AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins.

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

Atherosclerosis is an arterial disease that occurs due to the build-up of lipids, cholesterol and other substances in the arterial wall, collectively called plaque, leading to narrowing of the vessel lumen and, in time, disruption to the blood supply. The study of flow through atherosclerotic vessels is especially important since plaques not only cause a reduction in the vessel lumen but can rupture from the arterial wall, causing a blood clot in the vessel that may ultimately lead to heart attack or a stroke. Elevated level of oxidated low density lipoprotein (LDL) is a known risk factor associated with the genesis of atherosclerosis in arterial walls. Previous studies reported in literature have explored the transport of LDL through the arterial wall using analytical and mathematical models. In this work, we have presented a computational framework for the study of LDL transport in the lumen and the porous arterial wall. We have employed a four-layer arterial wall model and used governing equations to model the transport of LDL. We have used physiological parameters for the wall layers from literature and have validated the model based on the calculated filtration velocities and LDL concentration profiles in the arterial wall. We have further used this established model to study the effect of change in permeability and pressure on the LDL concentration. We have also studied the effect of pulsatile flow on the transport of LDL through the porous walls to examine the validity of the initial assumption of steady state and have seen that pulsation increases the time averaged net flux of LDL species by about 20%. We have next modeled a drug-eluting stent (DES), which is one of the popular remedies to cure atherosclerosis. The validation of DES model is followed by a combined study to analyze the effect of stent placement on LDL transport. Although there is no chemical reaction between the drug and LDL, we have noticed recirculation zones near the stent strut which result in accumulation of LDL molecules in the arterial wall. Future studies are aimed at incorporating variable porosity and permeability and a stenosed region in the geometry. The deformation of arterial wall due to pulsatile blood flow may lead to alteration of wall properties, which can give a realistic view of macromolecular transport.

Magister Pharmaceuticae - MPharm / Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins. This study was aimed at contributing to the generation of knowledge regarding the effect of niacin in reducing LDL levels through PCSK9 interaction. The aims/objectives of this study were achieved by utilizing two approaches, which included animal intervention with niacin followed by genetic screening of five prioritized genes involved in cholesterol synthesis and regulation. For animal intervention, 16 vervet monkeys were divided into two groups of eight animals consisting of a control and an experimental (niacin) group. The control group was given a normal standard diet of pre-cooked maize meal throughout the study, while the experimental group received the same diet supplemented with 100 mg/kg of niacin (SR) for 12 weeks. During the niacin intervention, blood was collected at baseline, every four weeks during the treatment period and the end of the washout period. The collected blood was used for biochemical analysis (total cholesterol, triglycerides, LDL-C, and HDL-C) and downstream genetic applications. The second phase included the screening of PCSK9, LDLR, SREBP-2, CETP and APOB-100 using genotyping and gene expression. Niacin administration produced statistically significant increases in plasma HDL-C at fourtime points (T1, T2, T3 and T4), which resulted in an overall increase in plasma HDL-C. Additionally, niacin administration resulted in a slight reduction in LDL-C and total cholesterol levels. Furthermore, the genotyping analysis revealed 13 sequence variants identified in PCSK9, LDLR, SREBP-2, CETP and APOB-100 genes. Five of these variants were predicted to be disease-causing and correlated with gene expression patterns. Three identified PCSK9 variants (H177N, R148S, G635G) were categorized as LOF mutations, and this was supported by a decline in gene expression in animals harbouring these variants. The LDLR also had LOF variants that were the reason for its decreased mRNA expression. Additionally, SREBP-2 proved to be a key mediator of cholesterol pathways. Therefore, the findings of the study conclusively suggest that niacin does increase HDL-C and decrease LDL-C and total cholesterol. Moreover, an interaction between niacin administration and PCSK9 was observed which resulted in decreased gene expression.

Sample preparation as an essential step in mass spectrometry-based analysis, plays a critical role in proteomics studies. Magnetic nanoparticles (MNPs) have been widely used in protein and peptide sample preparation due to their magnetic properties, biocompatibility, easy synthesis and surface functionalization. MNPs loaded with analyte or analyte modification reagent can be easily separated from the reaction medium by an externally applied magnetic field. The small size of MNPs provides high analyte loading and extraction capacity. Additionally, MNP can be decorated with different functional groups to achieve selective modification or extraction of analyte. In this study we have utilized silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) for protein and peptide sample preparation. Fluorescence-based methods were utilized for quantitative and qualitative characterization of N-hydrosucccinimidyl (NHS) ester groups on the surface of Fe3O4@SiO2 MNPs. Fluorophore Dansylcadaverine was conjugated to NHS ester functional groups. Fluorometric measurement of cleaved dansylcadaveine was employed to determine the number of NHS ester groups per MNPs that was found to be 2.6 × 102 and 3.4 × 103for 20 nm and 100 nm Fe3O4@SiO2 MNPrespectively. The efficiency of labeling native bovine serum albumin (BSA) by NHS ester coated Fe3O4@SiO2 MNPs was also explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each MNP. Lysine residues of apolipoprotein B-100 (apoB-100) on the surface of intact human low density lipoprotein (LDL) were labeled by NHS ester modified Fe3O4@SiO2 MNPs in aqueous solvents at room temperature. The MNP labeledapoB-100 was treated by SDS to remove lipids and then digested using trypsin. Tryptic peptides were eluted from MNPs by cleaving disulfide linkage between labeled peptides and MNPs. LC-MS/MS analysis found 28 peptides containing labeled lysine residues. These lysine residues should be on the solvent exposed surface of LDL since the large size of MNPs prevents contact of the labeling reagent to those lysines embedded inside the structure of LDL. TCEP- immobilized Fe3O4@SiO2MNPs were fabricated and utilized for reduction of disulfide bonds in bovine pancreas insulin and two different cyclic peptides. Disulfide bonds were efficiently cleaved at room temperature in both organic and aqueous solvents confirmed by LC-MS/MS analysis of reduced/alkylated protein and peptides. Disulfide reduction and alkylation reactions was performed in one step and the reducing agent was simply separated from peptide and protein solution by magnetic separation.

CONTEXTE: L'inefficacité de captation des acides gras libres (AGL) par le tissu adipeux blanc (TAB) est connue pour favoriser la résistance à l'insuline (RI) dans les tissus périphériques, mais dans le foie, elle favorise également la production accrue de lipoprotéines contenant l'apolipoprotéine B100 (lipoprotéines apoB). Nous avons récemment démontré que les femmes post-ménopausées obèses avec un nombre élevé de lipoprotéines apoB à jeun (apoB plasmatique > 1,2 g/L) avaient plus de RI que les femmes avec un taux d’apoB normal. Notre objectif était donc d'examiner si l'inefficacité de captation des AGL pourrait être un mécanisme expliquant la RI, in vivo dans cette population. HYPOTHÈSES: Les femmes ménopausées en surpoids/obèses avec un taux d’apoB élevé ont moins d'efficacité à capter les AGL par le TAB que les femmes avec un taux d’apoB faible. MÉTHODES/RÉSULTATS: L'efficacité de captation des AGL a été examinée dans 22 femmes non diabétiques en surpoids/obèses. La population a été séparée selon la médiane d’apoB (0.9 g/L) en 2 groupes; les femmes ayant un taux d’apoB inférieur vs supérieur à la médiane (N=11/groupe). L'efficacité de captation des AGL par le TAB a été indirectement évaluée en suivant le sort d'un repas riche en gras (0.0162g 13C-trioléine/g de matières grasses, 66% de gras, 47g gras/m2 surface corporelle) marqué au 13C-trioléine, sur 6h en circulation ([13C]TG et [13C]AGL plasmatiques) et en oxydation (13CO2 dans l’air expiré [AE]). L’enrichissement en 13C des échantillons d’AE et du plasma a été mesuré par spectrométrie de masse pour ratio isotopique. Les femmes ayant un apoB élevé avaient une clairance plasmatique totale postprandiale des TG (p <0,05) réduite sans diminuer de la clairance plasmatique totale des AGL, par rapport aux femmes ayant un faible taux d’apoB. Cependant, en examinant le sort du 13C-trioléine, les femmes ayant un apoB élevé avait une réduction de la clairance des [13C]TG plasmatiques (44,78 μM vs 7,81 μM; p <0,05) et des [13C]AGL plasmatiques (2,64 uM vs 0,06 uM; p <0,05) à 6h, sans aucune différence en % récupéré de 13C-trioléine dans le CO2 de l’AE. Ces données suggèrent que les femmes ayant un taux d’apoB élevé ont une réduction postprandiale de la clairance et de la captation de 13Ctrioléine par le TAB. CONCLUSION: La captation inefficace des AGL par le TAB des femmes post-ménopausées en surpoids et obèses avec un surplus d’apoB peut être un mécanisme sousjacent à la RI chez ces sujets. / BACKGROUND: Inefficiency of fatty acid (FA) trapping in white adipose tissue (WAT) is known to promote insulin resistance (IR) in peripheral tissue, but in the liver, it also promotes increased secretion of apolipoprotein B100-lipoproteins (apoB-lipoproteins). We recently demonstrated that post-menopausal obese women with high numbers of fasting apoB-lipoproteins (plasma apoB> 1.2 g/L) had higher IR than women with normal apoB. Our aim was thus to examine whether inefficiency of FA trapping was a mechanism explaining IR in vivo in this population. HYPOTHESES: Postmenopausal overweight and obese women with high apoB have lower efficiency of FA trapping in WAT than women with low apoB. METHODS/RESULTS: The efficiency of FA trapping was examined in 22 non-diabetic overweight and obese women, and the group was separated around median apoB (0.9 g/L) into women with higher vs lower apoB (N=11 per group). The efficiency of FA trapping in WAT was indirectly assessed by following the fate of a 13C-triolein labelled high-fat meal (0.0162g 13C-triolein/g of fat, 66% fat, 47 g fat/m2 body surface area) over 6 hours in circulation (plasma [13C]TG and [13C]FA) and oxidation (breath 13CO2). 13Cenrichment of breath and plasma samples was measured by isotope ratio mass spectrometry. Women with higher apoB had delayed total postprandial plasma TG clearance (p<0.05) but not total plasma free FA clearance compared to women with lower apoB. However, examining the fate of 13C-triolein revealed that women with higher apoB had delayed plasma [13C]-TG clearance (44.78 μM vs 7.81 μM; p<0.05) and plasma [13C]-FA clearance (2.64 μM vs 0.06 μM; p<0.05) at 6 hours, without any differences in % recovered 13C-triolein in breath CO2. This data suggests that women with higher apoB have lower postprandial 13C-triolein clearance and trapping in WAT. CONCLUSION: Ineffective FA trapping in WAT in post-menopausal overweight and obese women with higher apoB may be a mechanism underlying IR in these subjects.

No description available.

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) des fluctuations plasmatiques des molécules endogènes, mais aussi exogènes, et notamment des molécules à potentiel thérapeutique. L’imperméabilité de la BHE est compensée par la présence de mécanismes qui assurent le transport transendothélial des nutriments nécessaires au tissu nerveux, parmi lesquels la transcytose relayée par différents récepteurs. Dans le but d’améliorer le transfert d’agents thérapeutiques à travers la BHE, nous développons des « vecteurs » qui se lient à certains de ces récepteurs. Au cours de notre thèse, nous avons développé et optimisé des modèles in vitro de BHE et barrière sang-moelle épinière (BSME) syngéniques de rats et souris, basés sur la co-culture de cellules endothéliales microvasculaires (CEMs) cérébrales (CEMCs) ou spinales (CEMSs) et d'astrocytes. Parmi les récepteurs étudiés, nous montrons que le LDLR est exprimé à la membrane plasmique apicale des CEMCs et qu’il est impliqué dans la transcytose du LDL tout en évitant le compartiment lysosomal, confirmant l’intérêt de son ciblage dans nos approches. Nous montrons que nos vecteurs, conjugués à une molécule organique ou à un cargo protéique, sont endocytés par les CEMCs de façon LDLR-dépendante, évitent le compartiment lysosomal et franchissent la monocouche de CEMCs. Nous avons également mis en place des modèles in vitro de BHE et BSME enflammés, sachant que l’inflammation des CEMs est associée à de nombreuses pathologies du SNC. Ces modèles seront utiles pour évaluer des stratégies de vectorisation ciblant préférentiellement les structures du SNC en situation pathologique. / The blood-brain barrier (BBB) protects the central nervous system (CNS) from plasma fluctuations of endogenous, but also exogenous molecules, including therapeutic molecules. The BBB’s restrictive properties are compensated by the presence of different mechanisms that provide transport of nutrients across the BBB, including transcytosis of endogenous ligands mediated by receptors. Our objective is to improve drug delivery across the BBB and we developed “vectors” that target different recpetors. During our thesis we developed and optimized cellular tools and approaches, in particular syngeneic in vitro models of the BBB and blood-spinal cord barrier (BSCB) from both rat and mouse, based on the co-culture of brain (BMECs) or spinal cord (SCMECs) microvascular endothelial cells (MECs) and astrocytes. Among the receptors we studied, we show that the LDL receptor (LDLR) is expressed at the apical plasma membrane of BMECs and confirmed that it is involved in transcytosis of LDL through the vesicular compartment, while avoiding the lysosomal compartment, further establishing its interest as a target receptor. We show that our vectors conjugated to an organic molecule or to a protein cargo are endocytosed by BMECs in a LDLR-dependent manner, avoid the lysosomal compartment and cross the BMEC monolayers. Finally, we developed BBB and BSCB in vitro models in inflammatory conditions, considering that MECs inflammation is associated with many CNS lesions and pathologies. These models will be useful to better understand the inflammatory processes of CNS endothelial cells and to evaluate vectorization strategies preferentially targeting CNS structures in pathological condition.

Contexte : La dysfonction du tissu adipeux blanc (TAB) favorise les facteurs de risque de diabète de type 2 (DbT2), c’est-à-dire la résistance à l’insuline (RI), l’hyper sécrétion d’insuline glucostimulée (SIGS), le délai de clairance des gras et les concentrations élevées d’apoBlipoprotéines (apoB plasmatique) incluant les lipoprotéines de faible densité (LDL). De récentes études de notre laboratoire et d’autres suggèrent que le niveau élevé d’apoB plasmatique (hyperapoB) est une cause et non seulement une conséquence de la dysfonction du TAB. De plus, une internalisation augmentée d’apoB-lipoprotéines via les récepteurs tels que le récepteur aux LDLs (LDLR) et le cluster de différenciation 36 (CD36), favorise le risque de DbT2. Cependant, les mécanismes sous-jacents de même que les interventions nutritionnelles pour les cibler demeurent incertains. L'activation de la voie de l’inflammasome NLRP3/ interleukine (IL) -1β favorise la dysfonction du TAB et les facteurs de risque de DbT2 et est activée par les LDLs oxydées dans les cellules immunitaires. L'acide eicosapentaénoïque (AEP) et l'acide docosahexaénoïque (ADH) réduisent l'hyperapoB, l'activité de l’inflammasome NLRP3 dans les cellules immunitaires et les facteurs de risque de DbT2 chez l’humain. Ils sont synthétisés de façon endogène par l’entremise des désaturases d’acides gras δ-5 (D5D) et δ-6 (D6D). Chez l’humain, de faibles niveaux d’AEP et d’ADH circulants et d’activité de la D5D et une activité élevée de la D6D prédisent l'incidence de DbT2 et la RI par des mécanismes inconnus. Objectifs : L'hypothèse de ma thèse est que l'AEP et l’ADH améliorent les facteurs de risque de DbT2, soit la dysfonction du TAB, le délai de clairance des gras, la RI et l’hyper SIGS, ceci via une baisse de l'apoB plasmatique et de l’activité de l’inflammasome NLRP3 dans le TAB. Les objectifs sont d'examiner si: 1) les associations entre les activités de la D5D et de la D6D et les facteurs de risque de DbT2 dépendent de l'apoB plasmatique; 2) la supplémentation en AEP+ADH réduit l'apoB plasmatique, l'expression du LDLR et du CD36 dans le TAB, l'activité de l’inflammasome NLRP3 dans le TAB et les facteurs de risque de DbT2; 3) l’AEP+ADH inhibe la sécrétion d'IL-1β par le TAB humain stimulée par des signaux canoniques ou les LDLs natives. Méthodes: Des hommes et des femmes postménopausées normoglycémiques ont été testés à l’état basal et après une supplémentation en AEP (1,8 g/jour) et ADH (0,9 g/jour) de 12 semaines. Les activités de la D5D et de la D6D ont été estimées à partir des acides gras produits/précurseurs dans les phospholipides plasmatiques. Nous avons mesuré la SIGS, la RI et le disposition index lors d’un clamp Botnia. Après un repas à 66% de gras, le délai de clairance des gras a été mesuré par l’aire sous la courbe (sur 6 h) des triglycérides (TG) ou de l’apoB48 (chylomicrons) plasmatiques. Ex vivo dans une biopsie de TAB, nous avons mesuré l'expression de surface du LDLR et du CD36 par immunohistochimie, l'ARNm de NLRP3 et IL1B par RT-qPCR et la sécrétion d'IL-1β par alpha-LISA en l’absence ou en présence d’une stimulation par le lipopolysaccharide (LPS), l'adénosine triphosphate (ATP) et/ou les LDLs humaines natives et lors d’une co-incubation avec l’AEP+ADH. Résultats: À l’état basal (N=98), l'activité de la D5D corrélait négativement avec l'apoB plasmatique, la 2e phase de SIGS, la RI et le délai de clairance des chylomicrons et ces associations étaient dépendantes de l'apoB plasmatique. Inversement, l'activité de la D6D corrélait positivement avec la SIGS, la RI et le délai de clairance des chylomicrons indépendamment de l'apoB plasmatique. Chez les sujets ayant complété la supplémentation en AEP+ADH (N=30), on notait une amélioration de la 1e phase de SIGS, du disposition index et de la clairance des TGs. Des niveaux initiaux plus élevés d'apoB plasmatique, de TGs postprandiaux plasmatiques et de RI, et dans le TAB d'expression du LDLR et du CD36, de sécrétion d’IL-1β et d'ARNm de NLRP3 prédisaient une plus grande réduction de ces paramètres. En comparaison à l'acide palmitique, l’AEP+ADH inhibait la sécrétion d'IL-1β par le TAB, en l’abscence ou en présence d’une stimulation par le LPS, l'ATP et/ou les LDLs natives de ces sujets. Conclusion: Les associations inverses entre l'activité de la D5D avec les facteurs de risque de DbT2 sont dépendantes de l'apoB plasmatique. Les meilleurs répondants à la supplémentation en AEP et ADH, en termes de réduction d'apoB plasmatique, d’expression du LDLR et du CD36 dans le TAB, d'activité de l’inflammasome NLRP3 dans le TAB, de TGs postprandiaux et de RI, sont les sujets avec des niveaux initiaux élevés de ces paramètres. L’AEP et l’ADH inhibent directement la sécrétion d'IL-1β par le TAB humain induite par les LDLs natives ou d'autres signaux. Nous proposons que la supplémentation en AEP et ADH puisse cibler l'activité de l’inflammasome NLRP3 dans le TAB, induite par un niveau élevé d’apoB-lipoprotéines plasmatiques ou internalisées par les récepteurs, et ainsi aider à prévenir le DbT2. / Background: White adipose tissue (WAT) dysfunction promotes risk factors for type 2 diabetes (T2D), namely insulin resistance (IR), high glucose-stimulated insulin secretion (GIIS), delayed fat clearance and high concentrations of apoB-lipoproteins (measured as plasma apoB) including low density lipoproteins (LDL). Recent studies from our lab and others suggest that high plasma apoB (hyperapoB) is a cause and not only a consequence of WAT dysfunction. Moreover, upregulated receptor-mediated uptake of apoB-lipoproteins via LDL receptor (LDLR) and cluster of differentiation 36 (CD36), promotes the risk for T2D. However, underlying mechanisms as well as nutritional interventions to target them remain unclear. Activation of the NLRP3 inflammasome/interleukin (IL)-1β pathway promotes WAT dysfunction and risk factors for T2D and is activated by oxidized LDLs in immune cells. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) reduce hyperapoB, NLRP3 inflammasome activity in immune cells and risk factors for T2D in humans. They are synthesized endogenously through δ-5 (D5D) and δ-6 (D6D) fatty desaturases. In humans, low levels of circulating EPA and DHA and D5D activity and high D6D activity predict the incidence of T2D and IR by unknown mechanisms. Objectives: The hypothesis of my thesis is that EPA and DHA improve T2D risk factors, namely WAT dysfunction, delayed fat clearance, IR and high GIIS, this via a reduction of plasma apoB and WAT NLRP3 inflammasome activity. The objectives are to examine whether: 1) the associations between the levels of D5D and D6D activities and the risk factors for T2D are dependent on plasma apoB; 2) supplementation with EPA+DHA reduces plasma apoB, WAT LDLR and CD36 expression, WAT NLRP3 inflammasome activity and T2D risk factors; 3) EPA+DHA directly inhibits IL-1β secretion from human WAT stimulated by canonical signals or native LDLs. Methods: Normoglycemic men and postmenopausal women were tested at baseline and after supplementation with EPA (1.8 g/day) and DHA (0.9 g/day) for 12 weeks. The activities of D5D and D6D were estimated from the product/precursor fatty acids in plasma phospholipids. We measured GIIS, IR and disposition index by a Botnia clamp. Following a 66% fat meal, delayed fat clearance was measured as the area under the curve (over 6 h) of plasma triglycerides (TG) or apoB48 (chylomicrons). Ex vivo in a WAT biopsy, we measured LDLR and CD36 surface expression by immunohistochemistry, NLRP3 and IL1B mRNA by RT-qPCR, and IL-1β secretion by alpha-LISA either unstimulated or stimulated by lipopolysaccharide (LPS), adenosine triphosphate (ATP), and/or native human LDLs, and during co-incubation with EPA+DHA. Results: At baseline (N=98), D5D activity correlated negatively with plasma apoB, 2nd phase GIIS, IR and delayed chylomicron clearance and these associations were dependent on plasma apoB. Conversely, D6D activity correlated positively with GIIS, IR, and delayed chylomicron clearance independently of plasma apoB. In subjects who completed the EPA+DHA supplementation (N=30), there was an amelioration in 1st phase GIIS, disposition index and TG clearance. Higher baseline levels of plasma apoB, plasma postprandial TGs, IR, WAT LDLR and CD36 surface expression, WAT IL-1β secretion and WAT NLRP3 mRNA predicted a greater reduction of these parameters. In comparison with palmitic acid, EPA+DHA inhibited IL-1β secretion from WAT, either unstimulated or stimulated by LPS, ATP and/or subjects’ native LDLs. Conclusion: The negative associations of D5D activity with risk factors for T2D are dependent on plasma apoB. Best responders to EPA and DHA supplementation to reduce plasma apoB, WAT LDLR and CD36 expression, WAT NLRP3 inflammasome activity, delayed TG clearance, and IR are subjects with elevated baseline levels of these parameters. EPA and DHA directly inhibit IL-1β secretion from human WAT induced by native LDLs or other signals. We propose that EPA and DHA supplementation may target upregulated WAT NLRP3 inflammasome activity induced by high plasma concentrations, or receptor-mediated uptake, of apoB-lipoproteins, and thus help prevent T2D.