Lars Bo Nielsen

Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice

A deficiency in microsomal triglyceride transfer protein (MTP) causes the human lipoprotein deficiency syndrome abetalipoproteinemia. However, the role of MTP in the assembly and secretion of VLDL in the liver is not precisely understood. It is not clear, for instance, whether MTP is required to move the bulk of triglycerides into the lumen of the endoplasmic reticulum (ER) during the assembly of VLDL particles. To define MTPs role in hepatic lipoprotein assembly, we recently knocked out the mouse MTP gene (Mttp). Unfortunately, achieving our objective was thwarted by a lethal embryonic phenotype. In this study, we produced mice harboring a floxed Mttp allele and then used Cre-mediated recombination to generate liver-specific Mttp knockout mice. Inactivating the Mttp gene in the liver caused a striking reduction in VLDL triglycerides and large reductions in both VLDL/LDL and HDL cholesterol levels. The Mttp inactivation lowered apo B-100 levels in the plasma by >95% but reduced plasma apo B-48 levels by only approximately 20%. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Ultrastructural studies of wild-type mouse livers revealed numerous VLDL-sized lipid-staining particles within membrane-bound compartments of the secretory pathway (ER and Golgi apparatus) and few cytosolic lipid droplets. In contrast, VLDL-sized lipid-staining particles were not observed in MTP-deficient hepatocytes, either in the ER or in the Golgi apparatus, and there were numerous cytosolic fat droplets. We conclude that MTP is essential for transferring the bulk of triglycerides into the lumen of the ER for VLDL assembly and is required for the secretion of apo B-100 from the liver.

Transfer of low density lipoprotein into the arterial wall and risk of atherosclerosis

The aim of the review is to summarize the present knowledge on determinants of transfer of low density lipoprotein (LDL) into the arterial wall, particularly in relation to the risk of development of atherosclerosis. The flux of LDL into the arterial wall (in moles of LDL per surface area per unit of time) has two major determinants, i.e. the LDL concentration in plasma and the arterial wall permeability. LDL enters the arterial wall as intact particles by vesicular ferrying through endothelial cells and/or by passive sieving through pores in or between endothelial cells. Estimates in vivo of the LDL permeability of a normal arterial wall vary between 5 and 100 nl/cm2/h. In laboratory animals, the regional variation in the arterial wall permeability predicts the pattern of subsequent dietary induced atherosclerosis. Moreover, mechanical or immunological injury of the arterial wall increases the LDL permeability and is accompanied by accelerated development of experimental atherosclerosis. This supports the idea that an increased permeability to LDL, like an increased plasma LDL concentration, increases the risk of atherosclerosis. Hypertension, smoking, genetic predisposition, atherosclerosis, and a small size of LDL may all increase the arterial wall permeability to LDL and in this way increase the risk of accelerated development of atherosclerosis. The hypothesis that atherosclerosis risk can be reduced by improving the barrier function of the arterial wall towards the entry of LDL remains to be investigated; agents which directly modulate the LDL permeability of the arterial wall in vivo await identification.

Isolation and characterization of human apolipoprotein M-containing lipoproteins

Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; ∼5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDLapoM+) contained significantly more free cholesterol than HDL lacking apoM (HDLapoM−) (5.9 ± 0.7% vs. 3.2 ± 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDLapoM+ inhibited Cu2+-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDLapoM−. In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.

Effect of Apolipoprotein M on High Density Lipoprotein Metabolism and Atherosclerosis in Low Density Lipoprotein Receptor Knock-out Mice

To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM–/–) mice. Plasma apoM was predominantly associated with 10–12-nm α-migrating HDL particles. Human apoM overexpression (11-fold) increased plasma cholesterol concentration by 13–22%, whereas apoM deficiency decreased it by 17–21%. The size and charge of apoA-I-containing HDL in plasma were not changed in apoM-Tg or apoM–/– mice. However, in plasma incubated at 37 °C, lecithin:cholesterol acyltransferase-dependent conversion of α- to pre-α-migrating HDL was delayed in apoM-Tg mice. Moreover, lecithin: cholesterol acyltransferase-independent generation of pre-β-migrating apoA-I-containing particles in plasma was increased in apoM-Tg mice (4.2 ± 1.1%, p = 0.06) and decreased in apoM–/– mice (0.5 ± 0.3%, p = 0.03) versus controls (1.8 ± 0.05%). In the setting of low density lipoprotein receptor deficiency, apoM-Tg mice with ∼2-fold increased plasma apoM concentrations developed smaller atherosclerotic lesions than controls. The effect of apoM on atherosclerosis may be facilitated by enzymatic modulation of plasma HDL particles, increased cholesterol efflux from foam cells, and an antioxidative effect of apoM-containing HDL.

Genes for Apolipoprotein B and Microsomal Triglyceride Transfer Protein Are Expressed in the Heart Evidence That the Heart Has the Capacity to Synthesize and Secrete Lipoproteins

BACKGROUNDnExpression of both the apolipoprotein B (apoB) gene and the microsomal triglyceride transfer protein (MTP) gene is required for the assembly and secretion of triglyceride-rich lipoproteins in the liver and intestine. Both genes have been assumed to be silent in the heart.nnnMETHODS AND RESULTSnNorthern blot and RNase protection analyses showed that the apoB and MTP genes were expressed in the hearts of mice and humans. In situ hybridization studies revealed that the apoB mRNA was produced in cardiac myocytes. Electron microscopy of human cardiac myocytes revealed lipid-staining particles of relatively small diameter (approximately 250 A) within the Golgi apparatus.nnnCONCLUSIONSnThese studies strongly suggest that the heart synthesizes and secretes apoB-containing lipoproteins.

Familial Hypercholesterolemia and Atherosclerosis in Cloned Minipigs Created by DNA Transposition of a Human PCSK9 Gain-of-Function Mutant

A transgenic pig model of familial hypercholesterolemia can be used for translational atherosclerosis research. A Model of We hope to inherit our parents’ good features, like blue eyes or musical talent, but not their high cholesterol. Familial hypercholesterolemia, which is passed down in families, results in high levels of “bad” cholesterol [low-density lipoprotein (LDL)] and early onset of cardiovascular disease. To further translational research in this area, Al-Mashhadi and coauthors created a large-animal model of this genetic disease, showing that these pigs develop hypercholesterolemia and atherosclerosis much like people do. The D374Y gain-of-function mutation in the PCSK9 gene (which is conserved between pig and human) causes a severe form of hypercholesterolemia and, ultimately, atherosclerosis. Al-Mashhadi and colleagues engineered transposon-based vectors to express D374Y-PCSK9. After confirming function in human liver cancer cells, the authors cloned minipigs that expressed the mutant gene. On a low-fat diet, these pigs had higher total and LDL cholesterol than their wild-type counterparts. Breeding the male transgenic pigs with wild-type sows produced offspring that also had higher plasma LDL levels compared with normal, healthy pigs. A high-fat, high-cholesterol diet induced severe hypercholesterolemia in these animals as well as accelerated development of atherosclerosis that has human-like lesions. Other large-animal models only develop hypercholesterolemia when placed on the right diet, and small-animal models cannot recapitulate human-like pathology. The PCSK9 transgenic pigs created by Al-Mashhadi et al. develop hypercholesterolemia even on low-fat diets, and thus reflect the inherited human disease. This large-animal model will be important for better understanding the pathogenesis of familial hypercholesterolemia and for testing new therapeutics and imaging modalities before moving into human trials. Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.

Effects of high-volume plasmapheresis on ammonia, urea, and amino acids in patients with acute liver failure.

OBJECTIVE:In acute liver failure (ALF), urea production is severely impaired, and detoxification of ammonia by glutamine synthesis plays an important protective role. The aim of this study was to examine the effects of therapeutic high-volume plasmapheresis (HVP) on arterial concentrations and splanchnic exchange rates of ammonia, urea, and amino acids—in particular, glutamine.METHODS:A quantity of 8 L of plasma was exchanged over the course of 7 h in 11 patients with ALF after development of hepatic encephalopathy grade III–IV. Splanchnic exchange rates of ammonia, urea, and amino acids were measured by use of liver vein catheterization.RESULTS:HVP removed ammonia and glutamine at a rate of 1 μmol/min and 27 μmol/min, respectively. Arterial ammonia decreased from 160 ± 65 to 114 ± 50 μmol/L (p < 0.001). In contrast, arterial glutamine was only minimally changed from 1791 ± 1655 to 1764 ± 1875 μmol/L (NS). This implied that the rate of systemic glutamine synthesis was increased by 27 μmol/min. Splanchnic exchange rates (before vs after HVP) were as follows: for ammonia, −93 ± 101 versus− 70 ± 80 μmol/min (NS); urea-nitrogen, 0.08 ± 1.64 versus− 0.31 ± 0.45 mmol/min (NS); alanine, −73 ± 151 versus 12 ± 83 μmol/min (p < 0.05); and glutamine: 132 ± 246 versus 186 ± 285 μmol/min (NS), with negative values denoting release.Conclusions:Arterial ammonia decreased during HVP in patients with ALF. The data suggest that this effect of HVP could be explained by increased hepatic urea synthesis and possibly by increased glutamine synthesis in muscle tissue.

Atherogenecity of lipoprotein(a) and oxidized low density lipoprotein: insight from in vivo studies of arterial wall influx, degradation and efflux

The accumulation of atherogenic lipoproteins in the arterial intima is pathognomonic of atherosclerosis. Modification of LDL by covalent linkage of apo(a) (resulting in the formation of Lp(a)) or oxidation probably enhances its atherogenecity. Although the metabolism of LDL in arterial intima has been rather extensively characterized, little has been known about the interaction of Lp(a) and oxidized LDL (ox-LDL) with the arterial wall. The present paper reviews a series of recent in vivo studies of the interaction of Lp(a) and ox-LDL with the arterial wall. The results have identified several factors that affect the accumulation of Lp(a) and ox-LDL in the arterial intima and have provided fresh insight into unique metabolic characteristics of Lp(a) and ox-LDL that may explain the large atherogenic potential of these modified LDL species.

Human Apolipoprotein B Transgenic Mice Generated with 207- and 145-Kilobase Pair Bacterial Artificial Chromosomes EVIDENCE THAT A DISTANT 5′-ELEMENT CONFERS APPROPRIATE TRANSGENE EXPRESSION IN THE INTESTINE

We reported previously that ∼80-kilobase pair (kb) P1 bacteriophage clones spanning either the human or mouse apoB gene (clones p158 and p649, respectively) confer apoB expression in the liver of transgenic mice, but not in the intestine. We hypothesized that the absence of intestinal expression was due to the fact that these clones lacked a distant DNA element controlling intestinal expression. To test this possibility, transgenic mice were generated with 145- and 207-kb bacterial artificial chromosomes (BACs) that contained the human apoB gene and more extensive 5′- and 3′-flanking sequences. RNase protection, in situ hybridization, immunohistochemical, and genetic complementation studies revealed that the BAC transgenic mice manifested appropriate apoB gene expression in both the intestine and the liver, indicating that both BACs contained the distant intestinal element. To determine whether the regulatory element was located 5′ or 3′ to the apoB gene, transgenic mice were generated by co-microinjecting embryos with p158 and either the 5′- or 3′-sequences from the 145-kb BAC. Analysis of these mice indicated that the apoB gene’s intestinal element is located 5′ to the structural gene. Cumulatively, the transgenic mouse studies suggest that the intestinal element is located between −33 and −70 kb 5′ to the apoB gene.

The role of the kidney in lipid metabolism.

Purpose of review Cellular uptake of plasma lipids is to a large extent mediated by specific membrane-associated proteins that recognize lipid–protein complexes. In the kidney, the apical surface of proximal tubules has a high capacity for receptor-mediated uptake of filtered lipid-binding plasma proteins. We describe the renal receptor system and its role in lipid metabolism in health and disease, and discuss the general effect of the diseased kidney on lipid metabolism. Recent findings Megalin and cubilin are receptors in the proximal tubules. An accumulating number of lipid-binding and regulating proteins (e.g. albumin, apolipoprotein A-I and leptin) have been identified as ligands, suggesting that their receptors may directly take up lipids in the proximal tubules and indirectly affect plasma and tissue lipid metabolism. Recently, the amnionless protein was shown to be essential for the membrane association and trafficking of cubilin. Summary The kidney has a high capacity for uptake of lipid-binding proteins and lipid-regulating hormones via the megalin and cubilin/amnionless protein receptors. Although the glomerular filtration barrier prevents access of the large lipoprotein particles to the proximal tubules, the receptors may be exposed to lipids bound to filtered lipid-binding proteins not associated to lipoprotein particles. Renal filtration and receptor-mediated uptake of lipid-binding and lipid-regulating proteins may therefore influence overall lipid metabolism. The pathological mechanisms causing the pronounced atherosclerosis-promoting effect of uremia may involve impairment of this clearance pathway.