8 Dr. Niesor also demonstrated that CETP-induced pre-␤-HDL for- mation in vitro in human plasma was unchanged by dalcetrapib Please cite this article in press as: Bernini F, et al. Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing, 6–9 September2010. Atherosclerosis (2011), ARTICLE IN PRESS
F. Bernini et al. / Atherosclerosis xxx (2010) xxx–xxx ≤3 ␮M and increased at 10 ␮M. Differently, a dose-dependent 7. Session VI: metabolism and function of lipoproteins
inhibition of pre-␤-HDL formation by torcetrapib and anacetrapib (chaired and summarized by Paolo Parini (Stockholm,
(0.1–10 ␮M) was measured, suggesting that dalcetrapib modulates Sweden) and Athina Kalopissis (Paris, France))
CETP activity. In hamsters injected with [3H]-cholesterol-labeledautologous macrophages, and given dalcetrapib, torcetrapib or The session was opened by the invited lecture of John Parks anacetrapib, only dalcetrapib significantly increased faecal elim- (Winston-Salem, NC, USA) on “The role of hepatic ABCA1 in ination of both [3H]-neutral sterols and [3H]-bile acids while lipoprotein metabolism”. An overview on the role of ABCA1 in all compounds increased plasma HDL-[3H]-cholesterol. Based on the formation of HDL particles was followed by the presen- these findings he raised the possibility that modulation of CETP tation of studies on liver specific ABCA1−/− mice and by the activity by dalcetrapib which does not inhibit CETP-induced pre- identification of hepatic ABCA1 as major determinant of the circu- ␤-HDL formation may more efficiently increase reverse cholesterol lating HDL-cholesterol levels. The observation that Tangier Disease subjects have elevated plasma triglycerides (TG) and decreased Patrick CN Rensen (Leiden, The Netherlands) discussed the role LDL-cholesterol levels prompted the study of the role of hepatic of CETP in the HDL-increasing effect of hypolipidemic drugs. Pre- ABCA1 expression on apoB lipoprotein metabolism. Using McArdle vious studies done by the same group have shown that, using cells and liver specific ABCA1−/− mice, it as possible to demon- APOE*3-Leiden.CETP mice, a unique mouse model for human-like strate that lack of hepatic ABCA1 leads to increased plasma TG lipoprotein metabolism, lipid lowering drugs increased HDL-C lev- levels, VLDL secretion, and VLDL particle size. Reductions in post- els by reducing the hepatic lipid content and thereby decreasing heparin LPL and HL activity and of PI3 kinase activation were also hepatic CETP expression and plasma CETP levels. Based on these observed. Challenging of liver-specific ABCA1−/− mice with a high observations this study was aimed to extrapolate these findings fat diet resulted in systemic insulin resistance, defective hepatic from mice to humans. Since thiazolidinediones reduce hepatic insulin signaling, and increased liver inflammation. In conclusion, steatosis and increase HDL-C levels whereas metformin does not elimination of ABCA1 in hepatocytes reproduces the plasma lipid influence hepatic lipid levels and HDL-C, the aim of the study was phenotype of Tangier Disease. Hepatic overproduction of TG-rich to assess the effects of pioglitazone and metformin on hepatic TG, VLDL may be a mechanism for the increased plasma TG levels plasma CETP mass and HDL-C in patients with type 2 diabetes (T2D).
observed in Tangier Disease. Furthermore, hepatic ABCA1 seems The study included 78 men with T2D randomized to treatment with to play an important role in hepatic insulin and inflammatory sig- pioglitazone (30 mg/day) or metformin (2000 mg/day) and match- ing placebos. Plasma HDL-C, TG levels and CETP mass and hepatic The session continued with the presentation of Onno G. Holle- TG content were measured at baseline and after 24 weeks of treat- boom (Amsterdam, The Netherlands) on the impaired tissue ment. The results presented by Dr. Rensen showed that pioglitazone cholesterol efflux in carriers of mutations in APOA1 and ABCA1.
was able to reduce hepatic TG; this effect was correlated with a Reverse cholesterol transport (RCT) was studied in vivo in 5 carri- decrease of plasma CETP mass and TG, and with an increase of ers of mutations in APOA1, in 2 patients homozygous for mutations plasma HDL-C. Whereas metformin similarly reduced plasma TG, in ABCA1 and in 6 unaffected controls by constant infusion of 13C- it did not change hepatic TG, plasma CETP or HDL-C. This study, cholesterol. To calculate the RCT fluxes, a 4-compartment SAAM II in accordance with recent studies on APOE*3-Leiden.CETP mice, fit was applied to plasma free cholesterol, cholesterol ester and ery- shows that in patients with T2D a decrease in hepatic lipid content throcyte enrichment profiles. Tissue cholesterol efflux was reduced associates with a decrease in plasma CETP mass and an increase in by 19% in carriers compared to controls. In contrast, total fecal sterol excretion and the fecal recovery of tracer were comparable in carri- Bo Angelin (Stockolm, Sweden) presented the experience with ers and controls. The data substantiate that HDL contributes to the eprotirome, a liver selective thyroid agonist, in preclinical and efflux of tissue cholesterol in humans, although total fecal sterol clinical studies. The development of this class of drugs is of par- excretion may not reflect this efflux.
ticular relevance, because they exert beneficial effects of thyroid The studies presented by Gemma Brufau (Groningen, The hormones at hepatic level (i.e. stimulation of reverse cholesterol Netherlands) reported the effects of plant sterol supplementa- transport), without the side effects on other organs. The adminis- tion on transintestinal cholesterol excretion in mice (TICE). Using tration of the liver specific, thyroid receptor-beta selective thyroid D5 and D7-labeled cholesterol, whole body cholesterol fluxes and hormone analogue eprotirome to hypercholesterolemic subjects balance were determined in Abcg5−/− mice and their wild-type leads to a dose-dependent decrease in LDL-cholesterol, as well as littermates fed either plant sterol supplemented or control diet.
to reductions of apoB, triglycerides, non HDL-cholesterol and Lp(a) Plant sterol feeding resulted in a decrease in cholesterol absorp- levels. When eprotirome is combined with statins, additive lipid- tion, and induction of neutral sterol excretion in both genotypes. No lowering effects occur, in the absence of undesirable side-effects.
changes were observed in biliary cholesterol and bile salt secretion Paolo Parini (Stockolm, Sweden) discussed the effects of after plant sterol feeding. Plant sterols induced a massive increase atorvastatin on the regulation of carbohydrate and triglyceride in TICE in both genotypes, although to a lesser extent in Abcg5−/− metabolism. Previous works reported the efficacy of this drug in mice. Brufau’s data suggests that the cholesterol-lowering proper- reducing triglyceride levels in addition to the reduction of choles- ties of plant sterols are mainly explained by an induction of TICE terol concentration. This study aimed to investigate the molecular and partly dependent on the expression of Abcg5.
mechanisms exerting this activity in patients treated with high The session was concluded by Arnold von Eckardstein (Zurich, dose of atorvastatin for 4 weeks. In this subjects the expected reduc- Switzerland) who presented the association of monogenic disor- tion of plasma lipids were accompanied by significant modulation ders of HDL metabolism with changes in the lipidome of plasma of expression of genes involved in carbohydrate and triglyceride and lipoproteins. The studies investigated whether changes in metabolism, such as sterol regulatory element binding protein 1c, HDL metabolism affect plasma and lipoprotein levels of 27- glucokinase, angiopoietin like protein 3, glucose 6 phosphatase.
hydroxycholesterol (27-OHC), bile acids (BAs) and the levels of These results provide a possible mechanism contributing to the sphingosine-1-phosphate (S1P). 27-OHC is mainly transported by reduction of plasma triglyceride upon treatment with atorvas- HDL, extensively esterified by LCAT, and its concentration in HDL tatin and suggest an interesting option for the treatment of fatty and plasma are influenced by ABCA1 and ABCG1. Also, BA concen- liver in humans, in the absence of negative effects on the insulin- trations in plasma are influenced by HDL metabolism. A consistent part of BAs are transported by HDL and deficiency in ABCA1 is Please cite this article in press as: Bernini F, et al. Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing, 6–9 September2010. Atherosclerosis (2011), ARTICLE IN PRESS
F. Bernini et al. / Atherosclerosis xxx (2010) xxx–xxx associated with decreased BA levels in plasma. Finally, S1P concen- role of hyperinflammatory hematopoietic cells in hepatic steato- trations in HDL are decreased in patients with defects which cause sis by transplanting C57Bl/6J mice with bone marrow from wild low HDL-cholesterol but not increased in patients with defects that type (WT) or TNFRns heterozygous (+/−) mice. Genes involved in cause high HDL-cholesterol. No correlations between S1P and apoM inflammation and lipogenesis in liver (MCP-1, TNF-alpha, SREBP1c and FASN) were significantly upregulated in chimeric TNFRns (+/−) Ernst Steyrer (Graz, Austria) used primary intestinal mucosa mice fed a diabetogenic diet (36% fat and 0.03% cholesterol) for 8 fractions from phosphatidylethanolamine (PEMT)+/+ (WT) and weeks compared with chimeric WT mice. Although, plasma levels PEMT−/− (KO) mice. Upon stimulation with oleic acid, PEMT of cholesterol and triglycerides were unaltered, liver lipids were expression and activity increased 4-fold in WT samples, secreted significantly increased by 50% in the TNFRns (+/−) chimeric mice lipoproteins were larger and the ratio of secreted/cell associated compared with chimeric WT mice. This finding was confirmed by triglycerides was 4-fold higher compared to KO enterocyte frac- electron microscopy showing that the hepatocytes accumulated tions. Apo B accumulated with neutral lipids at apical sites within considerably more fat droplets. These data indicate that hyper- enterocytes of KO but not of WT mice. Thus, PEMT is expressed in stimulation of leukocyte TNFR1 leads to fat accumulation in the enterocytes and its absence impairs chylomicron secretion, result- Wolfgang Schneider (Vienna, Austria) showed that the cell- Apo M, mainly carried by HDL, is antiatherogenic in vitro.
surface derived soluble form of the receptor LR11/sorLA is Christina Christoffersen (Copenhagen, Denmark) showed that associated with cardiovascular disease in humans and intima- plasma apoM increased 2.1-fold in mice lacking LDL-receptors medial thickness after cuff injury in a mouse model. Evidence was (Ldlr−/−) and 1.5-fold in mice expressing dysfunctional LDL- provided that LR11 is required for the migratory response to AngII receptor-related protein 1 (Lrp1n2/n2), but was unaffected in apo E−/− mice. Overexpression of human apo M increased (50–70%) and apo M deficiency decreased (25%) plasma VLDL/LDL choles- ApoE−/−Dgat1−/− double knockout mouse model had reduced terol in (Ldlr−/−) mice only. Plasma clearance of apo M enriched plasma lipids and lower numbers of inflammatory macrophages.
VLDL/LDL was slower than that of control VLDL/LDL in Ldlr−/− and The data presented showed increased cholesterol efflux from Lrp1n2/n2 mice. Apo M overexpression decreased atherosclerosis ApoE−/−Dgat1−/− macrophages and reduced cholesterol absorp- (60%) in apo E−/− mice, but less so in Ldlr−/− mice. Thus, defec- tion and aortic plaque formation in the double knockout mouse tive LDLR function leads to higher plasma apo M concentration and Laura Calabresi (Milan, Italy) reported that genetically deter- Proprotein convertase subtilisin/kexin type-9 (PCSK9) modu- mined low LCAT activity in Italian families is not associated lates the number of LDL receptors by triggering their degradation.
with increased plasma soluble cell adhesion molecule concen- Lena Persson (Stockholm, Sweden) measured PCSK9, lathosterol and trations, despite low HDL cholesterol levels. Isolated HDL from cholesterol in serum from 85 humans during different conditions.
LCAT mutation carriers was more effective than control HDL in She showed that circulating PCSK9 has a diurnal rhythm in parallel down-regulating TNF␣-induced VCAM-1 expression in vitro. These with cholesterol synthesis, both being strongly reduced by fast- data suggest that HDL from LCAT deficient subjects maintains its ing. The acute depletion of hepatic cholesterol by cholestyramine results in drastic increases in PCSK9 and cholesterol synthesis, dis- Ruth Frikke-Schmidt (Copenhagen, Denmark) presented data rupting normal diurnal variations. This suggests that serum PCSK9 from the Copenhagen City Heart Study which showed that individ- and cholesterol synthesis are regulated by hepatic cholesterol lev- uals with soluble CD163 levels in the 96–100th centile compared to those in the 0–33rd centile had a five fold increased risk of type OSBP-related protein 1L (ORP1L) is an oxysterol binding 2 diabetes. In overweight men of higher age with a sCD163 level in homolog interacting with Rab7 and regulating late endosome (LE) the 96–100th centile this could translate to a 10 year absolute risk positioning in a cholesterol dependent manner. Terhi Vihervaara (Helsinki, Finland) showed that the deletion mutant ORP1L D560-563 fails to bind oxysterols. Its overexpression results in a scattered Acknowledgements
distribution of LE, due to enhanced LE–endoplasmic reticulum (ER)interactions. LE decorated by overexpressed ORP1L and ORP1L The 33rd ELC meeting was generously supported by its D560-563 fail to accept endocytosed dextran. Silencing of ORP1L longstanding main sponsor Roche (Switzerland), and additional results in inhibition of [3H]cholesterol efflux to apo A-I medi- sponsorship by AstraZeneca (Sweden), Boehringer Ingelheim ated by ABCA1. Thus, LE motility and functions in protein and (Germany); Genfit (France), Merck-Sharpe & Dohme (Switzerland) lipid transport are regulated by ORP1L and its ligand-binding sta- and Mabtech (Sweden). We also gratefully acknowledge Dr.
Joachim Ziegenhorn for the local organization.
The 34th ELC meeting is scheduled 5–8th September 2011 in 8. Session topic VII: Varia (chaired and summarized by Dilys
Tutzing, Germany. For information about the preliminary program Freeman (Glasgow, UK))
and abstract forms, please contact Dr. Arnold von Eckardstein,chairman of the ELC Updates and The prize for the best poster was given to Marcela Aparicio- forms will be published on the website of the ELC: Vergara from Groningen (The Netherlands). She investigated the Please cite this article in press as: Bernini F, et al. Report of the 33rd Annual Conference of the European Lipoprotein Club, Tutzing, 6–9 September2010. Atherosclerosis (2011),

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