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. 2013 Feb;62(2):382-91.
doi: 10.2337/db12-0390. Epub 2012 Sep 6.

Apolipoprotein E2 accentuates postprandial inflammation and diet-induced obesity to promote hyperinsulinemia in mice

David G Kuhel  1 , Eddy S Konaniah, Joshua E Basford, Courtney McVey, Colleen T Goodin, Tapan K Chatterjee, Neal L Weintraub, David Y Hui
Affiliations

Apolipoprotein E2 accentuates postprandial inflammation and diet-induced obesity to promote hyperinsulinemia in mice

David G Kuhel et al. Diabetes. 2013 Feb.

Abstract

Genetic studies have revealed the association between the ε2 allele of the apolipoprotein E (apoE) gene and greater risk of metabolic diseases. This study compared C57BL/6 mice in which the endogenous mouse gene has been replaced by the human APOE2 or APOE3 gene (APOE2 and APOE3 mice) to identify the mechanism underlying the relationship between ε2 and obesity and diabetes. In comparison with APOE3 mice, the APOE2 mice had elevated fasting plasma lipid and insulin levels and displayed prolonged postprandial hyperlipidemia accompanied by increased granulocyte number and inflammation 2 h after being fed a lipid-rich meal. In comparison with APOE3 mice, the APOE2 mice also showed increased adiposity when maintained on a Western-type, high-fat, high-cholesterol diet. Adipose tissue dysfunction with increased macrophage infiltration, abundant crown-like structures, and inflammation were also observed in adipose tissues of APOE2 mice. The severe adipocyte dysfunction and tissue inflammation corresponded with the robust hyperinsulinemia observed in APOE2 mice after being fed the Western-type diet. Taken together, these data showed that impaired plasma clearance of apoE2-containing, triglyceride-rich lipoproteins promotes lipid redistribution to neutrophils and adipocytes to accentuate inflammation and adiposity, thereby accelerating the development of hyperinsulinemia that will ultimately lead to advanced metabolic diseases.

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Figures

FIG. 1.
FIG. 1.
Plasma lipid levels in APOE2 and APOE3 mice. A: Fasting triglyceride levels. B: Fasting cholesterol levels in APOE2 and APOE3 mice fed chow diet (filled bars) and after feeding the Western-type diet for 4 weeks (open bars). Bars with different letters were different at P < 0.05 (n = 6). C: Postprandial clearance of [14C]lipids from plasma after feeding APOE2 (open symbols) and APOE3 (filled symbols) mice an olive oil meal containing [14C]triolein. D: Postprandial plasma triglyceride levels in chow-fed APOE2 (open symbols) and APOE3 (filled symbols) 2 h after oral feeding a lipid-rich meal. The data in C and D represent mean ± SD from four mice in each group, with * and # indicating significant differences from the APOE3 mice at P < 0.05 and P < 0.01, respectively. E and F: FPLC profiles from APOE2 (open symbols) and APOE3 mice (filled symbols) under both fasting (circle symbols) and postprandial (triangle symbols) conditions. The insets show Western blots of apoB100, apoB48, and apoE bands in VLDL (fractions 3–6) and IDL/LDL (fractions 18–21) of fasted (F) and postprandial (P) APOE2 (2F and 2P) and APOE3 (3F and 3P) mice. Note that the apoB proteins in APOE3 mice were barely detectable by Western blots.
FIG. 2.
FIG. 2.
Flow cytometry analysis of blood leukocytes in APOE2 and APOE3 mice. A and B: Representative plots of blood cells showing forward and side scattering of lymphocytes (L), monocytes (M), and granulocytes (G) from APOE2 and APOE3 mice, respectively, 2 h after feeding a lipid-rich meal. C: Bar graph showing the average number of each cell type ± SD in four APOE2 (open bars) and six APOE3 (filled bars) mice after overnight fast and 2 h after feeding a lipid-rich meal. *P < 0.05, difference from fasted mice of the same genotype; #P < 0.05, difference from APOE3 mice under same treatment conditions (n = 6 in each group). D and E: Representative histogram of granulocytes after staining with the fluorescent neutral lipid stain LipidTox. F: Bar graph showing mean neutral lipid staining intensity ± SD from four APOE2 (open bars) and eight APOE3 (filled bars) mice before (fasted) and 2 h after lipid meal feeding (postprandial). *P < 0.05, difference from the APOE3 mice. G: Inducible NOS (iNOS)–positive neutrophils. H: CD11b and Ly6G double-positive cells as the mean percentage of total leukocytes ± SD in the blood of four APOE2 (open bars) and six APOE3 (filled bars) mice before (fasted) or 2 h after feeding a lipid-rich meal (fed). *P < 0.05, difference from APOE3 mice under same feeding conditions (n = 6).
FIG. 3.
FIG. 3.
Body weights and adiposity of APOE2 and APOE3 mice. Bar graphs showing mean body weights ± SD (A) and percent body fat ± SD (B) in 10-week-old male APOE2 and APOE3 mice fed either chow diet (filled bars) or 4 weeks after feeding the Western-type diet (open bars). *P < 0.05, difference from chow-fed mice (n = 6).
FIG. 4.
FIG. 4.
Blood glucose and insulin levels. A: Mean fasting blood glucose ± SD. B: Fasting plasma insulin levels ± SD. C: Calculations of homeostasis model assessment of insulin resistance (HOMA) index in chow- (filled bars) and Western diet–fed (open bars) APOE2 and APOE3 mice. Bars with different letters denote significant differences at P < 0.05, whereas bars with similar letters were not statistically different (n = 6). Glucose tolerance tests with inset showing area under the curve (AUC) analysis (D) and insulin sensitivity tests (E) in APOE2 (open symbols) and APOE3 (filled symbols) mice. The data represent the mean from six mice in each group. *P < 0.05, difference from the APOE3 mice. A.U., arbitrary units.
FIG. 5.
FIG. 5.
Adipose tissue histology. A: Histological analysis of subcutaneous (inguinal) and visceral (gonadal) adipose tissues of APOE2 and APOE3 mice fed either chow diet or after feeding the Western diet for 4 weeks. Arrows point to representative crown-like structures in each image. Bars, 50 µm. B: Distribution of adipocyte cell sizes in subcutaneous (inguinal) and visceral (gonadal) adipose tissues of chow- and Western diet–fed APOE2 (dotted lines) and APOE3 (solid lines) mice. The data were obtained by measuring areas of 480 random adipocytes from three random fields from each mouse (n = 4 APOE2 and 6 APOE3 mice). C: Number of crown-like structures in subcutaneous and visceral adipose tissues of chow- and Western diet–fed APOE2 (open bars) and APOE3 (filled bars) mice. The data were obtained by counting the total number in each section compared with the total number of adipocytes and reported as mean ± SE. *P < 0.05, significant difference from the APOE3 mice; #P < 0.01, significant difference from the APOE3 mice.
FIG. 6.
FIG. 6.
Gene expression in adipose tissues. Total mRNA was isolated from subcutaneous and visceral adipose tissues of chow- (filled bars) and Western diet–fed (open bars) APOE2 and APOE3 mice and used for quantification by qPCR. Gene expression levels were normalized to levels of cyclophilin gene expression. A: Leptin expression levels. B: Adiponectin expression levels. C: MCP-1 expression levels. D: MIP-1α expression levels. E: Macrophage marker gene F4/80 expression levels. F: TNF-α expression levels. G: Inducible NOS2 expression levels. H: MRC2 expression levels. The data were reported as mean ± SD from six mice in each group. *P < 0.05, difference from mice with the same genotype fed chow diet; #P < 0.05, difference from mice with different genotype fed the same diet.
FIG. 7.
FIG. 7.
Plasma cytokine levels. Plasma was obtained from APOE2 (open bars) and APOE3 (filled bars) to determine the levels of adiponectin (A), leptin (B), interleukin-6 (C), and plasminogen activator inhibitor-1 (PAI-1) (D). The data represent mean ± SD from six mice in each group. *P < 0.05, significant difference from APOE3 mice.
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