apoB Production Research Articles

C-reactive protein levels are inversely correlated with the apolipoprotein B-48-containing triglyceride-rich lipoprotein production rate in insulin resistant men

The pro-inflammatory state and elevated plasma levels of post-prandial triglycerides (TG) are associated with increased cardiovascular disease risk. Recent studies suggested that the increase in the production rate of post-prandial lipoproteins observed in patients with insulin resistance (IR) may be caused, at least in part, by the dysregulation of intestinal insulin sensitivity triggered by inflammation. ObjectiveThe objective of the present study was to evaluate the association between IR, plasma C-reactive protein (CRP) levels and the kinetics of TG-rich lipoprotein (TRL) containing apolipoprotein (apo) B-48 in a large sample of insulin sensitive (IS) and IR men. MethodsThe in vivo kinetics of TRL apoB-48 were measured in 151 men following a primed-constant infusion of l-[5,5,5-D3]leucine. IR subjects (n=91) were characterized by fasting TG levels ≥1.5mmol/L and an index of homeostasis model assessment of IR (HOMA-IR)≥2.5 or type 2 diabetes, while IS subjects (n=24) were characterized by an HOMA-IR index <2.5 and TG levels <1.5mmol/L. ResultsIR subjects had higher TRL apoB-48 production rate (+202%; P<0.0001) and CRP levels (+51%; P=0.01) than IS subjects. TRL apoB-48 production rate and CRP levels were inversely correlated in IR subjects (r=−0.32; P=0.002). IR subjects with CRP levels above the median (2.20mg/L) had lower TRL apoB-48 production rate than IR subjects with CRP levels below the median (Δ=−24%; P<0.05). ConclusionOur results confirm that IR is associated with increased TRL apoB-48 secretion and suggest that a higher inflammatory status is associated with decreased TRL apoB-48 secretion among IR subjects.

3,5,3'-Triiodo-L-Thyronine- and 3,5-Diiodo-L-Thyronine- Affected Metabolic Pathways in Liver of LDL Receptor Deficient Mice.

3,5,3′-triiodo-L-thyronine (T3) and 3,5-diiodo-L-thyronine (T2), when administered to a model of familial hypercholesterolemia, i.e., low density lipoprotein receptor (LDLr)-knockout (Ldlr−/−) mice fed with a Western type diet (WTD), dramatically reduce circulating total and very low-density lipoprotein/LDL cholesterol with decreased liver apolipoprotein B (ApoB) production. The aim of the study was to highlight putative molecular mechanisms to manage cholesterol levels in the absence of LDLr. A comprehensive comparative profiling of changes in expression of soluble proteins in livers from Ldlr−/− mice treated with either T3 or T2 was performed. From a total proteome of 450 liver proteins, 25 identified proteins were affected by both T2 and T3, 18 only by T3 and 9 only by T2. Using in silico analyses, an overlap was observed with 11/14 pathways common to both iodothyronines, with T2 and T3 preferentially altering sub-networks centered around hepatocyte nuclear factor 4 α (HNF4α) and peroxisome proliferator-activated receptor α (PPARα), respectively. Both T2 and T3 administration significantly reduced nuclear HNF4α protein content, while T2, but not T3, decreased the expression levels of the HNFα transcriptional coactivator PGC-1α. Lower PPARα levels were found only following T3 treatment while both T3 and T2 lowered liver X receptor α (LXRα) nuclear content. Overall, this study, although it was not meant to investigate the use of T2 and T3 as a therapeutic agent, provides novel insights into the regulation of hepatic metabolic pathways involved in T3- and T2-driven cholesterol reduction in Ldlr−/− mice.

Abstract 12151: PCSK9 Mediates Atherogenesis

Background: Proprotein convertase subtilisin kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR), resulting in increased LDL cholesterol levels. We have suggested that PCSK9 regulates apolipoprotein B (apoB) metabolism via an autophagic process that is independent of the LDLR. Furthermore, whether PCSK9 plays a causal role in atherogenesis independent of LDLR has been controversial. Here, we investigated the molecular mechanisms by which PCSK9 modulates autophagy and the LDLR-independent effects of PCSK9 on atherogenesis. Methods and Results: Deletion of the PCSK9 gene from atherosclerosis prone LDb ( Ldlr-/-Apobec1-/-) mice resulted in substantially less atherosclerosis (&gt; 4-fold), as well as a substantial reduction of plasma apoB, lipids, and triglyceride-rich lipoproteins, but increased HDL particles. The triple knockout mice had a decreased rate of apoB secretion resulting from increased autophagic flux. The deletion of PCSK9 also had profound influences on aortic endothelial cell response to exposure to LDL. LDL associated with PCSK9 increased the expression of TLR2, Lox-1, ICAM-1, CCL2, CCL7, IL-6, IL-1β, Beclin-1, p62, and TRAF6, compared to LDL without PCSK9. Conclusion: Deletion of PCSK9 markedly decreases apoB production by increasing autophagic flux. The absence of PCSK9 results in decreased lipid levels and marked reduction of atherosclerosis, independent of LDLR. The influence on atherogenesis is in part due to the direct effects of PCSK9 on endothelial cell receptors and on proinflammatory and autophagy molecules. These findings suggest that there may be clinical benefits of PCSK9 inhibition due to mechanisms unrelated to increased LDLR activity.

Intravenous Glucose Acutely Stimulates Intestinal Lipoprotein Secretion in Healthy Humans.

Increased production of intestinal triglyceride-rich lipoproteins (TRLs) contributes to dyslipidemia and increased risk of atherosclerotic cardiovascular disease in insulin resistance and type 2 diabetes. We have previously demonstrated that enteral glucose enhances lipid-stimulated intestinal lipoprotein particle secretion. Here, we assessed whether glucose delivered systemically by intravenous infusion also enhances intestinal lipoprotein particle secretion in humans. On 2 occasions, 4 to 6 weeks apart and in random order, 10 healthy men received a constant 15-hour intravenous infusion of either 20% glucose to induce hyperglycemia or normal saline as control. Production of TRL-apolipoprotein B48 (apoB48, primary outcomes) and apoB100 (secondary outcomes) was assessed during hourly liquid-mixed macronutrient formula ingestion with stable isotope enrichment and multicompartmental modeling, under pancreatic clamp conditions to limit perturbations in pancreatic hormones (insulin and glucagon) and growth hormone. Compared with saline infusion, glucose infusion induced both hyperglycemia and hyperinsulinemia, increased plasma triglyceride levels, and increased TRL-apoB48 concentration and production rate (P<0.05), without affecting TRL-apoB48 fractional catabolic rate. No significant effect of hyperglycemia on TRL-apoB100 concentration and kinetic parameters was observed. Short-term intravenous infusion of glucose stimulates intestinal lipoprotein production. Hyperglycemia may contribute to intestinal lipoprotein overproduction in type 2 diabetes. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02607839.

Selective PPAR modulators (SPPARs) may fill the need for treatment of the atherogenic dyslipidemia of insulin resistance and type 2 diabetes: Can they reduce the associated cardiac risk?

Selective PPAR modulators (SPPARs) may fill the need for treatment of the atherogenic dyslipidemia of insulin resistance and type 2 diabetes: Can they reduce the associated cardiac risk?

Lipoprotein metabolism in an apoB-80 familial hypobetalipoproteinemia heterozygote

ObjectiveFamilial hypobetalipoproteinemia (FHBL) is characterized by mutations in APOB, the majority of these causing protein truncations, and low plasma levels of apolipoprotein (apo) B. The hypobetalipoproteinemia may be due to enhanced clearance and possibly reduced production of apoB-containing lipoproteins; the mechanism may depend on the length of the apoB truncation. We studied fasting lipoprotein metabolism in an FHBL subject heterozygous for a mutation causing a truncated apoB, apoB-80. Design and MethodsVery low density lipoprotein (VLDL)-, intermediate density lipoprotein (IDL)-, and low density lipoprotein (LDL)-apoB kinetics were determined in the fasting state using stable isotope methods and compartmental modeling. ResultsCompared with lean normolipidemic controls the apoB-80 FHBL subject had an elevated VLDL-apoB fractional catabolic rate and lower LDL production. ApoB production rates and IDL- and LDL-apoB fractional catabolic rates were not different. ConclusionFHBL subjects heterozygous for a mutation truncating apoB to 80% of full-length are able to produce VLDL-apoB normally, but have rapid clearance of these particles, resulting in low levels of circulating apoB.

Novel APOB missense variants, A224T and V925L, in a black South African woman with marked hypocholesterolemia

One genetic cause of markedly low plasma concentrations of apolipoprotein (apo) B and low density lipoprotein (LDL)-cholesterol is familial hypobetalipoproteinemia. We aimed to determine the molecular basis for the marked hypocholesterolemia consistent with heterozygous familial hypobetalipoproteinemia in a black female subject of Xhosa lineage. Coding regions of APOB, MTTP, PCSK9,ANGPTL3, SAR1B and APOC3 were sequenced, and APOE was genotyped. COS-7 cells were transfected with plasmids containing apoB variants. Western blotting was used to detect cellular and secreted apoB, and co-immunoprecipitation performed to assess binding with the microsomal triglyceride transfer protein (MTP). Sequence analysis of the APOB gene revealed her to be heterozygous for two novel variants, c.751G>A (A224T) and c.2854G>C (V925L). She was also homozygous for the APOEε2 allele, and did not carry a PCSK9 loss-of-function mutation. Although Ala(224) is within the postulated MTP binding region in apoB, it is not conserved among mammalian species. Subsequent genotyping showed that Ala224Thr is found in a southern African population (n=654) with an allele frequency of 1.15% and is not associated with plasma lipid levels. Val(925), like Ala(224), is within the N-terminal 1000 amino acids required for lipoprotein assembly, but was not found in the population screen. However, in vitro studies showed that apoB V925L did not affect apoB48 production or secretion nor have a deleterious effect on MTP interaction with apoB. Taken together, this suggests that the hypocholesterolemia in our case may be a result of being homozygous for APOEε2 with a low baseline cholesterol.

Abstract 14589: Adiponectin Inhibits Hepatic Production of Triglyceride-Rich Apolipoprotein B Lipoproteins Through Both LDL-Receptor Dependent and Independent Mechanisms

Background and Rationale: Increasing plasma concentrations of the anti-inflammatory protein, adiponectin, are significantly and independently associated with a lower risk of coronary heart disease (CHD), while hypoadiponectinemia, conversely, is a strong risk factor for CHD. The mechanisms by which adiponectin mediates its cardioprotective role are not clearly understood. Here, we have elucidated a key cause of the cardioprotective effect of adiponectin, involving a direct inhibitory action on hepatic production of pro-atherogenic triglyceride-rich apolipoprotein B (apoB) lipoproteins, mediated through both LDL-receptor dependent mechanisms, involving PCSK9, and LDL-receptor independent mechanisms. Methods and Results: Cultured human hepatocytes (HepG2 cells) and primary human hepatocytes were treated with physiological concentrations of purified recombinant human adiponectin (10 μg/mL) for 24 hours. Adiponectin treatment reduced endogenous apoB cellular expression and secretion markedly by 60% and 30%, respectively, (P&lt;0.01). Adiponectin also reduced apoB production stimulated by prior treatment of cells with oleate or with pro-inflammatory obese human serum (25% reduction for both, P&lt;0.01). Adiponectin-mediated inhibition of cellular apoB production occurred through a substantial increase in levels of hepatocyte LDL-receptors (70%, P&lt;0.01), which promote proteasome-mediated apoB degradation, and which resulted from a 50% reduction in cellular PCSK9 levels (P&lt;0.01 for both). Multiple LDL-receptor independent effects of adiponectin involved mechanisms which reduce cellular apoB stability: (1) a decrease in cellular microsomal triglyceride transfer protein (MTP) (by 25%, (P&lt;0.01); (2) an increase in AMP kinase (by 40%, P&lt;0.01); and (3) marked stimulation of the Akt/Erk insulin signaling pathways. Conclusions: Adiponectin has a direct mitigating effect on hepatic apoB production through mechanisms mediated in part by the LDL-receptor and PCSK9, MTP and the insulin signaling pathway. These findings indicate that that inhibition of apoB may explain in large part adiponectin’s cardioprotective role. Furthermore, targeted adiponectin-based therapies may be a novel approach to inhibit hepatic apoB production.

Effects of extended-release niacin on the postprandial metabolism of Lp(a) and ApoB-100-containing lipoproteins in statin-treated men with type 2 diabetes mellitus.

The effects of extended-release niacin (ERN; 1-2 g/d) on the metabolism of lipoprotein(a) (Lp(a)) and apolipoprotein (apo) B-100-containing lipoproteins were investigated in 11 statin-treated white men with type 2 diabetes mellitus in a randomized, crossover trial of 12-weeks duration. The kinetics of Lp(a) and very low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) apoB-100 were determined following a standardized oral fat load (87% fat) using intravenous administration of D3-leucine, gas chromatography-mass spectrometry, and compartmental modeling. ERN significantly decreased fasting plasma total cholesterol, LDL cholesterol, and triglyceride concentrations. These effects were achieved without significant changes in body weight or insulin resistance. ERN significantly decreased plasma Lp(a) concentration (-26.5%) and the production rates of apo(a) (-41.5%) and Lp(a)-apoB-100 (-32.1%); the effect was greater in individuals with elevated Lp(a) concentration. ERN significantly decreased VLDL (-58.7%), intermediate-density lipoprotein (-33.6%), and LDL (-18.3%) apoB-100 concentrations and the corresponding production rates (VLDL, -49.8%; intermediate-density lipoprotein, -44.7%; LDL, -46.1%). The number of VLDL apoB-100 particles secreted increased in response to the oral fat load. Despite this, total VLDL apoB-100 production over the 10-hour postprandial period was significantly decreased with ERN (-21.9%). In statin-treated men with type 2 diabetes mellitus, ERN decreased plasma Lp(a) concentrations by decreasing the production of apo(a) and Lp(a)-apoB-100. ERN also decreased the concentrations of apoB-100-containing lipoproteins by decreasing VLDL production and the transport of these particles down the VLDL to LDL cascade. Our study provides further mechanistic insights into the lipid-regulating effects of ERN.

Lipoprotein Metabolism in APOB L343V Familial Hypobetalipoproteinemia.

Familial hypobetalipoproteinemia (FHBL) is a codominant disorder of lipoprotein metabolism characterized by decreased plasma concentrations of low-density lipoprotein (LDL)-cholesterol and apolipoprotein B (apoB). The objective was to examine the effect of heterozygous APOB L343V FHBL on postprandial triglyceride-rich lipoprotein (TRL) and fasting lipoprotein metabolism. Plasma incremental area under the curve apoB-48 and apoB-48 kinetics were determined after ingestion of a standardized oral fat load using compartmental modeling. Very low-density lipoprotein (VLDL)-, intermediate-density lipoprotein (IDL)-, and LDL-apoB kinetics were determined in the fasting state using stable isotope methods and compartmental modeling. The postprandial incremental area under the curve (0-10 h) in FHBL subjects (n = 3) was lower for large TRL-triglyceride (-77%; P < .0001), small TRL-cholesterol (-83%; P < .001), small TRL-triglyceride (-88%; P < .001), and for plasma triglyceride (-70%; P < .01) and apoB (-63%; P < .0001) compared with controls. Compartmental analysis showed that apoB-48 production was lower (-91%; P < .05) compared with controls. VLDL-apoB concentrations in FHBL subjects (n = 2) were lower by more than 75% compared with healthy, normolipidemic control subjects (P < .01). The VLDL-apoB fractional catabolic rate (FCR) was more than 5-fold higher in the FHBL subjects (P = .07). ApoB production rates and IDL- and LDL-apoB FCRs were not different between FHBL subjects and controls. We conclude that when compared to controls, APOB L343V FHBL heterozygotes show lower TRL production with normal postprandial TRL particle clearance. In contrast, VLDL-apoB production was normal, whereas the FCR was higher in heterozygotes compared with lean control subjects. These mechanisms account for the marked hypolipidemic state observed in these FHBL subjects.

Ceramide as a mediator of non-alcoholic Fatty liver disease and associated atherosclerosis.

Cardiovascular disease (CVD) is a serious comorbidity in nonalcoholic fatty liver disease (NAFLD). Since plasma ceramides are increased in NAFLD and sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD, the role of ceramides in dyslipidemia was assessed using LDLR-/- mice, a diet-induced model of NAFLD and atherosclerosis. Mice were fed a standard or Western diet (WD), with or without myriocin, an inhibitor of ceramide synthesis. Hepatic and plasma ceramides were profiled and lipid and lipoprotein kinetics were quantified. Hepatic and intestinal expression of genes and proteins involved in insulin, lipid and lipoprotein metabolism were also determined. WD caused hepatic oxidative stress, inflammation, apoptosis, increased hepatic long-chain ceramides associated with apoptosis (C16 and C18) and decreased very-long-chain ceramide C24 involved in insulin signaling. The plasma ratio of ApoB/ApoA1 (proteins of VLDL/LDL and HDL) was increased 2-fold due to increased ApoB production. Myriocin reduced hepatic and plasma ceramides and sphingomyelin, and decreased atherosclerosis, hepatic steatosis, fibrosis, and apoptosis without any effect on oxidative stress. These changes were associated with decreased lipogenesis, ApoB production and increased HDL turnover. Thus, modulation of ceramide synthesis may lead to the development of novel strategies for the treatment of both NAFLD and its associated atherosclerosis.

Abstract 527: Decay of Hepatic and Serum Apolipoprotein(a) Levels is Slower than ApolipoproteinB Following Inhibition of Lipid Synthesis

The association of Lipoprotein(a) (Lp(a)) levels and increased cardiovascular risk is substantiated by human epidemiology, genetics and interventional apheresis studies. Lp(a) is a plasma lipoprotein consisting of a Low Density Lipoprotein (LDL) particle with one molecule of apolipoprotein B100 (ApoB100) covalently linked to one molecule of apolipoprotein(a). Published studies show that apo(a) synthesis and secretion from hepatoma cells is coupled to triglyceride synthesis and secretion, illustrating a point of control for Lp(a) production. The objective of this study is to compare Lp(a), apo(a) and apoB production, secretion and half-life in vivo to understand mechanisms controlling circulating levels of Lp(a). Since rodent species do not express endogenous apo(a), human apo(a) transgenic mice were created and bred with human apoB100 transgenic mice to generate Lp(a)-producing mice. The circulating level of Lp(a) in the transgenic mice is ~3.7 mg/dL that is comparable to 5.4 mg/dL measured in a healthy human subject. To inhibit triglyceride and VLDL secretion in liver, mice were treated with a microsomal triglyceride transfer protein inhibitor (MTPi), and plasma was collected in a time-course. This treatment allowed measurement of plasma Lp(a), apo(a) and apoB half-lives since secretion of nascent VLDL and LDL was blocked. MTPi caused an 81% reduction in cholesterol and 68% reduction in triglycerides after 5 days of treatment. ApoB decreased significantly within 6 hours of treatment and remained low for 5 days. The calculated half-life was 4.6 hours. By contrast, apo(a) and Lp(a) decreased significantly after 3 days of treatment with half-lives of 2.4 and 2.5 days, respectively, illustrating delayed catabolism of apo(a)/Lp(a). Hepatic Lp(a), apo(a) and apoB were decreased significantly after 5 days dosing, indicating that the apolipoproteins were likely degraded and not accumulated in the liver. Treatment of stable HepG2 cells expressing apo(a) with MTPi caused similar difference in Lp(a) and apoB half-lives. These models illustrate rapid decay of apoB, but not apo(a), when lipoprotein particle assembly is blocked, and they provide methods for future mechanistic studies of apo(a) assembly into Lp(a) and its catabolism.

Abstract 380: Vascular Pcsk9: A Mediator for Atherogenesis Independent of LDL Receptor

PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases the LDL levels by binding to hepatocyte LDL receptors (LDLR) and subjects it to degradation. We show that PCSK9 regulates apolipoprotein B (apoB) production by inhibiting its degradation process via the autophagic pathway, irrespective of the presence of LDLR. In addition to the role of PCSK9 in promoting hyperlipidemia, we hypothesized that vascular-PCSK9 in endothelial cells (EC) plays a role in initiating atherogenesis, irrespective of the presence of LDL receptor. Our laboratory has generated double knockout mice lacking both LDLR and Apobec1 (apoB mRNA editing enzyme), named LDb, Ldlr-/-Apobec1-/-. They have the lipoprotein phenotype mimics human with hyperlipidemia; elevated levels of VLDL and LDL with low levels of HDL. They develop atherosclerotic lesions spontaneously. To investigate the role of PCSK9 in atherogenesis, we deleted Pcsk9 gene from LDb mice to generate the triple knockout mice (named LTp, Ldlr-/-Apobec1-/-Pcsk9-/-). In comparison to LDb mice (n=14), the LTp mice (n=8) had significantly decreased levels of cholesterol (387±10 vs. 313±14 mg/dl; p&lt;0.0008) and triglyceride (304±15 vs. 204±2.3 mg/dl; p&lt;0.0002). However, despite their high cholesterol levels at over 300 mg/dl, the atherosclerotic lesions in LTp mice were significantly decreased in comparison to LDb mice (8.8%±3.5 vs. 24%±3.3, p=0.004, n=5 vs. 5). We hypothesized that vascular PCSK9 regulates the development of atherosclerosis. We incubated LDL containing PCSK9 (LDL/PCSK9) on primary aortic endothelial cells (EC) obtained from LDb or LTp to study the effects of LDL/PCSK9 on inflammation. We show that LDL/PCSK9 could not induce the expressions of Lox-1, TLR-2, or ICAM-1 in EC from LTp, resulting in absence responses on proinflammatory markers (CCL2 and CCL7) and autophagic molecules (p62 and TRAF6). In conclusion, our results suggest that vascular PCSK9 play an essential role in atherogenesis.

Abstract 313: Perinatal Growth Restriction Decreases Hepatic RNA Editing of Apolipoprotein B in Male Rat Offspring

Introduction: Individuals born with intrauterine growth restriction develop cardiovascular disease. We employed hepatic microarray-based expression profiling of male rat offspring born to mothers subjected to combined intrauterine and postnatal calorie restriction (IPCR) as a discovery driven experiment to interrogate intrauterine growth restriction. These studies identified that the expression of Apobec1 and Apobec1 complementation factor (A1CF), genes known to edit apolipoprotein B (ApoB) mRNA through the epigenetic process of cytosine deamination, are decreased in IPCR. Editing of ApoB mRNA at position 666 changes the ribonucleotide base from a cytosine to a uracil (U) and controls the production of ApoB100 and ApoB48 lipoproteins. Hypothesis: IPCR decreases hepatic ApoB mRNA editing in male offspring through transcriptional regulation of Apobec1 and A1CF. Methods: Pyrosequencing designed to interrogate ApoB mRNA editing was used to quantify hepatic ApoB100 and ApoB48 transcripts in an experiment employing the male offspring (litters culled to 6 male offspring for each mother) from 4 control fed mothers and 4 mothers subjected to 50% calorie restriction from embryonic day 11 to postnatal day 21 (IPCR). RT-qPCR and western blotting were employed to quantify the hepatic expression of Apobec1 and A1CF. Serum ApoB100 and Apo48 were quantified by ELISA. Results: ApoB transcripts in the control fed offspring were edited, with a U base call of 55 ± 2% at position 666, whereas IPCR offspring possessed decreased editing with a U base call of 12 ± 1% (n=12 for each group; F=368; p&lt;0.0001). The RT-qPCR relative quotient (RQ) in the IPCR group was 0.2 ± 0.1 (F=107;p&lt;0.0001) and 0.4 ± 0.1 (F=25;p&lt;0.0001) for Apobec1 and A1CF, respectively (RQ=1 in the control fed offspring for both Apobec1 and A1CF). Relative to control, IPCR treatment significantly decreased hepatic Apobec1 and A1CF proteins by 45% ± 10 and 27 % ± 9.2 respectively. IPCR treatment increased serum ApoB100 by 1.5 fold ± 0.43 (p= 0.051) and significantly decreased ApoB48 by 0.8 fold ± 0.1 (n=6) compared to control. Conclusion: These data define a novel association of IPCR and ApoB editing through a nutritionally controlled epigenetic process working through transcriptional control of Apobec1 and A1CF.

Abstract 521: Impact of Insulin-resistance, Body Mass Index and Dietary Fat Intakes on Apolipoprotein B-48 Kinetic

Substantial evidence exists indicating that elevated plasma levels of apolipoprotein (apo) B-48-containing lipoproteins are associated with increased cardiovascular disease risk. Overaccumulation of apoB-48-containing lipoproteins of intestinal origin observed in patients with insulin-resistance (IR) is thought to be attributable to both elevated intestinal production and reduced clearance of these lipoproteins. The objective of this study was to assess the relative contribution of IR, anthropometry, and dietary fat content on triglyceride (TG)-rich lipoprotein (TRL) apoB-48 kinetic. The in vivo kinetic of TRL apoB-48 was measured in 131 men with primed-constant infusion of L-[5,5,5-D3]leucine. TRL apoB-48 production rate (PR) and fractional catabolism rate (FCR) were determined by multi-compartmental modeling, while TRL apoB-48 pool size (PS) was measured by ELISA. IR was defined by TG levels &gt; 1.5 mmol/L and Homeostasis Model Assessment of IR (HOMA-IR) &gt; 2.5. Age was inversely associated with TRL apoB-48 FCR (ρ=-0.21; P=0.02), while BMI was positively correlated with TRL apoB-48 PR (ρ=0.33; P=0.0001) and PS (ρ=0.44; P&lt;0.0001). Fat intake was associated with TRL apoB-48 PR (ρ=0.34; P&lt;0.0001) and PS (ρ=0.34; P&lt;0.0001). HOMA-IR was positively associated with TRL apoB-48 PR (ρ=0.26; P=0.005) and PS (ρ=0.35; P=0.0001). Furthermore, when adjusted for fat intake, TRL apoB-48 PR was significantly higher in IR subjects (n=73) than in insulin-sensitive subjects (Δ=+64%; P&lt;0.0001), and TRL apoB-48 FCR was lower (Δ=-13%; P=0.005). Multiple linear regression analysis showed that IR was the most significant constitutive predictor of TRL apo B-48 PR variance (6.6%; P=0.01), while age and BMI were not significantly associated with PR. In conclusion, these results suggest that IR is a major factor contributing to TRL apoB-48 overaccumulation, possibly through increased production and decreased clearance, independent of age, BMI and fat intake.

Abstract 125: Small Dense LDL ApoB Is Catabolized More Slowly Than Large LDL ApoB in Subjects With Combined Hyperlipidemia, and Rosuvastatin Enhances Its Clearance

LDL consists of particles varying in size, density, and composition. Small dense LDL (sdLDL) cholesterol (C) has been shown to be a better predictor of coronary heart disease risk than total LDL-C. Intensive statin therapy significantly lowers sdLDL-C as well as large buoyant LDL (lbLDL)-C. We tested the hypotheses that lbLDL and sdLDL apoB100 have different catabolic rates, and that statin therapy enhances the fractional catabolism of apoB100 in both particles. Six subjects (3 men and 3 women; age 63±5 years [mean±SEM]; BMI 25.5±1.5 kg/m 2 ) with combined hyperlipidemia (triglycerides [TG] 191±50 mg/dl; LDL-C 160±16 mg/dl; HDL-C 43±6 mg/dl) received placebo and rosuvastatin 40 mg/day for 8 weeks each in sequential order. At the end of each phase, the kinetics of apoB100 in triglyceride rich lipoproteins ([TRL] d &lt;1.019 g/ml), lbLDL ( d =1.019-1.044 g/ml), and sdLDL ( d =1.044-1.063 g/ml) were determined using stable isotope methodology, gas chromatography-mass spectrometry, and multicompartmental modeling. Compared to placebo, rosuvastatin markedly decreased the plasma levels of LDL-C (52%; P =0.0002), TG (32%; P =0.06), and total apoB (42%; P &lt;0.0001). ApoB100 pool size in TRL, lbLDL, and sdLDL decreased 32±6%, 39±5%, 41±4%, respectively. These changes were attributable to significant ( P &lt;0.05) increases in apoB100 fractional catabolic rates in TRL (45±16%), lbLDL (131±66%), and sdLDL (97±32%). During both the placebo and rosuvastatin phases, sdLDL apoB100 was catabolized more slowly than lbLDL apoB100 ( P =0.01 and 0.004, respectively). Rosuvastatin did not alter apoB100 production rate or the percent of TRL apoB100 converted to each LDL subfraction. Proteomic analysis of the LDL particles indicated that rosuvastatin increased apoD abundance in lbLDL and decreased apoAIV abundance in sdLDL. No significant effects on other apolipoproteins were noted. These data indicate that sdLDL apoB100 is catabolized significantly more slowly than lbLDL apoB100 in dyslipidemic subjects on placebo or statin therapy. Maximal dose rosuvastatin decreases plasma apoB concentrations by enhancing apoB100 catabolism in TRL, lbLDL, and sdLDL and alters the distribution of minor apolipoproteins in LDL subfractions.

Abstract 129: Effects of a Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitor, Alirocumab, on Lipid and Lipoprotein Metabolism in Normal Subjects

Background: Proprotein convertase subtilisin/kexin type (PCSK9) inhibitors are promising new drugs for the treatment of hypercholesterolemia. They inhibit the binding of PCSK9 to the low density lipoprotein (LDL) receptor that, in turn, decreases lysosomal degradation of LDL receptors and increases their numbers on the cell surface. In Phase 2/3 studies, alirocumab significantly lowered plasma levels of LDL-cholesterol (C) and apolipoprotein B (apoB). The mechanism underlying the LDL-C lowering effects of PCSK9 inhibition has not been reported. Method: We enrolled 10 healthy volunteers (4 male, 6 female), into a Phase 1, placebo-controlled, single-blind, single-sequence study to examine the effects of alirocumab, 150 mg administered subcutaneously every two weeks, on lipid and lipoproteins levels and the metabolism of apoB in very low density (VLDL), intermediate density (IDL) and LDL. Subjects received 2 doses of placebo followed by 5 doses of alirocumab. At the end of each treatment period, fasting lipids and lipoprotein levels were measured, and stable isotope studies of the apoB turnover in VLDL, IDL and LDL were performed. Results: Alirocumab significantly reduced plasma levels of total-C by 37% (178.4±34 to 112.7±29 mg/dL), LDL-C by 59% (110.2±25 to 45.5±26 mg/dL), and apoB by 51% (93.6±25 to 45.5±13 mg/dL) compared to placebo. Plasma triglycerides (TG) and HDL-C did not change. Levels of LDL-C, LDL-TG, and LDL-apoB fell by 55.8±10%, 33.9±13%, and 56.0±11%, respectively (all p&lt;0.0001), on alirocumab. The reductions in LDL apoB were explained by a dramatic increase in the fractional clearance rate (FCR) of LDL apoB from 0.50±0.18 on placebo to 1.02±0.35 pools/day on alirocumab (p&lt;0.001) and a trend toward lower LDL apoB production rates on alirocumab (15.1±4.6 vs 12.9±3.3 mg/kg/day; p=0.10). Levels of IDL-C, IDL-TG and IDL-apoB were also reduced significantly and there was a trend toward an increase in IDL apoB FCR on alirocumab (placebo: 9.2±4 vs alirocumab: 10.8±3 pools/day; p=0.06). Additional kinetic parameters will be presented at the meeting. Summary: Alirocumab treatment significantly reduced the levels of IDL and LDL, and these changes were due to increases in the FCRs of these lipoproteins, particularly LDL.

Simultaneous Quantitation of Lipid and Apo‐Protein Synthesis: Diet‐Mediated Reduction in De Novo Lipogenesis is Associated with a Stimulation of ApoB and A1 Production In Vivo

We recently demonstrated the ability to simultaneously quantify lipid and protein flux in vivo using oral D2O dosing. The uniqueness of using D2O is the rapid equilibration with total body water, use of oral and not intravenous dosing, and ease to measure the true precursor enrichment (either plasma, urine, or saliva). To examine whether dietary modulation of de novo fatty acid and cholesterol synthesis would lead to parallel changes in apoB and/or A1 production, non‐human primates were either fed a standard carbohydrate‐based diet or a high‐fat, high‐cholesterol diet for several months. Lipid and protein kinetics were studied by giving a single oral bolus of D2O and sampling blood for ~ 96 hours thereafter. The dietary perturbation altered the plasma lipid profiles. The analyses of lipid labeling (measured via GC‐IRMS) demonstrated a marked suppression of fatty acid and cholesterol synthesis with high‐fat feeding. Despite these changes we observed a substantial increase in both apoB and A1 synthesis (measured via LC‐MS). In total, we demonstrate that diet‐induced modulation of pathways involving de novo lipid synthesis do not necessarily translate into a comparable protein‐flux phenotype. Our data suggest that the dietary lipid, as compared to de novo synthesized lipid, has a pronounced effect on circulating apo‐protein flux. The methodology applied here can be readily adapted for studies in other pre‐clinical models (e.g. rodents) as well as applied in humans. This protocol design provides a translational biomarker approach for interrogating modulations that affect dyslipidemia.

Insulin Resistance Causes Posttranslational Downregulation of Hepatic Sortilin 1 in Diabetic Mice

Dyslipidemia increases the risk of cardiovascular diseases in diabetes. Hepatic Sortilin 1 decreases hepatic apoB production, and its liver expression is associated with plasma LDL cholesterol and cardiovascular disease risk in humans. The goal of this study is to investigate the insulin regulation of hepatic Sortilin 1 in diabetes. Insulin treatment rapidly induced Sortilin 1 protein, but not mRNA, in AML12 cells. A PI3K inhibitor or an AKT inhibitor treatment rapidly reduced, while overexpression of a constitutively active AKT induced Sortilin 1 protein in AML12 cells. LC-MS/MS analysis showed that Sortilin 1 was phosphorylated at serine 825. Previous studies have shown that phosphorylation of S825 by casein kinase II promotes the cellular trafficking function of Sortilin 1 via enhancing its interaction with other trafficking adaptor proteins. Interestingly, abolishing S825 phosphorylation by S825A mutation or by a casein kinase 2 inhibitor prevented insulin induction of Sortilin1 in AML12 cells. High fat diet-fed obese mice and streptozocin-treated mice showed decreased hepatic Sortilin 1 protein. Treating mice with a PI3K inhibitor rapidly decreased hepatic Sortilin 1 protein and increased plasma LDL-cholesterol and triglyceride levels. Furthermore, over-expression of Sortilin 1 in mouse livers with adenovirus-mediated gene delivery prevented PI3K inhibitor inhibition of hepatic Sortilin 1. This prevented the PI3K inhibitor-induced elevation of plasma triglyceride, but not cholesterol in mice, suggesting that Sortilin 1-independent mechanisms also mediate the cholesterol-raising effect of PI3K inhibitor. In summary, insulin regulation of Sortilin 1 may be a novel link between insulin resistance and increased hepatic apoB production in insulin resistance.

Estrogen enhances secretion of apolipoprotein B-100 containing lipoproteins by BeWo cells.

Although the early human embryo is capable of covering its cholesterol demand by endogenous synthesis, during later stages of development the fetus may become dependent on transplacental cholesterol transport. On one hand, this conclusion is based on the severe developmental abnormalities of embryos with mutations in the gene specifying the enzyme catalyzing the last step of cholesterol synthesis, 7-dehydrocholesterol reductase, causing Smith-Lemli-Opitz Syndrome. On the other hand, increased total maternal plasma cholesterol levels may reflect the requirement by the growing fetus and/or the placenta for cholesterol. Various molecules and complexes must cross the placental barrier consisting of trophoblasts and fetal endothelial cells to reach the fetal circulation. The de novo synthesis of apolipoprotein B (apoB)-containing lipoproteins coupled to secretion from trophoblasts towards the fetal side is one efficient pathway for cholesterol supply. ApoB and the microsomal triglyceride transfer protein (MTP) are essential components for the assembly of apoB-containing lipoproteins. The aim of this study was to evaluate functional properties of the human placental cell line BeWo as an invitro model for placental synthesis of apoB-containing lipoproteins by focusing on components required for lipoprotein assembly and secretion. We demonstrate mRNA and protein production of apoB-100, MTP, and protein disulfide isomerase (PDI) in BeWo cells. In addition, metabolic radiolabeling and apoB-immunoprecipitation of cell extracts and media revealed that synthesis and secretion of apoB-containing lipoproteins are enhanced by estrogen. The expression of apoB-100, MTP, and PDI, and the estrogen-stimulated lipoprotein secretion by BeWo cells suggest that these cells are a useful system to study aspects of lipoprotein metabolism at the placental barrier.