Differential modulation of diet-induced obesity and adipocyte functionality by human apolipoprotein E3 and E4 in mice

Abstract

Objective: Apolipoprotein E (apoE), a key protein in lipid metabolism, is highly expressed in adipose tissues. Studies have shown that human APOE*4 is associated with a lower body mass index but with a greater risk of coronary heart disease compared with other APOE alleles. To define the isoform-specific role of apoE in regulating the expandability and functionality of adipose tissues, we investigated the effects of diet-induced obesity in mice whose endogenous Apoe gene has been replaced by either the human APOE*3 or APOE*4 allele.

Results: After 8 weeks on a Western-type high-fat diet, male APOE4 mice displayed impaired tolerance to glucose and fat overload compared with APOE3 mice. Subcutaneous fat tissues in APOE4 and APOE3 mice after high fat feeding were not different. In contrast, although epididymal fat tissues in APOE4 mice gained 30% less weight during the high fat feeding than in APOE3 mice, they showed impaired insulin-stimulated glucose uptake ex vivo. Epididymal APOE4 adipocytes were larger in size than APOE3 adipocytes, and expressed reduced levels of mRNA for peroxisome proliferator-activated receptor gamma2 and adiponectin, important markers of adipocyte functionality. Adenoviral expression of apoE3 in apoE-null culture adipocytes induced adiponectin mRNA in a dose-dependent manner, but the induction was significantly blunted in cells overexpressing apoE4. However, in contrast to the apoE3-expressing cells, Glut1, but not Glut4, expression levels were positively correlated with increased apoE4 mRNA, suggesting that apoE4 expression in adipocyte interferes in insulin-sensing pathways.

Conclusion: Dysfunctional epididymal adipose tissues contribute to the accelerated impairment of glucose tolerance in APOE4 mice fed a Western-type diet. Our results underscore the importance of functionality of individual fat depots rather than total fat mass as a determinant for metabolic disturbance during diet-induced obesity.

Figure 1

The effect of Western-type diet (WD) on body weight in APOE3 (○) or APOE4 (■) mice over the experimental period (a), fecal fat excreted (b) and food intake (c). Weights of liver (d), subcutaneous adipose tissue (e) and epididymal adipose tissue (f) of 4-month-old mice fed regular chow (RC) or WD. (a) Statistical analysis was carried out by repeated measurements analysis of variance (ANOVA) using unweighted median analysis. Inset displays the number of mice weighed at each time point, APOE3 (upper row), APOE4 (bottom row). Analysis between subjects showed difference between alleles (P = 0.02). (b–f) Data are means±s.e.; n = 12–16, *P = 0.03 for the difference between groups.

Figure 2

Adipose tissue in APOE4 mice. (a) Morphology of epididymal adipose tissues from mice fed regular chow (RC) or Western-type diet (WD) over 8 weeks (8 week WD). Magnification ×40. (b) Size distribution of adipocytes in inguinal (upper panel) and epididymal fat (lower panel) from APOE3 (○) or APOE4 (●) mice fed RC, 4 weeks WD and 8 weeks WD (c) Cross-sectional areas of adipocytes from subcutaneous (left panel) and epididymal adipose tissues (right panel) of APOE3 (white bars) or APOE4 mice (black bars) fed different diets. Data are means±s.e.; *P = 0.05, n = 5–8 mice; 200–300 cells were randomly selected and scored in each fat depot of each mice. All mice were 12–15 weeks of age.

Figure 3

Plasma distribution of cholesterol (a), triglycerides (b) and apolipoprotein E (c) in APOE3 and APOE4 mice. Postprandial lipoproteins of mice fed Western-type diet (WD) were fractionated by fast protein liquid chromatography (FPLC) and results are presented as micrograms of lipids in each fraction. Fractions 11–17 corresponded to TRL (very low-density lipoprotein (VLDL) and Chylomicrons), 17–25 to LDL and TRL remnants and 25–31 to high-density lipoprotein (HDL). (d) Variation of plasma triglyceride (TG) levels measured during lipid challenge in 5-month-old APOE3 (○) or APOE4 (■) mice fed 3 months WD. Data are the means±s.e.; n = 4–7, *P<0.05 for the difference between genotypes in each condition.

Figure 4

Plasma glucose (a), insulin (b), non-esterified free fatty acid (NEFA) (c), change of NEFA (d) and NEFA to insulin ratio (e). Plasma was isolated from overnight fasted APOE3 (○) or APOE4 (■) mice fed Western-type diet (WD) at 1 h after a single high-fat meal. Data are the means±s.e.; n = 8–10, *P<0.05 for the difference between genotypes in each condition.

Figure 5

Oral glucose tolerance tests (OGTTs) (a, b), area under the glucose curves (AUC) (c) and intraperitoneal insulin tolerance test (ITT) (d) in 4-month-old APOE3 (○) or APOE4 (■) mice fed regular chow (a) or Western-type diet (WD) for 2 months. (b, d). Data are the means±s.e., n = 5–8. *P< 0.05 for the difference between genotypes in each condition.

Figure 6

Explants of subcutaneous inguinal (SC) and epididymal (EPID) fat were incubated for 24 h at 37 °C in triplicate, with or without insulin, and glucose uptake was determined as described in Methods. Results are milligram of glucose taken up by milligram of tissue protein. Values are means±s.e.; n = 5, *P ≤ 0.05 vs for the difference between groups.

Figure 7

Effects of APOE allele and high-fat feeding on expression of PPARγ2 (a), ADIPONECTIN (b) or APOE (c) genes in subcutaneous (SC) inguinal fat (left panels) and epididymal (EPID) visceral fat (right panels) in 4-month-old APOE3 (○) or APOE4 (■) mice. Mice were fed regular chow (RC) or Western-type diet (WD) during 2 months. Values are means±s.e.; n = 6–8. Group comparisons were by Mann–Whitney U-test; *P ≤ 0.05 for the difference between genotypes in each condition.

Figure 8

mRNA levels of APOE (a), interleukin-6 (IL6) (b), PPARγ2 (c), ADIPONECTIN (d), GLUT1 (e) and GLUT4 (f) genes in adipocytes differentiated in vitro. Embryonic fibroblasts isolated from apoE-deficient mouse were treated with 0, 1×10⁷ (1×) and 2×10⁷ (2×) p.f.u. of adenoviruses encoding APOE3 (○) or APOE4 (■). Values are means±s.e.; n = 6 replicates in each condition and genotype. Group comparisons were by Mann–Whitney U-test; *P ≤ 0.05 vs for the difference between genotypes in each condition. Relationships between relative expressions of GLUT1 (g) and GLUT4 (h).