Protective effects of maternal immunization with naturally oxidized LDL (nLDL) on male offspring fed 0.5% cholesterol diet.

<p>(A) Glucose response in an OGTT in offspring after 29 weeks on diet. (B) Insulin responses in the same OGTT. AUCs for glucose and insulin are shown as insets; statistical significances for individual time points are not indicated. n = 26.</p

Overview of the experimental design and aims.

<p>Overview of the experimental design and aims.</p

Evidence of an eosinophil trafficking defect in HFD mice.

<p>(A) Ileal sections from normal chow or 7 day HFD mice were stained for in situ apoptotic cells using TdT-Fluor staining protocol. Minimal apoptosis was detected in either condition relative to nuclease treated positive control. (B) Bone marrow and (C) peripheral blood leukocytes were isolated from 5 week HFD mice and proportions of eosinophils determined by flow cytometry. D) Expression of CCL11 and CCL24 genes in the small intestinal mucosa of 1 week HFD or normal chow mice. In all graphs each data point represents an individual mouse from one experiment. *p<0.05, **p<0.01, ***p<0.001 Mann-Whitney U test.</p

Protective effects of maternal OxLDL immunization in male offspring.

<p>(<b>A,B</b>) Glucose and insulin responses during an OGTT in male offspring of mothers immunized with OxLDL or PBS and non-immunized control mothers, after 28 weeks on 60% sucrose diet (n = 26). AUCs are shown as insets. (<b>C–H</b>) Pancreas immunohistochemistry of the same animals. Alpha cells identified by glucagon-staining in offspring of OxLDL-immunized (<b>C</b>) and control mothers (<b>D</b>). No-primary-antibody control (<b>E</b>). Beta cells staining for insulin in offspring of (<b>F</b>) OxLDL-immunized, (<b>G</b>) PBS-immunized and (<b>H</b>) control mothers (<b>I–K</b>)<b>.</b> An additional group of male offspring of OxLDL-immunized and control mothers was fed sucrose for 38 weeks and then subjected to hyperinsulinemic euglycemic clamp (n = 16). Body weights (I) and fasting plasma glucose levels (J) were not significantly different, whereas the amounts of glucose infused during the euglycemic phase was markedly greater in offspring of OxLDL-immunized mothers (K).</p

Protective effects of nLDL immunization in euglycemic female mice fed regular chow.

<p>To assess direct effects of immunization on glucose responses, OGTTs were performed on separate groups of female mice 40 and 250 days after the primary immunization. Controls were non-immunized. (<b>A</b>) Body weights at both time points. (<b>B</b>) Glucose responses after 40 days. (<b>C,D</b>) Glucose and insulin responses after 250 days. AUC, Area under the curve. n = 24 (40 days) and 25 (250 days).</p

Characterization of experimental diets.

<p>(<b>A</b>) Effects of regular chow, 0.5% cholesterol diet and 60% sucrose diet on (<b>A</b>) body weight, (<b>B</b>) lipoprotein profiles and (<b>C,D</b>) glucose response in male mice after 78 days on diet (n = 31). FPLC analysis was performed on pooled plasma samples from 2–3 mice per group. Glucose responses were assessed by OGTT) on 31 mice (C) and differences in the area under the curve (AUC) determined by unpaired T-test (D). (<b>E–I</b>) Plasma concentrations of major free fatty acids (FFAs) in male offspring of OxLDL-immunized (OxLDL) and non-immunized mothers (controls) after 24 weeks on 60% sucrose or regular diet (n = 32). (<b>J</b>) Total FFAs.</p

Effect of maternal OxLDL immunization on hepatic expression of genes of relevance for diabetes.

<p>mRNA expression of genes known to be regulated in diabetic conditions was assessed by commercial gene array in the liver of male offspring of OxLDL-immunized and control mice after 28 weeks on 60% sucrose diet (experiment #4). Only genes significantly regulated more than 1.5 fold are shown. IL-10 is included even though it failed to reach significance.</p

Effects of maternal OxLDL immunization on the activity of antioxidant enzymes and glutathion concentration in offspring.

<p>Hepatic activity of (A) cytosolic superoxide dismutase (SOD), (B) total SOD, (C) glutathione peroxidase (GPx), (D) catalase, and (E) mitochondrial SOD. (F) Hepatic glutathion concentration. Ventricular activities of mitochondrial SOD (G) and (H) GPx.</p

Intestinal permeability compared in HFD and Ob/Ob mice.

<p>(A) 4kDa Fitc-dextran flux across jejunum or ileum of normal chow (n = 4–6), HFD (n = 4) or Ob/Ob (n = 4) mice measured in Ussing chambers. (B) FITC-dextran concentration in the plasma of Ob/Ob mice and wild-type littermates following oral administration each data point represents an individual mouse (n = 8 per group) from one experiment. (C) Transepithelial conductance across jejunum or ileum of normal chow (n = 4–6), HFD (n = 4) or Ob/Ob (n = 4) mice measured in Ussing chambers D) Fecal albumin concentration in Ob/Ob mice, wildtype littermates, normal chow and HFD mice. Each data point represents and individual mouse from one experiment. *p<0.05, **p<0.01, ***p<0.001 by one way ANOVA (Ussing chamber analysis) or Mann-Whitney U test (<i>in vivo</i> FITC dextran or fecal albumin assays).</p

Protective effects of OxLDL immunization in male mice fed 60% sucrose.

<p>To assess direct effects of immunization with extensively oxidized LDL in immunized mice exposed to more obesogenic conditions, 4 month old male LDLR^-/- mice immunized with OxLDL or PBS and controls were fed 60% sucrose for 38 weeks. Age-matched non-immunized mice fed regular chow served as additional control. (<b>A</b>) Body weights. (<b>B,C</b>) OGTT glucose response curves and AUCs. (<b>D,E</b>) Corresponding OGTT insulin curves and AUCs. n = 22.</p