Apolipoprotein B (apoB) is the main protein of very-low-density lipoprotein (VLDL). As apoB is translated and moves through the secretory pathway, lipids from cytoplasmic lipid droplets (LDs) are added to form VLDL particles. Without adequate lipid availability, apoB is misfolded and undergoes proteasomal degradation; however, evidence now shows that apoB can be degraded through autophagy. Inhibiting autophagy decreased apoB localization to autophagosomes in HepG2 cells, but also decreased apoB recovered from cells and media. Inducing autophagy increased apoB localization to autophagosomes and decreased apoB recovery. LDs are also degraded through autophagy however LDs were not affected by autophagy modulation in HepG2 cells. In primary hamster hepatocytes, inhibiting autophagy reduced apoB-autophagosome co-localization and increased LD numbers. These data suggest that autophagy may play a complex role in VLDL assembly by regulating degradation of both apoB and LDs. This dual role is more evident in primary hepatocytes indicating a potential physiological role.

Apolipoprotein B (apoB) is the main protein of very-low-density lipoprotein (VLDL). As apoB is translated and moves through the secretory pathway, lipids from cytoplasmic lipid droplets (LDs) are added to form VLDL particles. Without adequate lipid availability, apoB is misfolded and undergoes proteasomal degradation; however, evidence now shows that apoB can be degraded through autophagy. Inhibiting autophagy decreased apoB localization to autophagosomes in HepG2 cells, but also decreased apoB recovered from cells and media. Inducing autophagy increased apoB localization to autophagosomes and decreased apoB recovery. LDs are also degraded through autophagy however LDs were not affected by autophagy modulation in HepG2 cells. In primary hamster hepatocytes, inhibiting autophagy reduced apoB-autophagosome co-localization and increased LD numbers. These data suggest that autophagy may play a complex role in VLDL assembly by regulating degradation of both apoB and LDs. This dual role is more evident in primary hepatocytes indicating a potential physiological role.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

A new variant of human apolipoprotein B has been identified in a subject with decreased low density lipoprotein cholesterol and apolipoprotein B concentrations. Although no clinical signs of fat malabsorption were observed the subject was diagnosed, on the basis of his low cholesterol and apolipoprotein B level, as having hypobetalipoproteinaemia. The variant of apolipoprotein B was first identified by Western blot analysis. The analysis revealed an abnormal low molecular weight form of apolipoprotein B as well as normal apolipoprotein B-100 indicating that the subject was heterozygous for a truncated form of apolipoprotein B. The new variant (apo B-32) was a result of a C→T transition at nucleotide 4548 in exon 26 of the apolipoprotein B gene. This mutation changes a CAG codon which codes for glutamine into a TAG stop codon resulting in translation of a truncated apolipoprotein B protein approximately 32% the length of normal apolipoprotein B-100. Although only 32% of the length of apolipoprotein B-100, apo B-32 was still capable of forming lipoprotein particles as indicated by its presence in both the low density and high density lipoprotein fractions. This density distribution is unique since apo B-32 is the shortest known truncated apolipoprotein B to be found in the low density lipoprotein fraction. This finding clearly indicates that the region of apo B-32 is important in the lipid binding characteristics of apolipoprotein B-100. The binding of apo B-32 to heparin confirmed three heparin binding sites previously predicted to be in the amino-terminal 30% of apolipoprotein B-100. Isolated lipoproteins formed from apo B-32 appeared to be similar to high density lipoproteins in size and composition. However, unlike high density lipoproteins, the apo B-32 lipoproteins in plasma were partially precipitated by polyanionlcation reagents normally used to precipitate very low density and low density lipoproteins. The presence of both apolipoproteins Al and E on the apo B-32 lipoproteins suggested that apolipoprotein Al or E may mediate the metabolism of apo B-32 since apo B-32 does not posses the receptor binding region for the low density lipoprotein receptor. Four further subjects were identified as having reduced low density lipoprotein and apolipoprotein B concentrations. However a lack of any truncated apolipoprotein B in their plasma made it difficult to link their hypobetalipoproteinaemia with the apolipoprotein B gene. The cause of the hypobetalipoproteinaemia in these subjects remains uncharacterised although future linkage analysis studies in these individuals and family members will at least establish whether their hypobetalipoproteinaemia is related to the apo B gene.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Individer med homozygot familjär hyperkolesterolemi(FH), har höga halter av Low Density Lipoprotein (LDL) vilket leder till ökad risk för kardiovaskulära sjukdomar. Behandling av dessa individer kan göras med extrakorporal elimination av LDL med hjälp av specifika reningskolonner. Syftet med studien var att utvärdera några agarosmodifierade adsorbenter för denna applikation. Adsorbenterna, modifierad polyakrylat (DALI), agaros (Zetaros), direkt sulfateradZetarose och taurin immobiliserad Zetarose, inkuberades med humant plasma spädd med PBS, och en volyms förhållande mellan matris och plasman på 1:5. Inkubering utfördes i rumstemperatur under 60 min med kontinuerlig blandning i rotator. Efter inkubation centrifugerades proverna och LDL bestämdes i såväl supernatant som pellet. Totalmängd adsorberade proteiner analyserades också i eluat från erhållen pellet. LDL bestämdes indirekt med hjälp av Friedewaldsformel (LDL = totalkolesterol (TC) –highdensitylipoprotein (HDL) - (0,45 x Triglycerider(TG)). TC och TG bestämdes enzymatiskt medan HDL kvantifierades som TC efter utfällning av LDL med dextransulfat. Resultaten visar tydligt att DALI har god adsorptionsförmåga.Dock uppvisar de modifierade Zetaroserna begränsad adsorptionskapacitet för LDL. Vid desorption av adsorbenterna visar SDS en bättre elueringsförmåga än NaCl relaterad till protein, vilket tyder hydrofoba proteiner. Metodiken som används i studien är lämplig för vidare studier av andra adsorbenter som förväntas användas i kliniska applikationer för elimination av LDL hos FH patienter. / Individuals that suffer from homozygote Familiar Hyperkolesterolemia (FH), has increased amounts of Low Density Lipoproteins (LDL) which leads to a higher risk of cardiovascular diseases. Treatment of these individuals can be achieved by extracorporeal elimination of LDL using specific columns. The aim of this study was to evaluate different agarose-modified adsorbents ability to adsorb LDL from human plasma. The adsorbents (DALI, Zetarose, sulphonated Zetarose and taurine immobilized onto Zetarose) were incubated for 60 minutes with human plasma diluted with PBS, in a ratio of 1:5 between the matrix and the plasma during rotation with a rotator. After incubation the samples were centrifuged and the LDL content was determined in both the supernatant and the pellet. The amount proteins adsorbed were assayed by eluting the pellets. LDL was determined indirectly using Friedwalds equation; LDL= Total cholesterol (TC) - High density lipoprotein (HDL)-(0,45x Triglycerides (TG). The values of TC and TG in the sample were determined enzymatically, whilst HDL was quantified as TC after LDL-precipitation by dextran sulfate. The results clearly show that DALI has good adsorption capacity, but none of the modified Zetaroses shows any capacity to absorb LDL from human plasma. Desorption of the adsorbents using SDS gave higher amounts of eluated protein compared to NaCl elution, indicating hydrofobic proteins. However, the methods used in this study could be used to evaluate new adsorbents for LDL-elimination applications in patients with chronic hyperlipemia.

Les maladies cardiovasculaires constituent la première cause de décès dans le monde et l’athérosclérose est le premier facteur causal de ces maladies. Parmi les multiples facteurs de risque athéromateux, un facteur est souvent décrit :la modification des lipoprotéines de basse densité (LDLs). Bien que le phénomène d’athérogénèse ne soit pas encore complètement résolu, il est actuellement admis que les LDLs natives passent la paroi vasculaire et s’accumulent au niveau sous-endothélial où elles sont oxydées et endocytées par les macrophages. Une théorie plus récente indique que les LDLs peuvent être également modifiées dans la circulation.<p>Néanmoins, le processus par lequel ces lipoprotéines sont modifiées reste hautement controversé. Depuis quelques années, le modèle de modification des LDLs par la myéloperoxydase est apparu comme un modèle physiopathologique contrairement au modèle longuement utilisé de l’oxydation des LDLs par le cuivre. La myéloperoxydase est une enzyme présente dans les granules primaires des neutrophiles mais qui lors d’inflammations chroniques, comme dans l’athérosclérose, peut se retrouver dans le milieu extracellulaire et former un oxydant puissant qui attaque les protéines, les lipides ou les acides nucléiques. Les LDLs modifiées par la myéloperoxydase ne sont plus reconnues par le récepteur membranaire spécifique pour les LDLs. De plus, très peu d’études ont décrit à ce jour les modifications apportées par la myéloperoxydase aux LDLs.<p>Dans ce contexte, nous avons étudié la spécificité de la myéloperoxydase à modifier les LDLs. Dans ce modèle, la partie protéique de la lipoprotéine est majoritairement touchée. C’est pourquoi nous avons développé et optimisé des méthodes d’analyse par spectrométrie de masse de l’apolipoprotéine B-100, la seule protéine de la LDL. De plus, l’activité de la myéloperoxydase à la surface des LDLs a également été investiguée. <p>Les résultats de ce travail montrent que la myéloperoxydase s’attaque de manière spécifique aux LDLs et que le modèle chimique utilisant de l’acide hypochloreux pour mimer l’action de la myéloperoxydase n’est pas parfait. Enfin, nous avons également observé des changements dans l’activité enzymatique lorsque la myéloperoxydase est adsorbée à la surface des LDLs.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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This thesis describes two approaches for studying of lipoprotein metabolism in sheep. The first approach involves the assay of lipoprotein lipase (LPL) activity to determine the role of lipoprotein-triacylglycerol fatty acids in fat deposition in sheep. This enzyme is the rate limiting enzyme in the hydrolysis of fatty acids from lipoprotein-triacylglycerol. The second approach was to characterize and quantify in vivo lipoprotein metabolism using iodinated very low density lipoprotein (¹²⁵I-VLDL) and low density lipoprotein (¹³¹I-LDL). Cross-bred lambs were divided into two treatment groups and either weaned early at 5 weeks of age or remained suckling. Lambs were slaughtered at 12 or 23 weeks at which time the body composition and adipose tissue LPL activity were determined. The differences in rearing led to differences in body composition. The suckled lambs were larger and fatter than weaned lambs. The increased fatness in the suckled lambs was associated with increased LPL activity (U/mg protein) in subcutaneous adipose tissue and was reflected in higher LPL activity in post-heparin plasma (PHP) taken 2 days prior to slaughter. The role of insulin in the regulation of LPL activity was investigated by either infusing a subset of the weaned and suckled lambs with insulin for 7 or 18 weeks or using the euglycemic clamp technique to study the effect of short insulin infusions. The long term infusion of insulin had no significant effect on PHP LPL or on adipose tissue LPL (U/g tissue). However, after infusing insulin for 6h at 6.3 mU.kg⁻·⁷⁵.h⁻¹ during the euglycemic clamps, a two fold increase in LPL activity in biopsied subcutaneous adipose tissue was observed. In the second approach, in vivo lipoprotein metabolism was investigated in 4 lambs using apolipoprotein B as a marker. Following the simultaneous injection of ¹²⁵I VLDL and ¹³¹I VLDL, the specific activities of apoB in VLDL, IDL and LDL fractions were determined. ApoB specific activity curves demonstrated that VLDL is metabolised to IDL and subsequently to LDL. The turnover of VLDL-B (3.45mg.d⁻¹.kg⁻¹) and LDL-B (4.8mg.d⁻¹.kg⁻¹) was calculated by fitting the VLDL-¹²⁵I-B and LDL-¹³¹I-B specific activity data to a mono-exponential equation. The metabolism of lipoproteins, inferred from the study of apoB, was shown to be similar in sheep to that reported in other animals although the amount of lipoprotein synthesised was low. A model to describe the kinetics of apoB metabolism in sheep was developed using SAAM. The proposed model features a three pool delipidation chain for VLDL, and subsystems containing two pools for IDL and LDL. IDL may be catabolised to LDL or cleared directly from the plasma. The developed model can now be used to compare the metabolism of lipoproteins in different physiological states and to design new experiments to study lipoprotein metabolism further.

Les virus des hépatites B et C (VHB et VHC) entretiennent des liens étroits avec le métabolisme lipidique des hépatocytes. Ainsi, la réplication du VHB est dépendante de certains récepteurs nucléaires hépatiques, tels que HNF4α et PPARα, impliqués dans ce métabolisme. L’assemblage des particules virales du VHC dépend lui de la voie de synthèse des lipoprotéines de très faible densité (VLDL) et le virus circule dans le sang sous forme de lipo-viro-particules associé notamment à l’apolipoprotéine B, un composant essentiel des VLDL. Dans ce travail, nous avons d’abord étudié le rôle de FXRα, le récepteur nucléaire des sels biliaires, sur la réplication du VHB. Nous avons montré, in vitro, que les sels biliaires, via FXRα, activaient le promoteur de Core du VHB qui contrôle le niveau de réplication virale. Puis dans l’étude des liens entre les lipoprotéines et le VHC, nous avons montré que l’apoB présente sur certaines particules virales jouaient un rôle important dans l’infectiosité du virus in vitro, et que la protéine Cideb, présente en surface des gouttelettes lipidiques et impliquée dans l’assemblage des VLDL, était impliquée dans l’association du VHC avec l’apoB et influençait l’infectiosité des virions sécrétés. De plus nous avons mis en évidence l’existence de particules sub-virales chez les patients infectés, de nature lipoprotéique mais ne portant que les protéines d’enveloppes du VHC. Tous ces résultats renforcent l’idée d’une adaptation du VHB et du VHC au métabolisme lipidique hépatique. Les bénéfices éventuels qu’en retirent ces deux virus, ainsi que l’existence de possibles thérapeutiques anti-virales ciblant le métabolisme lipidique, restent à explorer / Hepatitis B and C viruses (HBV and HCV) infections are tightly linked with hepatic lipid metabolism. HBV replication depends on specific nuclear receptors, such as HNF4α and PPARα, both implicated in this metabolism. HCV assembly depends on the synthesis of Very-Low-Density Lipoproteins (VLDL), and the virus circulates in the blood as lipo-viral-particles associated in particular with apoB, an essential component of VLDL. In this study, we first studied the influence of FXRα, the nuclear receptor for bile acids, on HBV replication. We showed that, in vitro, bile acids, via FXRα, were able to activate the HBV Core promoter which controls the level of viral replication. Then, in the study of the interactions between HCV and lipoproteins, we demonstrated that apoB, which is associated with a proportion of viral particles, played an important role in HCV infectivity in vitro, and that Cideb, a protein involved in VLDL assembly, was implicated in the association between HCV and apoB and influenced the infectivity of secreted viral particles. Finally, we showed that, besides HCV infectious particles, sub-particles bearing only viral envelope glycoproteins circulated in the blood of infected patients. Interactions of HBV with the metabolism of bile acids, and of HCV with the metabolism of lipoproteins, are two examples of adaptation of a parasite to its host. The potential benefits from these interactions are still to be determined, as well as the possibility to develop anti-viral strategies targeting lipid metabolism