Sex Differences in Metabolic Indices and Chronic Neuroinflammation in Response to Prolonged High-Fat Diet in ApoE4 Knock-In Mice

Abstract

Late-onset Alzheimer's disease (LOAD) likely results from combinations of risk factors that include both genetic predisposition and modifiable lifestyle factors. The E4 allele of apolipoprotein E (ApoE) is the most significant genetic risk factor for LOAD. A Western-pattern diet (WD) has been shown to strongly increase the risk of cardiovascular disease and diabetes, conditions which have been strongly linked to an increased risk for developing AD. Little is known about how the WD may contribute to, or enhance, the increased risk presented by possession of the ApoE4 allele. To model this interaction over the course of a lifetime, we exposed male and female homozygote ApoE4 knock-in mice and wild-type controls to nine months of a high-fat WD or standard chow diet. At eleven months of age, the mice were tested for glucose tolerance and then for general activity and spatial learning and memory. Postmortem analysis of liver function and neuroinflammation in the brain was also assessed. Our results suggest that behavior impairments resulted from the convergence of interacting metabolic alterations, made worse in a male ApoE4 mice group who also showed liver dysfunction, leading to a higher level of inflammatory cytokines in the brain. Interestingly, female ApoE4 mice on a WD revealed impairments in spatial learning and memory without the observed liver dysfunction or increase in inflammatory markers in the brain. These results suggest multiple direct and indirect pathways through which ApoE and diet-related factors interact. The striking sex difference in markers of chronic neuroinflammation in male ApoE4 mice fed the high-fat WD suggests a specific mechanism of interaction conferring significant enhanced LOAD risk for humans with the ApoE4 allele, which may differ between sexes. Additionally, our results suggest researchers exercise caution when designing and interpreting results of experiments employing a WD, being careful not to assume a WD impacts both sexes by the same mechanisms.

Keywords: ApoE4; C57BL/6NTac mice; Western-pattern diet; diabetes; late-onset Alzheimer’s disease; liver; memory; modifiable lifestyle factors; neuroinflammation; obesity.

Figure 1

Body weights of WT and ApoE4 mice during the treatment period. Both male and female WT (A,B) and ApoE4 (C,D) mice gained weight at comparable rates throughout the study period. ^…WD, -CONTROL; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Experimental timeline (GTT = glucose tolerance testing). See Section 4: Methods for details.

Figure 3

Glucose tolerance test. Average residual blood glucose levels in the 60 min period post-administration were elevated in all groups fed the high-fat diet. However, this effect was more pronounced in both female WT (B) and ApoE4 mice (D) than in either male WT (A) or ApoE4 (C) animals * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 4

Liver weights. Male and female WT (A) and male ApoE4 (B) animals exposed to WD were found to have significantly larger livers than those on control. Female ApoE4 animals did not show this increase in liver weight. * p < 0.05, **** p < 0.0001.2.3. Selective Effects of WD on Liver Weights in ApoE4 Males and WT Animals.

Figure 5

Liver glutathione levels. Baseline glutathione levels are higher in ApoE4 animals, relative to WT, and were significantly reduced only in male Western diet ApoE4 mice, (p < 0.05) and not females, suggesting a possible protective effect on impaired liver function and oxidative stress in these animals. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 6

Chronic inflammatory cytokines in the brain. Expression of inflammatory cytokines was measured by ELISA. No effect of high-fat diet is evident in WT in any of the cytokines. In contrast, male (but not female) ApoE4 mice showed a significant increase in IL-1β in response to chronic exposure to the high-fat diet. * p < 0.05.

Figure 7

Behavioral assays. Both male and female WT (A) and ApoE4 (B) mice fed WD trended toward less activity in the open-field test. WT male and female mice fed WD (C,D) showed marginally slower latency to escape in the Barnes maze. In contrast, both male and female ApoE4 mice fed the WD (E,F) showed consistently longer latencies to escape, with this effect being significant and most pronounced in the male mice (E). It should be noted that control diet ApoE4 mice (E,F) showed consistently shorter latencies to escape than WT (C,D) control-diet-fed animals and non-significant differences in ambulation in the open-field where ApoE4 animals (B) ambulated more than WT animals (A), regardless of sex. * p < 0.05, ** p < 0.01.