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Journal: Journal of lipid research

168

Background: HDL-associated paraoxonase 1 (PON1) activity is associated with cardiovascular and other human diseases. As the role of genetic variants outside of the PON gene cluster on PON1 activity is unknown, we sought to identify common and rare variants in such loci. Methods: We typed 33,057 variants on the CVD chip in 1,362 subjects to test for their effects on adjusted-PON1 activity. Three novel genes (FTO, ITGAL, and SERPINA12) and the PON gene cluster had SNPs associated with PON1 arylesterase (AREase) activity. These loci were carried forward for rare-variant analysis using Exome chip genotypes in an overlapping subset of 1,051 subjects using sequence kernel association testing. Results: PON1 (p=2.24x10-4), PON3 (p=0.022), FTO (p=0.019), and SERPINA12 (p=0.039) had both common and rare-variants associated with PON1 AREase. ITGAL variants were associated with PON1 activity when using weighted-SKAT analysis (p=2.63x10-3). When adjusting for the initial common variants, SERPINA12 became marginally significant (p=0.09), while all other findings remained significant (p<0.05), suggesting independent rare-variant effects. Conclusions: We present novel findings that common and rare variants in FTO, SERPINA12, and ITGAL predict PON1 activity. These results further link PON1 to diabetes and inflammation and may inform the role of HDL in human disease.

Concepts: DNA, Gene, Genetics, Epidemiology, Disease, Infectious disease, PON1, Paraoxonase

167

Vitamin A or retinol is arguably the most multifunctional vitamin in the human body as it is essential from embryogenesis to adulthood. The pleiotropic effects of vitamin A are exerted mainly by one active metabolite, all-trans retinoic acid (atRA), which regulates the expression of a battery of target genes through several families of nuclear receptors (RARs, RXRs and PPARβ/δ), polymorphic retinoic acid (RA) response elements and multiple coregulators. It also involves extra nuclear and non-transcriptional effects such as the activation of kinase cascades, which are integrated in the nucleus via the phosphorylation of several actors of RA signaling. However, vitamin A itself proved recently to be active and RARs to be present in the cytosol to regulate translation and cell plasticity. All these new concepts expand the scope of the biologic functions of vitamin A and RA.

Concepts: Cell nucleus, Cell, Signal transduction, Retinol, Vitamin A, Retinoic acid, Tretinoin, Retinoid

167

Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation which could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptors to bleomycin-induced lung injury and measured efferocytosis, inflammation and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days post-injury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days post-injury, 51% increase in lung macrophages 14 days post-injury) and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days post-injury respectively). Reduced fibrosis in CD36-/- mice was associated with lower levels of pro-fibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1 and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 hours following zymosan administration in G2A-/- mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.

Concepts: Immune system, White blood cell, Monocyte, Fibrosis, Cell biology, Macrophage, Apoptosis, Idiopathic pulmonary fibrosis

167

The LDL receptor (LDLR) supports efficient uptake of both LDL and VLDL remnants by binding lipoprotein at the cell surface, internalizing lipoprotein through coated pits and releasing lipoprotein in endocytic compartments before returning to the surface for further rounds of uptake. While many aspects of lipoprotein binding and receptor entry are well understood, it is less clear where, when and how the LDLR releases lipoprotein. To address these questions, the current study employed quantitative fluorescence imaging to visualize the uptake and endosomal processing of LDL and the VLDL remnant, β-VLDL. We find that lipoprotein release is rapid with most release occurring prior to entry of lipoprotein into early endosomes. Published biochemical studies have identified two mechanisms of lipoprotein release: one that involves the β-propeller module of the LDLR and a second that is independent of this module. Quantitative imaging comparing uptake supported by the normal LDLR or by an LDLR variant incapable of β-propeller-dependent release show that the β-propeller-independent process is sufficient for release for both lipoproteins, but that the β-propeller process accelerates both LDL and β-VLDL release. Together these findings define where, when and how lipoprotein release occurs and provide a generalizable methodology for visualizing endocytic handling in situ.

Concepts: Cholesterol, Low density lipoprotein receptor gene family, Lipoproteins, Endocytosis, Endosome, Release, LDL receptor

166

Bile acid sequestrants (BASs) are cholesterol-lowering drugs that also affect hyperglycemia. The mechanism by which BASs exert these and other metabolic effects beyond cholesterol lowering remains poorly understood. The present study aimed to investigate the effects of a BAS, colestilan, on body weight, energy expenditure, and glucose and lipid metabolism and its mechanisms of action in high-fat-fed hyperlipidemic APOE*3 Leiden (E3L) transgenic mice. Mildly insulin resistant E3L mice were fed a high-fat diet with or without 1.5% colestilan for 8 weeks. Colestilan treatment decreased body weight, visceral and subcutaneous fat, and plasma cholesterol and triglyceride levels but increased food intake. Blood glucose and plasma insulin levels were decreased, and hyperinsulinemic-euglycemic clamp analysis demonstrated improved insulin sensitivity, particularly in peripheral tissues. In addition, colestilan decreased energy expenditure and physical activity, whereas it increased the respiratory exchange ratio, indicating that colestilan induced carbohydrate catabolism. Moreover, kinetic analysis revealed that colestilan increased [3H]-NEFA incorporation in biliary cholesterol and phospholipids and increased fecal lipid excretion. Gene expression analysis in liver, fat and muscle supported the above findings. In summary, colestilan decreases weight gain and improves peripheral insulin sensitivity in high-fat-fed E3L mice by enhanced NEFA incorporation in biliary lipids and increased fecal lipid excretion.

Concepts: Cholesterol, Metabolism, Nutrition, Insulin, Liver, Triglyceride, Bile, Carbohydrate

164

Cerebrotendinous xanthomatosis (CTX) is a rare, difficult to diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial since an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX compared to 5α-cholestanol. 7α12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX newborn DBS concentrations of this ketosterol (120-214 ng/ml) were around 10-fold higher than in unaffected newborn DBS (16.4±6.0 ng/ml), such that its quantification provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX, preventing morbidity and mortality.

Concepts: Infant, Cancer, Disease, Death, Prevention, Quantification, Newborn screening, Cerebrotendineous xanthomatosis

151

The high degree of size heterogeneity of apolipoprotein(a) [apo(a)], the distinct protein component of lipoprotein(a) [Lp(a)], renders more difficult the development and selection of specific antibodies directed to apo(a) and poses significant challenges to the development of immunoassays to measure its concentration in plasma or serum samples. Apo(a) is extremely variable in size not only between but also within individuals because of the presence of two different, genetically determined apo(a) isoform sizes. Therefore, the antigenic determinants per particle available to interact with the antibodies will vary in the samples and the calibrators, thus contributing to apo(a) size-dependent inaccuracy of different methods. The lack of rigorous validation of the immunoassays and common means of expressing Lp(a) concentrations hinder the harmonization of results obtained by different studies and contribute to the lack of common cut points for identification of individuals at risk for coronary artery disease or for interventions aimed at reducing Lp(a) levels. The aim of our review is to present and critically evaluate the issues surrounding the measurements of Lp(a), their impact on the clinical interpretation of the data and the obstacles we need to overcome to achieve the standardization of Lp(a) measurements.

Concepts: Immune system, Antibody, Angina pectoris, Coronary artery disease, Heart, Artery, Coronary circulation, Antigen

150

146

Long chain polyunsaturated fatty acids (LC-PUFA; C20-C22) are highly enriched in vertebrate retina and testes, where they are further elongated to very long chain PUFA (VLC-PUFA; C≥28) by Elongation of Very Long Chain Fatty Acids-4 (ELOVL4). These fatty acids play essential roles in modulating neuronal function and health. To better understand the role of LC-PUFA and VLC-PUFA in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes of rod- and cone-dominant retinas. Fatty acid methyl esters and glycerophospholipid molecular species were analyzed by GC-MS/GC-FID and ESI-MS/MS, respectively. We found that whole retinas and OS from rod-dominant animals have higher amounts of LC-PUFA and VLC-PUFA than do cone-dominant retinas. The retinas and OS from the cone-dominant retinas also have lower amounts of di-DHA (22:6/22:6) molecular species in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, than do rod-dominant retinas. Since PUFA are necessary for optimal G-protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

142

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs), are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent fatty acid substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain fatty acids were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, co-activators, inhibitors, protein interactions, and post-translational modification.

Concepts: DNA, Protein, Amino acid, Nutrition, Fatty acid, Enzyme, Cell membrane, Essential fatty acid