Nutritional regulation of adipose tissue apolipoprotein E expression

Apolipoprotein E (apoE) is a multifunctional protein that is highly expressed in human and murine adipose tissue. Endogenous adipocyte apoE expression influences adipocyte triglyceride turnover and modulates the expression of genes involved in lipid synthesis and oxidation. We now demonstrate the regulation of adipose tissue apoE expression by nutritional status in lean and obese mice. Obesity induced by high-fat diet, or by hyperphagia in ob/ob mice, produces significant reduction of adipose tissue apoE expression at the protein and messenger RNA level. Fasting in C57BL/6J mice for 24 h significantly increased apoE protein and messenger RNA levels. In ob/ob mice, transplantation of adipose tissue from lean littermate controls to restore circulating leptin levels produced significant weight loss over 12 wk and also produced an increase in adipose tissue apoE expression. The increase in adipose tissue apoE expression in this model, however, did not require leptin. Adipose tissue apoE was also significantly increased in ob/ob mice after a 48-h fast or after 7 days of caloric restriction. In summary, obesity suppresses adipose tissue apoE expression, whereas fasting or weight loss increases it. From our previous observations, these changes in adipose tissue apoE expression will have significant impact on adipose tissue lipid flux and lipoprotein metabolism. Furthermore, these results suggest adipose tissue apoE participates in defending adipose tissue and organismal energy homeostasis in response to nutritional perturbation.     

Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

Elimination of macrophage-specific apolipoprotein E reduces diet-induced atherosclerosis in C57BL/6J male mice

Apolipoprotein (apo)E is synthesized in atherosclerotic lesions by macrophages, however, its role in lesions is not known. Whereas apoE could exacerbate atherosclerosis by promoting macrophage uptake of cholesterol-rich lipoproteins or modulating protective inflammatory responses, it could also restrict lesion formation by facilitating cholesterol efflux out of lesions. The role of apoE was examined in lethally irradiated male C57BL/6J wild-type (WT) mice that were repopulated with bone marrow cells (BMT) from either identical C57BL/6J mice (WT+WT BMT) or C57BL/6J apoE-deficient mice (WT+E-/- BMT). This enabled us to compare normal mice with mice possessing macrophages that did not express apoE. The participation of macrophage-derived apoE in atherosclerosis was assessed by placing the mice on an atherogenic diet. Male WT+E-/- BMT mice had significantly reduced lesion area in the aortic valves (P < 0.01) compared with male WT+WT BMT mice ( approximately 22,000 vs. approximately 49,000 microm2/section, respectively). Further evaluation revealed that plasma cholesterol, lipoprotein cholesterol distribution, and plasma apoE were similar between the two groups, indicating that these known risk factors did not account for the differences in lesion area. However, the two groups were distinguished by the amount of apoE found in the lesions. ApoE antigen was expressed abundantly in WT+WT BMT lesions, whereas WT+E-/- BMT lesions contained little apoE. These findings indicate that the majority of apoE in lesions is synthesized locally by resident macrophages, and suggest that locally produced apoE can promote diet-induced atherosclerosis in male wild-type mice.     

Reduction of isoprostanes and regression of advanced atherosclerosis by apolipoprotein E

Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR(-/-)) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivo oxidant stress, we quantitated the isoprostane iPF(2 alpha)-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF(2 alpha)-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects.     

Retrovirus-mediated expression of apolipoprotein A-I in the macrophage protects against atherosclerosis in vivo

We have previously reported that the lack of apolipoprotein (apo) E expression by macrophages promotes foam cell formation in vivo. Because transgenic mice overexpressing human apoA-I from the liver (h-apoA-I TgN) are protected from the atherogenesis induced by apoE deficiency, we hypothesized that the presence of apoA-I in the vessel wall could reduce the negative effect of apoE deficiency on lesion growth. To address this issue, we used both retroviral transduction and transgenic approaches to produce in vivo systems where apoA-I is expressed from macrophages. In the retroviral transduction study, apoA-I-deficient (apoA-I(-/-)) mice reconstituted with apoE-deficient (apoE(-/-)) bone marrow cells that were infected with a retroviral vector expressing human apoA-I (MFG-HAI) had 95% lower atherosclerotic lesion area than that of recipients of apoE(-/-) bone marrow cells infected with the parental virus (MFG). To determine whether the protective effect of locally produced apoA-I was due to the lack of systemic apoA-I, we conducted a different experiment using h-apoA-I TgN mice as recipients of apoE(-/-) bone marrow with or without human apoA-I (driven by a macrophage-specific transgene defined as mphi-AI). Aortic lesion area in apoE(-/-)/mphi-AI --> h-apoA-I TgN mice was decreased by 85% compared with apoE(-/-) --> h-apoA-I TgN mice. These data demonstrate that expression of apoA-I from macrophages protects against atherogenesis without affecting plasma apoA-I and high density lipoprotein cholesterol levels.     

Deficiency of inducible NO synthase reduces advanced but not early atherosclerosis in apolipoprotein E-deficient mice

The inducible nitric oxide synthase (iNOS) is abundantly expressed by smooth muscle cells and macrophages in atherosclerotic lesions. Apolipoprotein E-deficient (apoE(-/-)) mice develop early and advanced atherosclerotic lesions. The role of iNOS in both early and advanced atherosclerotic formation was determined in apoE(-/-) mice. Mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 12 weeks of age on chow diet, iNOS(-/-)/apoE(-/-) mice developed comparable sizes of early atherosclerotic lesions in the aortic root as did iNOS(+/+)/apoE(-/-) mice (30,993+/-4746 vs. 26,648+/-6815 microm(2)/section; P=0.608). After being fed the Western diet for 12 weeks, iNOS(-/-)/apoE(-/-) mice developed significantly smaller advanced lesions than iNOS(+/+)/apoE(-/-) mice (458,734+/-14,942 vs. 519,570+/-22,098 microm(2)/section; P=0.029). This reduction in lesion formation could not be explained by differences in plasma lipid levels. To examine whether iNOS contributed to LDL oxidation, smooth muscle cells were isolated from the aorta, activated with TNF-alpha, and then incubated with native LDL in the absence or presence of N-Omega-nitro-L-arginine methyl ester (L-NAME), a specific NOS inhibitor. L-NAME significantly inhibited LDL oxidation by smooth muscle cells from iNOS(+/+)/apoE(-/-) mice (P=0.048), but it had no effect on LDL oxidation by cells from iNOS(-/-)/apoE(-/-) mice. iNOS(-/-)/apoE(-/-) mice had a significantly lower plasma lipoperoxide level on the Western diet (2.74+/-0.23 vs. 3.89+/-0.41 microM MDA; P=0.021) but not on chow diet (1.02+/-0.07 vs. 1.51+/-0.29 microM MDA; P=0.11). Thus, the absence of iNOS-mediated LDL oxidation may contribute to the reduction in advanced lesion formation of iNOS(-/-)/apoE(-/-) mice.     

Adrenalectomy in genetically obese ob/ob and db/db mice increases the proton conductance pathway

Adrenalectomy (ADX) prevents the excessive weight gain in the genetically obese ob/ob and db/db mice. To test the possibility that this results from increased energy expenditure due to increased thermogenesis in brown adipose tissue (BAT), we measured GDP binding to mitochondria from interscapular brown adipose tissue (BAT) in db/db and ob/ob mice and their lean controls after adrenalectomy, with and without corticosterone replacement. Both the vehicle treated and corticosterone treated db/db and ob/ob mice had lower body weights than the sham-operated mice GDP binding to mitochondria from IBAT was significantly lower in both the db/db and ob/ob mice than in their lean controls. Adrenalectomy significantly increased GDP binding in all mice compared to the respective sham-operated mice, but, the percentage increase was always greater in the db/db and ob/ob mice. Corticosterone treatment of adrenalectomized db/db, ob/ob or lean mice lowered GDP binding to sham levels. Our data confirm previous findings that adrenalectomy results in increased GDP binding to mitochondria from IBAT. Injections of corticosterone into adrenalectomized mice results in a decrease in GDP binding to values which are similar to values in sham-operated mice. Thus adrenalectomy may inhibit the development of obesity by increasing the thermic activity in IBAT.     

Effect of macrophage-derived apolipoprotein E on hyperlipidemia and atherosclerosis of LDLR-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice fed a Western diet exhibit severe hyperlipidemia and develop significant atherosclerosis. Apolipoprotein E (apoE) is a multifunctional protein synthesized by hepatocytes and macrophages. We sought to determine effect of macrophage apoE deficiency on severe hyperlipidemia and atherosclerosis. Female LDLR(-/-) mice were lethally irradiated and reconstituted with bone marrow from either apoE(-/-) or apoE(+/+) mice. Four weeks after transplantation, recipient mice were fed a Western diet for 8 weeks. Reconstitution of LDLR(-/-) mice with apoE(-/-) bone marrow resulted in a slight reduction in plasma apoE levels and a dramatic reduction in accumulation of apoE and apoB in the aortic wall. Plasma lipid levels were unaffected when mice had mild hyperlipidemia on a chow diet, whereas IDL/LDL cholesterol levels were significantly reduced when mice developed severe hyperlipidemia on the Western diet. The hepatic VLDL production rate of mice on the Western diet was decreased by 46% as determined by injection of Triton WR1339 to block VLDL clearance. Atherosclerotic lesions in the proximal aorta were significantly reduced, partially due to reduction in plasma total cholesterol levels (r=0.56; P<0.0001). Thus, macrophage apoE-deficiency alleviates severe hyperlipidemia by slowing hepatic VLDL production and consequently reduces atherosclerosis in LDLR(-/-) mice.     

A recycling pathway for resecretion of internalized apolipoprotein E in liver cells

We have investigated the recycling of apoE in livers of apoE(-)/- mice transplanted with wild type bone marrow (apoE(+/+) --> apoE(-)/-), a model in which circulating apoE is derived exclusively from macrophages. Nascent Golgi lipoproteins were recovered from livers of apoE(+/+) --> apoE(-)/- mice 8 weeks after transplantation. ApoE was identified with nascent d < 1.006 and with d 1.006-1.210 g/ml lipoproteins at a level approximately 6% that of nascent lipoproteins from C57BL/6 mice. Hepatocytes from apoE(+/+) --> apoE(-)/- mice were isolated and cultured in media free of exogenous apoE. ApoE was found in the media primarily on the d < 1.006 g/ml fraction, indicating a resecretion of internalized apoprotein. Secretion of apoE from C57BL/6 hepatocytes was consistent with constitutive production, whereas the majority of apoE secreted from apoE(+/+) --> apoE(-)/- hepatocytes was recovered in the last 24 h of culture. This suggests that release may be triggered by accumulation of an acceptor, such as very low density lipoproteins, in the media. In agreement with the in vivo data, total recovery of apoE from apoE(+/+) --> apoE(-)/- hepatocytes was approximately 6% that of the apoE recovered from C57BL/6 hepatocytes. Since plasma apoE levels in the transplanted mice are approximately 10% of control levels, the findings indicate that up to 60% of the internalized apoE may be reutilized under physiologic conditions. These studies provide definitive evidence for the sparing of apoE and its routing through the secretory pathway and demonstrate that internalized apoE can be resecreted in a quantitatively significant fashion.     

ApoE derived from adipose tissue does not suppress atherosclerosis or correct hyperlipidemia in apoE knockout mice

The synthesis of apoE by adipocytes has profound effects on adipose tissue lipid flux and gene expression. Using adipose tissue transplantation from wild-type (WT) to apoE knockout (EKO) mice, we show that adipose tissue also contributes to circulating apoE. Different from circulating apoE produced by bone marrow transplantation (BMT), however, adipose tissue-derived apoE does not correct hyperlipidemia or suppress atherosclerosis. ApoE secreted by macrophages has a more acidic isoform distribution, and it increases binding of reconstituted VLDL particles to hepatocytes and fibroblasts more effectively than apoE secreted by adipocytes. The incremental binding can be entirely accounted for by binding to the LDL receptor. After BMT into EKO hosts, plasma cholesterol and macrophage-derived apoE are largely within IDL/LDL- and HDL-sized particles. After adipose tissue transplantation, most cholesterol and adipocyte apoE remain in VLDL. After BMT, circulating apoE no longer demonstrates predominance of acidic isoforms compared with that circulating after fat transplantation. In conclusion, fat transplantation provides circulating apoE levels similar to those provided by bone marrow transplantation, but it does not suppress hyperlipidemia or atherosclerosis. A potential mechanism contributing to this difference is differential binding to cell surface lipoprotein receptors.     

Role of CC chemokine receptor 2 in bone marrow cells in the recruitment of macrophages into obese adipose tissue

The MCP-1 (monocyte chemoattractant protein-1)/CCR2 (CC motif chemokine receptor-2) pathway may play a role in macrophage infiltration into obese adipose tissue. Here we investigated the role of CCR2 in the recruitment of bone marrow-derived macrophages into obese adipose tissue in vitro and in vivo. Using the TAXIScan device, which can measure quantitatively the directionality and velocity of cell migration at time lapse intervals in vitro, we demonstrated that bone marrow cells (BMCs) from wild type mice migrate directly toward MCP-1 or culture medium conditioned by adipose tissue explants of genetically obese ob/ob mice, which are efficiently suppressed by pharmacological blockade of CCR2 signaling. The number of F4/80-positive macrophages was reduced in the adipose tissue from high fat diet-fed obese KKAy or ob/ob mice treated with a CCR2 antagonist propagermanium relative to vehicle-treated groups. We also found that the number of macrophages is reduced in the adipose tissue from ob/ob mice reconstituted with CCR2(-/-) BMCs (ob/ob + CCR2(-/-) BMCs) relative to those with CCR2+/+ BMCs (ob/ob + CCR2+/+ BMCs). Expression of mRNAs for CD11c and TLR4 (Toll-like receptor 4) markers of proinflammatory M1 macrophages was also decreased in the adipose tissue from ob/ob + CCR2(-/-) BMCs relative to ob/ob + CCR2+/+ BMCs, whereas mannose receptor and CD163, markers of anti-inflammatory M2 macrophages, were unchanged. This study provides in vivo and in vitro evidence that CCR2 in bone marrow cells plays an important role in the recruitment of macrophages into obese adipose tissue.     

Obesity modulates the expression of haptoglobin in the white adipose tissue via TNFalpha.

Increase in adipose mass results in obesity and modulation of several factors in white adipose tissue (WAT). Two important examples are tumor necrosis factor alpha (TNFalpha) and leptin, both of which are upregulated in adipose tissue in obesity. In order to isolate genes differentially expressed in the WAT of genetically obese db/db mice compared to their lean littermates, we performed RNA fingerprinting and identified haptoglobin (Hp), which is significantly upregulated in the obese animals. Hp is a glycoprotein induced by a number of cytokines, LPS (Lipopolysaccharide), and more generally by inflammation. A significant upregulation of WAT Hp expression was also evident in several experimental obese models including the yellow agouti (/) A(y), ob/ob and goldthioglucose-treated mice (10-, 8-, and 7-fold, respectively). To identify the potential signals for an increase in Hp expression in obesity, we examined leptin and TNFalpha in vivo. Wild type animals treated with recombinant leptin did not show any alteration in WAT Hp expression compared to controls that were food restricted to the level of intake of the treated animals. On the other hand, Hp expression was induced in mice transgenically expressing TNFalpha in adipose tissue. Finally, a significant downregulation of WAT Hp mRNA was observed in ob/ob mice deficient in TNFalpha function, when compared to the ob/ob controls. These results demonstrate that haptoglobin expression in WAT is increased in obesity in rodents and TNFalpha is an important signal for this regulation.     

Obesity causes very low density lipoprotein clearance defects in low-density lipoprotein receptor-deficient mice

We have reported that obese leptin-deficient mice (ob/ob) lacking the low-density lipoprotein receptor (LDLR(-/-)) develop severe hyperlipidemia and spontaneous atherosclerosis. In the present study, we show that obese leptin receptor-deficient mice (db/db) lacking LDLR have a similar phenotype, even in the presence of elevated plasma leptin levels. We investigated the mechanism for the hyperlipidemia in obese LDLR(-/-) mice by comparing lipoprotein production and clearance rates in C57BL/6, ob/ob, LDLR(-/-) and ob/ob;LDLR(-/-) mice. Hepatic triglyceride production rates were equally increased ( approximately 1.4-fold, P<.05) in both LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob mice. LDL clearance was decreased ( approximately 1.3- fold, P<.01) to a similar extent in LDLR(-/-) and ob/ob;LDLR(-/-) mice compared to C57BL/6 and ob/ob controls. While VLDL clearance was delayed in LDLR(-/-) compared to C57BL/6 and ob/ob mice (2-fold, P<.001), this delay was exaggerated in ob/ob;LDLR(-/-) mice (3.8-fold, P<001). The VLDL clearance defects were due to decreased hepatic uptake compared to C57BL/6 (54% and 26% for LDLR(-/-) and ob/ob;LDLR(-/-), respectively, P<.001). When VLDL was collected from C57BL/6, ob/ob, LDLR(-/-), and ob/ob;LDLR(-/-) donors and injected into LDLR(-/-) recipient mice, counts remaining in the liver were 1.4-fold elevated in mice receiving LDLR(-/-) VLDL and 2-fold increased in mice receiving ob/ob;LDLR(-/-) VLDL compared to controls receiving C57BL/6 VLDL (P<.01). Thus, the increase in plasma lipoproteins in ob/ob;LDLR(-/-) mice is caused by delayed VLDL clearance. This appears to be due to defects in both the liver and the lipoproteins themselves in these obese mice.     

Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin

Obesity and type 2 diabetes are associated with nonalcoholic steatohepatitis (NASH), but an obese/diabetic animal model that mimics human NASH remains undefined. We examined the induction of steatohepatitis and liver fibrosis in obese and type 2 diabetic db/db mice in a nutritional model of NASH and determined the relationship of the expressions of osteopontin (OPN) and leptin receptors to the pathogenesis of NASH. db/db mice and the corresponding lean and nondiabetic db/m mice were fed a diet deficient in methionine and choline (MCD diet) or control diet for 4 wk. Leptin-deficient obese and diabetic ob/ob mice fed similar diets were used for comparison. MCD diet-fed db/db mice exhibited significantly greater histological inflammation and higher serum alanine aminotransferase levels than db/m and ob/ob mice. Trichrome staining showed marked pericellular fibrosis in MCD diet-fed db/db mice but no significant fibrosis in db/m or ob/ob mice. Collagen I mRNA expression was increased 10-fold in db/db mice, 4-fold in db/m mice, and was unchanged in ob/ob mice. mRNA expressions of OPN, TNF-alpha, TGF-beta, and short-form leptin receptors (Ob-Ra) were significantly increased in db/db mice compared with db/m or ob/ob mice. Parallel increases in OPN and Ob-Ra protein levels were observed in db/db mice. Cultured hepatocytes expressed only Ob-Ra, and leptin stimulated OPN mRNA and protein expression in these cells. In conclusion, our results demonstrate the development of an obese/diabetic experimental model for NASH in db/db mice and suggest an important role for Ob-Ra and OPN in the pathogenesis of NASH.     

Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents.

Long chain fatty acid transport is selectively up-regulated in adipocytes of Zucker fatty rats, diverting fatty acids from sites of oxidation toward storage in adipose tissue. To determine whether this is a general feature of obesity, we studied [(3)H]oleate uptake by adipocytes and hepatocytes from 1) homozygous male obese (ob), diabetic (db), fat (fat), and tubby (tub) mice and from 2) male Harlan Sprague-Dawley rats fed for 7 weeks a diet containing 55% of calories from fat. V(max) and K(m) were compared with controls of the appropriate background strain (C57BL/6J or C57BLKS) or diet (13% of calories from fat). V(max) for adipocyte fatty acid uptake was increased 5-6-fold in ob, db, fat, and tub mice versus controls (p < 0.001), whereas no differences were seen in the corresponding hepatocytes. Similar changes occurred in fat-fed rats. Of three membrane fatty acid transporters expressed in adipocytes, plasma membrane fatty acid-binding protein mRNA was increased 9-11-fold in ob and db, which lack a competent leptin/leptin receptor system, but was not increased in fat and tub, i.e. in strains with normal leptin signaling capability; fatty acid translocase mRNA was increased 2.2-6.5-fold in tub, ob, and fat adipocytes, but not in db adipocytes; and only marginal changes in fatty acid transport protein 1 mRNA were found in any of the mutant strains. Adipocyte fatty acid uptake is generally increased in murine obesity models, but up-regulation of individual transporters depends on the specific pathophysiology. Leptin may normally down-regulate expression of plasma membrane fatty acid binding protein.     

ApoE-/-Fas-/- C57BL/6 mice: a novel murine model simultaneously exhibits lupus nephritis, atherosclerosis, and osteopenia

To establish a mouse model of accelerated atherosclerosis in lupus, we generated apolipoprotein E-deficient (apoE(-/-)) and Fas(lpr/lpr) (Fas(-/-)) C57BL/6 mice. On a normal chow diet, 5 month old apoE(-/-)Fas(-/-) mice had enlarged glomerular tuft areas, severe proteinuria, increased circulating autoantibody levels, and increased apoptotic cells in renal and vascular lesions compared with either single knockout mice. Also, double knockout mice developed increased atherosclerotic lesions but decreased serum levels of total and non-HDL cholesterol compared with apoE(-/-)Fas(+/+) littermates. Moreover, female apoE(-/-)Fas(-/-) mice had lower vertebral bone mineral density (BMD) and bone volume density (BV/TV) than age-matched female apoE(-/-)Fas(+/+) mice. Compared with apoE(-/-)Fas(+/+) and apoE(+/+)Fas(-/-) mice, apoE(-/-)Fas(-/-) mice had decreased circulating oxidized phospholipid (OxPL) content on apoB-100 containing lipoprotein particles and increased serum IgG antibodies to OxPL, which were significantly correlated with aortic lesion areas (r = 0.58), glomerular tuft areas (r = 0.87), BMD (r = -0.57), and BV/TV (r = -0.72). These results suggest that the apoE(-/-)Fas(-/-) mouse model might be used to study atherosclerosis and osteopenia in lupus. Correlations of IgG anti-OxPL with lupus-like disease, atherosclerosis, and bone loss suggested a shared pathway of these disease processes.     

Determination of the lower threshold of apolipoprotein E resulting in remnant lipoprotein clearance.

Apolipoprotein E (apoE) is the ligand for receptor-mediated clearance of remnant lipoproteins. ApoE at concentrations only 10% of normal, achieved through transplantation of wild-type marrow into apoE(-/-) mice, is sufficient for the maintenance of normal serum lipid and lipoprotein levels. The goal of the present study was to identify the minimal concentration of serum apoE still affecting cholesterol levels, and to determine whether any effects on remnant clearance below this level of apoE were detectable. ApoE(+/+) marrow was mixed with apoE(-/-) marrow in proportions of 1, 5, 10, and 25% to make chimeric mice with serum levels of apoE ranging from 0.005 to 0.46 mg/dl. Analysis of serum cholesterol and apoE levels demonstrated a positive correlation between apoE levels and cholesterol reduction (r = 0.83), with levels of 0.04 mg/dl representing the functional threshold level. There were no differences in lipoprotein profiles and clearance between apoE(-/-) mice and mice with serum apoE of less than 0.04 mg/dl, as assessed by FPLC, non-denaturing gel electrophoresis, and turnover studies. However, electron microscopy of negative stains showed fewer lipoprotein particles with a diameter of <30 nm in the serum of these mice compared to apoE(-/-) mice. These data demonstrate that the threshold of serum apoE resulting in cholesterol reduction is 0. 04 mg/dl, and indicate that apoE below this level affects lipoprotein size distribution possibly by accelerating the clearance of smaller remnants.     

Impairment of adipsin expression is secondary to the onset of obesity in db/db mice

The nature of the primary biochemical lesions in genetically obese mice, which might prove to be useful models for human obesity, remains totally obscure. The recent finding that the expression of adipsin was virtually suppressed in both db/db and ob/ob adult mice has opened new perspectives, suggesting a potential role for this defect in the pathogenesis of obesity. To be of etiological significance, adipsin deficiency must be present very early in life when excess fat storage starts to develop. We show here that at 10 days of age db/db pups exhibit significantly overdeveloped adipose tissue as compared with lean (+/db) pups but similar levels of both adipose tissue adipsin mRNA and serum adipsin. Adipsin expression was still normal in obese mice 15 days old but frankly deficient at 30 days of age when hyperinsulinemia has developed. Thus the defect in adipsin expression in db/db mice is a secondary feature which cannot be ascribed a role in the onset of obesity.