Effects of population density and sex on plasma cholesterol levels of Paigen diet-fed HypoE mice.

<p>HypoE mice were housed singly (1/cage, black bars; males, n = 16, females; n = 13) or in mixed genotype groups (HypoE and one or more SRBI^+/−ApoeR61^h/h littermates per group, 4–5/cage, gray bars; males, n = 19, females; n = 24). Beginning at two months of age the animals were switched from a normal chow diet to the Paigen diet. After 19 days of Paigen diet feeding, plasma was harvested and total cholesterol (TC), unesterified cholesterol (UC) and UC/TC ratios were determined from the HypoE mice. Data are means ± SD. Statistically significant differences were determined by unpaired Student’s t or Mann-Whitney test.</p

Effects of atherogenic diets on lipoprotein cholesterol profiles of HypoE mice.

<p>HypoE mice housed in groups (including one or more SRBI^+/−ApoeR61^h/h littermates per group, 4–5/cage) were fed Western (open/white symbols, n = 5), Paigen NC (gray symbols, n = 5) or Paigen (black symbols, n = 3) diets for one month starting at two months of age and then plasma was harvested and subjected to FPLC size fractionation. The data shown include only the HypoE mice. <b>A.</b> Averaged plasma lipoprotein total cholesterol (TC) profiles (mg/dL plasma). Brackets indicate the approximate elution positions of VLDL, intermediate-density lipoproteins (IDL)/LDL, and HDL. <b>(Inset)</b> Expanded scale for the IDL/LDL and HDL regions of the profiles. <b>B.</b> For each profile from individual mice, total cholesterol levels in the indicated pooled fractions corresponding to VLDL-, IDL/LDL- or HDL-size particles were summed and averages were calculated. Data are represented as mean ± SD. Statistically significant differences were determined by Kruskal-Wallis tests followed by the Dunn’s multiple comparison post-test. *p<0.05.</p

Population density (A) and mixed genotype housing (B) effects on Paigen diet-fed HypoE mouse survival.

<p>Mice were housed at the indicated population densities (1, 2–3 or 4–5 (group)/cage) with either uniform (HypoE) or mixed genotypes (HypoE and one or more SRBI^+/−ApoeR61^h/h littermates per group). Beginning at two months of age the animals were switched from a normal chow diet to the standard Paigen diet. The data shown include only the HypoE mice. <b>A</b> Kaplan-Meier survival curves for HypoE mice housed singly (1 mouse/cage, black line), or uniform genotype (HypoE only) groups of 2–3 (light gray dashed line), or 4–5 (light gray solid line) mice per cage with the indicated median survival times and number of animals per group (n). A notch in the 4–5 mice per cage line indicates a censored animal that was euthanized due to severe ulcerative dermatitis at 35 days after initiating Paigen diet feeding. <b>B</b> Kaplan-Meier survival curves for HypoE mice housed singly (black line) or in uniform genotype (HypoE only, light gray line) or mixed genotype (HypoE and SRBI^+/−ApoeR61^h/h E, gray line) group housing (4–5 mice/cage).</p

Effects of Atherogenic Diets, Population Density and Sex on Survival.

a<p>From two months of age HypoE mice of the indicated sexes were fed either the Paigen or Paigen NC diets and housed either singly (one/cage) or in groups of 4–5 mice per cage, with the groups containing HypoE and one or more SRBI^+/−ApoeR61^h/h littermates. The values of median survival are for HypoE mice only.</p>b<p>*, **p<0.0001 ^#, ^{# #}p<0.02.</p

Effects of atherogenic diets on plasma lipids.

a<p>Mice were housed in groups with mixed genotypes. HypoE mice were fed either a Western diet (n = 13), Paigen diet without cholate (Paigen NC) (n = 18), or Paigen diet (n = 6) for one month beginning at two months of age. Data represent mean ± SD. Statistically significant differences among normal chow and the three atherogenic diets were determined by Kruskal-Wallis tests followed by the Dunn’s multiple comparison post-test.</p>*<p>p<0.05 vs Paigen NC and Paigen,</p>**<p>p<0.05 vs Paigen. WT; wild type, TC; total cholesterol, UC; unesterified cholesterol.</p>b<p>The data for group-housed, wild-type and HypoE mice fed a normal chow diet are from Zhang S, et al (2)-.</p

Population density and sex effects on plasma corticosterone and oxytocin in Paigen diet-fed HypoE mice.

<p>HypoE mice were housed in mixed genotype groups (HypoE and one or more SRBI^+/−ApoeR61^h/h littermates per group, 4–5/cage, gray bars) or singly (1/cage, black bars). Beginning at two months of age the animals were switched from a normal chow diet to the Paigen diet. After 19 days of Paigen diet feeding, the mice were weighed, plasma and hearts were harvested, heart weights measured and plasma levels of corticosterone and oxytocin were determined. The values presented are from the HypoE mice only; they do not include the companion (group housing) SR-BI^+/−ApoeR61^h/h mice. <b>A</b> Plasma corticosterone levels: group housed males, n = 11; singly housed males, n = 14; group housed females, n = 16; singly housed females, n = 12. <b>B</b> Plasma oxytocin levels: group housed males, n = 8; singly housed males, n = 8; group housed females, n = 12; singly housed females, n = 11. Data are means ± SD. Statistically significant differences were determined by unpaired t-test. <b>C</b> Correlation of oxytocin levels and heart-to-body weight ratios for all HypoE mice (n = 39). Gray and black circles indicate group and single housing, respectively. Statistics were evaluated using Spearman’s rank correlation.</p

Effects of atherogenic diets on survival (A) and heart-to-body weight ratios (B) of HypoE mice.

<p>Mice were housed in groups with mixed genotypes (HypoE and one or more SRBI^+/−ApoeR61^h/h littermates per group, 4–5/cage) and, beginning at two months of age were switched from a normal chow diet to the indicated atherogenic diets. The data shown include only the HypoE mice. <b>A</b> Kaplan-Meier survival curves. Mice were fed either Paigen (gray solid line, n = 15), Paigen NC (gray dotted line, n = 14), or Western (black dashed line, n = 15) diets. <b>B</b> Heart-to-body weight ratios. For the indicated times (1–3 months), mice were fed the Paigen (n = 14), chow (n = 8), Western (n = 5 (1 month), n = 4 (3 months)), or Paigen NC (n = 6 (1 month), n = 4 (2 months)) diets. Gray and white bars represent those populations in which approximately 50% or 100% of the animals survived after the indicated times of feeding. Data are means ± SD. Statistically significant differences were determined by Kruskal-Wallis tests followed by the Dunn’s multiple comparison post-test. *p<0.05.</p

Contributions of a Disulfide Bond and a Reduced Cysteine Side Chain to the Intrinsic Activity of the High-Density Lipoprotein Receptor SR-BI

The high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), binds HDL and mediates selective cholesteryl ester uptake. SR-BI’s structure and mechanism are poorly understood. We used mass spectrometry to assign the two disulfide bonds in SR-BI that connect cysteines within the conserved Cys³²¹-Pro³²²-Cys³²³ (CPC) motif and connect Cys²⁸⁰ to Cys³³⁴. We used site-specific mutagenesis to evaluate the contributions of the CPC motif and the side chain of extracellular Cys³⁸⁴ to HDL binding and lipid uptake. The effects of CPC mutations on activity were context-dependent. Full wild-type (WT) activity required Pro³²² and Cys³²³ only when Cys³²¹ was present. Reduced intrinsic activities were observed for CXC and CPX, but not XXC, XPX, or XXX mutants (X ≠ WT residue). Apparently, a free thiol side chain at position 321 that cannot form an intra-CPC disulfide bond with Cys³²³ is deleterious, perhaps because of aberrant disulfide bond formation. Pro³²² may stabilize an otherwise strained CPC disulfide bond, thus supporting WT activity, but this disulfide bond is not absolutely required for normal activity. C³⁸⁴X (X = S, T, L, Y, G, or A) mutants exhibited altered activities that varied with the side chain’s size: larger side chains phenocopied WT SR-BI treated with its thiosemicarbazone inhibitor BLT-1 (enhanced binding, weakened uptake); smaller side chains produced almost inverse effects (increased uptake:binding ratio). C³⁸⁴X mutants were BLT-1-resistant, supporting the proposal that Cys³⁸⁴’s thiol interacts with BLT-1. We discuss the implications of our findings on the functions of the extracellular loop cysteines in SR-BI and compare our results to those presented by other laboratories

Plasma lipid levels and body weights of apoE KO and PDZK1/apoE dKO mice.

<p>Four month old animals were fasted for 4 hours prior to sample collection. Values are represented as mean±standard error from 8–25 animals (mean 20 animals) per group. Statistical significance was determined by pairwise comparisons of each value from PDZK1/apoE dKO mice with apoE KO controls by using unpaired Student's t test. The abbreviations and units used are TC (total plasma cholesterol, mg/dL), UC (plasma unesterified cholesterol, mg/dL), UC:TC (plasma unesterified cholesterol to total plasma cholesterol ratio), PL (plasma phospholipids, mg/dL), TG (plasma triglycerides, mg/dL) and Wt (body weights, g).</p>a, b, c, e<p>P<0.01</p>d<p>P<0.05</p

Immunoblot analysis of hepatic SR-BI expression.

<p>Mice with the indicated genotypes were fed a high fat/high cholesterol/cholate-containing “Paigen” diet for three months. Livers were harvested and subjected to immunoblotting using anti-SR-BI and anti-actin (loading control) antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008103#s2" target="_blank">Materials and Methods</a>.</p