The Effects of Apolipoprotein F Deficiency on High Density Lipoprotein Cholesterol Metabolism in Mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20–25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/−0.9 mg/dl vs. WT: 1.2+/−0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls

Lipoprotein Composition in ApoF KO mice.

<p>Lipoproteins were isolated from one milliliter of pooled mouse plasma by KBr density gradient ultracentrifugation as follows: VLDL d<1.006, IDL/LDL 1.006</p

The effect of cholesterol loading and depletion on plasma lipids in ApoF KO mice.

<p>ApoF wild type (WT) and knockout (KO) mice (9–10 weeks of age) were fed a chow diet supplemented with either 0.01% ezetimibe or 0.2% cholesterol for 11 days. Values are reported as mean +/− standard deviation.</p>*<p>P<0.05 relative to WT for each treatment group.</p

The effects of High fat feeding on plasma lipids in ApoF deficient mice.

<p>Female ApoF wild type (WT), heterozygous (Het), and knockout (KO) mice were bled for baseline lipid measurements and placed on a diet containing 45% kcal from fat for 9 weeks. Plasma was then collected after 2, 4, and 9 weeks on the diet. All mice were fasted 4 hours before bleeding at every time point. Values are reported as the mean +/− standard deviation for each group (WT n = 8, Het n = 10, KO n = 8).</p><p> <b><u>Two Way ANOVA Results:</u></b></p><p>Cholesterol: Time p<0.0001.</p><p>HDL-C: Time p<0.0001.</p><p>Non HDL-C: Time p<0.0001.</p><p>Triglycerides: Time n.s.,Genotype p = 0.0025 (Bonferroni vs WT: * p<0.05, ** p<0.01).</p><p>CE: Interaction p = 0.0428, Time p<0.0001.</p><p>Free Chol: Time p<0.0001, Genotype p = 0.0003 (Bonferroni WT vs KO *p<0.05).</p

X-Gal staining of tissues from ApoF KO mice harboring a beta-galactosidase reporter gene.

<p>The targeted ApoF allele contains a beta-galactosidase reporter gene in place of ApoF. X-gal staining was performed on a wild type (WT) and an ApoF KO mouse (KO) to examine ApoF expression. The wild type mouse is included for an estimation of background staining for each tissue in the absence of beta-galactosidase.</p

Blood Chemistry Parameters in ApoF KO mice.

<p>Blood was collected in Lithium-Heparin tubes and used to measure classical chemistry parameters. Sodium, Potassium, Creatinine, Blood Urea Nitrogen (BUN), Aspartate Aminotransferase (AST), and Alanine Aminotransferase (ALT) were measured on a clinical chemistry analyzer. Data is reported as the mean +/− standard deviation. A two-tailed t-test was used to compare between genotypes by sex, and no statistically significant differences were found.</p

HDL cholesterol content, size, and apoprotein composition in ApoF KO mice.

<p>A. Gel filtration chromatography of 150 microliters of pooled plasma from wild type (WT), and ApoF KO (KO) mice. B. HDL size of plasma from wild type (WT), heterozygous (Het) and homozygous ApoF KO (KO) mice as determined by NMR analysis. C. Denaturing SDS-PAGE gel of proteins from HDL (1.063</p

Glycosylation, processing and secretion of the mouse apo F protein.

<p><i>A.</i> Schematic of the mouse apo F precursor protein depicting the predicted boundaries of the signal peptide, proprotein region, and furin cleavage site (RAKR/S). The N-linked glycosylation site at N206 is shown with a diamond. <i>B.</i> Western blot for mouse apo F in cells (left) and media (right), from HEK293 cells transiently transfected with either green fluorescent protein (GFP), wild type mouse ApoF (mApoF), or mouse apo F with asparagine 206 mutated to alanine (N206A). <i>C.</i> Western blot of apo F in one microliter of plasma from mice overexpressing mApoF from a liver-specific AAV vector. Plasma was denatured with heat and then subjected with deglycosylation by PNGase F, Sialidase A, and O-glycanase. The mature and pro-fragment portions of the apo F protein are shown with arrows.</p

Tissue expression, processing and glycosylation of murine apolipoprotein F.

<p><i>A.</i> Real time RT-PCR analysis of total RNA from tissues of female mice: brain, heart, lung, liver, spleen, kidney, duodenum, jejunum, ileum, inguinal fat, brown fat, adrenals, thyroid, and ovaries. <i>B.</i> Real time RT-PCR analysis of total RNA from tissues of male mice: brain, heart, lung, liver, spleen, kidney, duodenum, jejunum, ileum, inguinal fat, brown fat, stomach and testes. Values are shown as the mean +/− standard deviation for each animal (n = 3 per group).</p