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How the kidney prevents urinary excretion of plasma proteins continues to be debated. Here, using unfixed whole-mount mouse kidneys, we show that fluorescent-tagged proteins and neutral dextrans permeate into the glomerular basement membrane (GBM), in general agreement with Ogston's 1958 equation describing how permeation into gels is related to molecular size. Electron-microscopic analyses of kidneys fixed seconds to hours after injecting gold-tagged albumin, negatively charged gold nanoparticles, and stable oligoclusters of gold nanoparticles show that permeation into the lamina densa of the GBM is size-sensitive. Nanoparticles comparable in size with IgG dimers do not permeate into it. IgG monomer-sized particles permeate to some extent. Albumin-sized particles permeate extensively into the lamina densa. Particles traversing the lamina densa tend to accumulate upstream of the podocyte glycocalyx that spans the slit, but none are observed upstream of the slit diaphragm. At low concentrations, ovalbumin-sized nanoparticles reach the primary filtrate, are captured by proximal tubule cells, and are endocytosed. At higher concentrations, tubular capture is saturated, and they reach the urine. In mouse models of Pierson's or Alport's proteinuric syndromes resulting from defects in GBM structural proteins (laminin β2 or collagen α3 IV), the GBM is irregularly swollen, the lamina densa is absent, and permeation is increased. Our observations indicate that size-dependent permeation into the lamina densa of the GBM and the podocyte glycocalyx, together with saturable tubular capture, determines which macromolecules reach the urine without the need to invoke direct size selection by the slit diaphragm.

OBJECTIVE We investigated the differential roles of apolipoprotein E (apoE) isoforms in modulating diabetic dyslipidemia—a potential cause of the increased cardiovascular disease risk of patients with diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced using streptozotocin (STZ) in human apoE3 (E3) or human apoE4 (E4) mice deficient in the LDL receptor (LDLR−/−). RESULTS Diabetic E3LDLR−/− and E4LDLR−/− mice have indistinguishable levels of plasma glucose and insulin. Despite this, diabetes increased VLDL triglycerides and LDL cholesterol in E4LDLR−/− mice twice as much as in E3LDLR−/− mice. Diabetic E4LDLR−/− mice had similar lipoprotein fractional catabolic rates compared with diabetic E3LDLR−/− mice but had larger hepatic fat stores and increased VLDL secretion. Diabetic E4LDLR−/− mice demonstrated a decreased reliance on lipid as an energy source based on indirect calorimetry. Lower phosphorylated acetyl-CoA carboxylase content and higher gene expression of fatty acid synthase in the liver indicated reduced fatty acid oxidation and increased fatty acid synthesis. E4LDLR−/− primary hepatocytes cultured in high glucose accumulated more intracellular lipid than E3LDLR−/− hepatocytes concomitant with a 60% reduction in fatty acid oxidation. Finally, the exaggerated dyslipidemia in diabetic E4LDLR−/− mice was accompanied by a dramatic increase in atherosclerosis. CONCLUSIONS ApoE4 causes severe dyslipidemia and atherosclerosis independent of its interaction with LDLR in a model of STZ-induced diabetes. ApoE4-expressing livers have reduced fatty acid oxidation, which contributes to the accumulation of tissue and plasma lipids.

Given the multiple differences between mice and men, it was once thought that mice could not be used to model atherosclerosis, principally a human disease. Apolipoprotein E-deficient (apoEKO) mice have convincingly changed this view, and the ability to model human-like plaques in these mice has provided scientists a platform to study multiple facets of atherogenesis and to explore potential therapeutic interventions. In addition to its well-established role in lipoprotein metabolism, recent observations of reduced adiposity and improved glucose homeostasis in apoEKO mice suggest that apoE may also play a key role in energy metabolism in peripheral organs, including adipose tissue. Finally, along with apoEKO mice, knockin mice expressing human apoE isoforms in place of endogenous mouse apoE have provided insights into how quantitative and qualitative genetic alterations interact with the environment in the pathogenesis of complex human diseases.

Objective— To examine the effects of apoB100 structure, specifically a mutation in the LDLr binding region, on the production of LDL and development of atherosclerosis in vivo. Methods and Results— Ldlr −/− Apobec1 −/− mice lacking the LDLR and apoB editing enzyme accumulated LDL in plasma and developed severe atherosclerosis when they had wild-type apoB100. In marked contrast, in Ldlr −/− Apobec1 −/− mice carrying the Apob100-β mutation, in the 2 putative LDLR-binding domains of apoB prevented both LDL accumulation and atherosclerosis. Intestinal absorption of lipids and triglyceride secretion from the liver were not affected. However, the VLDL particles with apoB100-β were larger in volume by about 70%, and carried approximately four times as much apoE per particle. ApoB100-β synthesis rate in the primary hepatocytes was normal, but its intracellular degradation was enhanced. Additionally, mutant apoB100 VLDL cleared from the circulation more quickly in vivo through apoE-LRP-mediated mechanism than VLDL with wild-type apoB100. In contrast, uptake of the 2 VLDL by macrophages were not different. Conclusion— While conformational change to apoB100 during conversion of VLDL to LDL exposes LDLR binding domains and facilitates LDLR-mediated lipoprotein clearance, it may also inhibit LRP-mediated VLDL uptake and contribute to LDL accumulation in familial hypercholesterolemia.

Objective— In humans, apolipoprotein (apo) E4 is associated with elevated plasma cholesterol levels and a high risk of developing atherosclerosis, whereas apoE2 is protective. Here we investigate the mechanism by which mice expressing human apoE isoforms recapitulate this association when they also express high levels of human low-density lipoprotein receptor (LDLR). Methods and Results— Primary hepatocytes from apoE4 mice secreted less apoE into the medium than hepatocytes from apoE2 mice. Increased LDLR expression decreased this secretion and increased degradation of apoE4. An apoE4-GFP fusion protein expressed in the liver of apoE-deficient mice accumulated on the hepatocyte surface bordering the space of Disse in an LDLR-dependent manner. Fluorescence-labeled very low–density lipoprotein (VLDL) remnants accumulated on the hepatocyte surface in apoE4 mice with high LDLR, but they were internalized poorly. In contrast, apoE2-GFP did not accumulate on the hepatocyte surface even when the LDLR expression was high, but apoE2 mice with high LDLR internalized the remnants avidly without sequestering them on the hepatocyte surface. Conclusions— The high affinity of apoE4 to the LDLR enhances VLDL sequestration on the hepatocyte surface but delays their internalization. This delay likely increases VLDL conversion to cholesterol-enriched remnants in apoE4 mice with high LDLR, and probably to LDL in humans with apoE4.

Air pollution is a complex mixture of gases (e.g., ozone), particulate matter, and organic compounds present in outdoor and indoor air. Dogs exposed to severe air pollution exhibit chronic inflammation and acceleration of Alzheimer's-like pathology, suggesting that the brain is adversely affected by pollutants. We investigated whether residency in cities with high levels of air pollution is associated with human brain inflammation. Expression of cyclooxygenase-2 (COX2), an inflammatory mediator, and accumulation of the 42-amino acid form of β-amyloid (A β42), a cause of neuronal dysfunction, were measured in autopsy brain tissues of cognitively and neurologically intact lifelong residents of cities having low (n:9) or high (n:10) levels of air pollution. Genomic DNA apurinic/apyrimidinic sites, nuclear factor- κB activation and apolipoprotein E genotype were also evaluated. Residents of cities with severe air pollution had significantly higher COX2 expression in frontal cortex and hippocampus and greater neuronal and astrocytic accumulation of A β42 compared to residents in low air pollution cities. Increased COX2 expression and A β42 accumulation were also observed in the olfactory bulb. These findings suggest that exposure to severe airpollution is associated with brain inflammation and A β 42 accumulation, two causes of neuronal dysfunction that precede the appearance of neuritic plaques and neurofibrillary tangles, hallmarks of Alzheimer's disease.

Low density lipoprotein receptor (LDLr) is critical for plasma lipoprotein clearance and a decreased LDLr expression has been thought to explain the increased plasma cholesterol and atherosclerosis risk in humans with apolipoprotein (apo) E4. However, in mice expressing human apoE4 instead of their mouse counterpart, increased LDLr expression caused accumulation of cholesterol-rich apoE-poor chylomicron remnants, and resulted in significant atherosclerosis. The production rates of chylomicrons and very low density lipoproteins (VLDL) were unchanged, as was the clearance rate of exogenously given apoE-deficient remnants. These data suggest an enhanced conversion of nascent lipoprotein particles to cholesterol-rich remnants that are poorly cleared. We hypothesize that the negative effects from increased LDLr are due to the high binding affinity of apoE4 for the receptor which reduces the availability of apoE4 for transfer to triglyceride-rich lipoproteins (TRL), thereby inhibiting their rapid internalization.

Reducing dilute aqueous HAuCl4 with NaSCN under alkaline conditions produces 2-3 nm diameter yellow nanoparticles without the addition of extraneous capping agents. We here describe two very simple methods for producing highly stable oligomeric grape-like clusters (oligoclusters) of these small nanoparticles. The oligoclusters have well-controlled diameters ranging from ∼5 to ∼30 nm, depending mainly on the number of subunits in the cluster. Our first ["delay-time"] method controls the size of the oligoclusters by varying from seconds to hours the delay time between making the HAuCl4 alkaline and adding the reducing agent, NaSCN. Our second ["add-on"] method controls size by using yellow nanoparticles as seeds onto which varying amounts of gold derived from "hydroxylated gold", Na(+)[Au(OH4-x)Clx](-), are added-on catalytically in the presence of NaSCN. Possible reaction mechanisms and a simple kinetic model fitting the data are discussed. The crude oligocluster preparations have narrow size distributions, and for most purposes do not require fractionation. The oligoclusters do not aggregate after ∼300-fold centrifugal-filter concentration, and at this high concentration are easily derivatized with a variety of thiol-containing reagents. This allows rare or expensive derivatizing reagents to be used economically. Unlike conventional glutathione-capped nanoparticles of comparable gold content, large oligoclusters derivatized with glutathione do not aggregate at high concentrations in phosphate-buffered saline (PBS) or in the circulation when injected into mice. Mice receiving them intravenously show no visible signs of distress. Their sizes can be made small enough to allow their excretion in the urine or large enough to prevent them from crossing capillary basement membranes. They are directly visible in electron micrographs without enhancement, and can model the biological fate of protein-like macromolecules with controlled sizes and charges. The ease of derivatizing the oligoclusters makes them potentially useful for presenting pharmacological agents to different tissues while controlling escape of the reagents from the circulation.

We have generated mice expressing the human apo E4 isoform in place of the endogenous murine apo E protein and have compared them with mice expressing the human apo E3 isoform. Plasma lipid and apolipoprotein levels in the mice expressing only the apo E4 isoform (4/4) did not differ significantly from those in mice with the apo E3 isoform (3/3) on chow and were equally elevated in response to increased lipid and cholesterol in their diet. However, on all diets tested, the 4/4 mice had approximately twice the amount of cholesterol, apo E, and apo B-48 in their VLDL as did 3/3 mice. The 4/4 VLDL competed with human LDL for binding to the human LDL receptor slightly better than 3/3 VLDL, but the VLDL clearance rate in 4/4 mice was half that in 3/3 mice. On an atherogenic diet, there was a trend toward greater atherosclerotic plaque size in 4/4 mice compared with 3/3 mice. These data, together with our earlier observations in wild-type and human APOE*2-replacement mice, demonstrate a direct and highly significant correlation between VLDL clearance rate and mean atherosclerotic plaque size. Therefore, differences solely in apo E protein structure are sufficient to cause alterations in VLDL residence time and atherosclerosis risk in mice.

Thiazolidinediones (TZDs) are insulin sensitizers used to treat patients with insulin resistance. To assess potential gene-drug interactions, mice expressing human apolipoprotein E3 or E4 (APOE3 or APOE4) were fed a Western-type high-fat diet for 12 wk, at which time they developed similarly impaired glucose tolerance. Supplementing the diet with rosiglitazone (1.5 mg/g body weight) for an additional 4 wk improved plasma lipid profiles in both APOE3 and APOE4 mice. However, glucose tolerance improved only in APOE3 mice. Induction of adipogenesis and lipogenesis was severely blunted in adipose tissues, but not in the livers, of APOE4 mice. Consequently, lipids were channeled to the liver, causing marked steatosis in these mice. Impaired glucose tolerance was not a prerequisite for this adverse effect, and long-term treatment with rosiglitazone altered liver enzymes and caused hepatic fibrosis in APOE4 mice. Finally, TZDs failed to stimulate PPARγ activation and adipocyte differentiation in preadipocytes and embryonic fibroblasts isolated from APOE4 mice compared to those from APOE3 mice. We conclude that the effects of TZDs are APOE isoform dependent, and that the metabolic damage observed in APOE4 mice is rooted in an impaired activation of the adipogenic program in the adipose tissues expressing APOE4.—Arbones-Mainar, J. M., Johnson, L. A., Altenburg, M. K., Kim, H.-S., Maeda, N. Impaired adipogenic response to thiazolidinediones in mice expressing human apolipoproteinE4.

Apolipoprotein E (apoE), a key protein in lipid metabolism, is highly expressed in adipose tissues. Studies have shown that human APOE*4 is associated with a lower body mass index but with a greater risk of coronary heart disease compared with other APOE alleles. To define the isoform-specific role of apoE in regulating the expandability and functionality of adipose tissues, we investigated the effects of diet-induced obesity in mice whose endogenous Apoe gene has been replaced by either the human APOE*3 or APOE*4 allele.After 8 weeks on a Western-type high-fat diet, male APOE4 mice displayed impaired tolerance to glucose and fat overload compared with APOE3 mice. Subcutaneous fat tissues in APOE4 and APOE3 mice after high fat feeding were not different. In contrast, although epididymal fat tissues in APOE4 mice gained 30% less weight during the high fat feeding than in APOE3 mice, they showed impaired insulin-stimulated glucose uptake ex vivo. Epididymal APOE4 adipocytes were larger in size than APOE3 adipocytes, and expressed reduced levels of mRNA for peroxisome proliferator-activated receptor gamma2 and adiponectin, important markers of adipocyte functionality. Adenoviral expression of apoE3 in apoE-null culture adipocytes induced adiponectin mRNA in a dose-dependent manner, but the induction was significantly blunted in cells overexpressing apoE4. However, in contrast to the apoE3-expressing cells, Glut1, but not Glut4, expression levels were positively correlated with increased apoE4 mRNA, suggesting that apoE4 expression in adipocyte interferes in insulin-sensing pathways.Dysfunctional epididymal adipose tissues contribute to the accelerated impairment of glucose tolerance in APOE4 mice fed a Western-type diet. Our results underscore the importance of functionality of individual fat depots rather than total fat mass as a determinant for metabolic disturbance during diet-induced obesity.

Factors that regulate apolipoprotein E (apoE) secretion by macrophages will have important effects on vessel wall lipid flux and atherosclerosis. Macrophages express the LDL receptor, which binds apoE with high affinity and could thereby affect the net secretion of apoE from macrophages. In these studies, we demonstrate that treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA with simvastatin, to increase LDL receptor activity, reduces the secretion of apoE. To further examine the relationship between LDL receptor expression and apoE secretion from macrophages, mouse peritoneal macrophages (MPMs) were isolated from mice with constitutively high expression of human LDL receptor to increase overall LDL receptor expression by 2- to 3-fold. Cells with increased LDL receptor expression also showed reduced apoE secretion compared with MPMs with basal LDL receptor expression. The effect of changes in LDL receptor expression on apoE secretion was isoform-specific, with greater reduction of apoE4 compared with apoE3 secretion and no reduction of apoE2 secretion, paralleling the known affinity of each isoform for LDL receptor binding. The effect of the LDL receptor on apoE secretion for each isoform was further reflected in LDL receptor-dependent changes in apoE-mediated cholesterol efflux. These results establish a regulatory interaction between two branches of macrophage sterol homeostatic pathways that could facilitate a rapid response to changes in macrophage sterol content relative to need.

Background Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion. Methods In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia. Results Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice. Conclusion These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile.

Differences in affinity of human apolipoprotein E (apoE) isoforms for the low density lipoprotein receptor (LDLR) are thought to result in the differences in lipid metabolism observed in humans with different APOE genotypes. Mice expressing three common human apoE isoforms, E2, E3, and E4, in place of endogenous mouse apoE were used to investigate the relative roles of apoE isoforms in LDLR- and non-LDLR-mediated very low density lipoprotein (VLDL) clearance. While both VLDL particles isolated from mice expressing apoE3 and apoE4 bound to mouse LDLR with affinity and Bmax similar to VLDL containing mouse apoE, VLDL with apoE2 bound with only half the Bmax. In the absence of the LDLR, all lines of mice expressing human apoE showed dramatic increases in VLDL cholesterol and triglycerides (TG) compared to LDLR knockout mice expressing mouse apoE. The mechanism of the hyperlipidemia in mice expressing human apoE isoforms is due to impairment of non-LDL-receptor-mediated VLDL clearance. This results in the severe atherosclerosis observed in mice expressing human apoE but lacking the LDLR, even when fed normal chow diet. Our data show that defects in LDLR independent pathway(s) are a potential factor that trigger hyperlipoproteinemia when the LDLR pathway is perturbed, as in E2/2 mice.

The primary receptor mediating clearance of apolipoprotein (apo)E- and apoB100-containing lipoproteins from the circulation is the low density lipoprotein (LDL) receptor. Reduced expression of the LDLR is believed to be a precipitating factor in the pathogenesis of type III hyperlipoproteinemia (HLP) in some humans homozygous for the apoE2 allele (APOE*2). To test the effect of genetic changes in LDL receptor expression on the pathogenesis of type III HLP, we have generated a variant allele at the endogenous mouse Ldlr locus that expresses the human LDL receptor transcript. Transcription of the human LDLR minigene is regulated by the endogenous mouse promoter sequence, but a truncation of 3'-untranslated region results in increased mRNA stability. Consequently, in liver of heterozygotes, steady state levels of mouse and human LDLR transcripts are 50 and 180% the levels of total transcript in wild type mice, respectively. Overall, the 2.3-fold normal level of LDLR message in heterozygotes completely ameliorates type III HLP caused by the homozygosity for the human APOE*2 allele, normalizing their plasma lipoprotein profile. We conclude that a modest increase in expression of the LDLR through message stabilization is sufficient to prevent precipitation of type III HLP in mice.