Loss of the actin remodeler Eps8 causes intestinal defects and improved metabolic status in mice

Abstract

Background: In a variety of organisms, including mammals, caloric restriction improves metabolic status and lowers the incidence of chronic-degenerative diseases, ultimately leading to increased lifespan.

Methodology/principal findings: Here we show that knockout mice for Eps8, a regulator of actin dynamics, display reduced body weight, partial resistance to age- or diet-induced obesity, and overall improved metabolic status. Alteration in the liver gene expression profile, in behavior and metabolism point to a calorie restriction-like phenotype in Eps8 knockout mice. Additionally, and consistent with a calorie restricted metabolism, Eps8 knockout mice show increased lifespan. The metabolic alterations in Eps8 knockout mice correlated with a significant reduction in intestinal fat absorption presumably caused by a 25% reduction in intestinal microvilli length.

Conclusions/significance: Our findings implicate actin dynamics as a novel variable in the determination of longevity. Additionally, our observations suggest that subtle differences in energy balance can, over time, significantly affect bodyweight and metabolic status in mice.

Figure 1. Reduced bodyweight in Eps8KO mice.

A–B. Age- (A) and high fat diet-induced (B) obesity in Eps8KO and wild-type mice (KO and WT, respectively, in this and all subsequent figures). HFD, high fat diet. C. Soxhlet analysis. D. Weight of individual organs. Ing., inguinal white fat; Epid., epididymal white fat; Sca., scapular brown fat; Kidn., kidney. Values are expressed as percent bodyweight. In all panels (and in all subsequent figures): n, number of mice tested per condition (in D, 13–17 mice were used, depending on the analyzed organ). In all panels (and in all subsequent figures), values are expressed as means ± SEM. Two-tailed t-test was used to assess statistical significance: *, P<0.05; **, P<0.01; ***, P<0.005; n.s., not significant (in this and in all subsequent figures).

Figure 2. Increased insulin sensitivity in Eps8KO mice.

A. Insulin tolerance, measured as blood glucose after insulin injection. B. Glucose tolerance, measured as blood glucose after glucose injection. C. Plasma insulin levels after overnight fast. HFD, high fat diet.

Figure 3. Reduced intestinal fat absorption in Eps8KO mice.

A. Food intake. B. Feces production. C. Feces energy content. D. Left, intestinal permeability, determined fluorometrically in the plasma 4 hr after an oral gavage of Dextran-FITC (4 kD). Right, in vivo intestinal transit, determined as the time (transit time) between oral gavage of Carmin Red and the appearance of red feces. E. Absorption of ¹⁴C-oleic acid determined by the fecal dual isotope method. F. Intestinal fat absorption determined by the plasma dual isotope method. Please note that while differences are readily detectable between genotypes, no difference in absorption is observable between the two substrates, within genotypes. This indicates that the breakdown of triglycerides at the intestinal level is not impaired. G. Intestinal uptake of a fatty acid (left), peptidic (middle), and sugar (right) substrate determined by the everted intestinal sac model. Grey bars depict background values after competition with excess cold substrate: OA, 40 mM cold oleic acid; SG, 20 mM cold sarcosyl-glycine, MDG, 100 mM cold methyl-glucopyranoside. In each experiment 3 sacs/mouse were prepared from the number of mice/experimental condition indicated in the figure. In all panels: ND, normal diet; HFD, high fat diet.

Figure 4. Energy expenditure of Eps8 KO mice.

A. Oxygen consumption (VO₂) determined at ambient temperature (RT), and at 30°C (TNZ, thermal neutral zone). B–C. Locomotor activity determined on a 24 hr cycle (B) or represented as the average of day and night time (C). In panel B (and in the following panel D), time points, that differed significantly between WT and Eps8KO mice are indicated by horizontal lines. Red and black lines indicate values that were higher or lower, respectively, in Eps8KO vs. WT mice. D. Core body temperature, determined telemetrically in WT and Eps8KO mice, and presented as a 24 hr cycle (top) or as the average of day and night time (bottom). E. Scatter plot of the correlation between core body temperature and bodyweight. F. Core body temperature before and after 8 weeks on normal chow (ND) or on high fat diet (HFD). In all graphs n = 7 for WT and 8 for KO.

Figure 5. Eps8KO mice display a CR-like phenotype.

A. The 20 genes differentially expressed in the livers of Eps8KO mice vs. WT (See Table S2) were compared to published studies that analyzed differential gene expression in the livers of CR mice compared to controls [CR-I, ; CR-II, ; CR-III, [23]]. In the study by Pohjanvirta et al., two conditions of starvation (4 days and 10 days) were used, and both are reported in the comparison. In the study by Bauer et al., several conditions were used and we report the longest one (48 hs) in the comparison. “Genes differ. expr. (P<0.05)”, number of significantly (P<0.05) differentially expressed genes in this study (Eps8KO) and in the other studies (CR) (the number of Eps8KO genes vary because not all genes that we analyzed were present on the chips used in the other studies). “Overlap (concord.)”, genes overlapping between the indicated datasets and concordantly regulated (upregulated or downregulated); the observed (Obs.) overlapping genes are reported in comparison to the expected (Exp.) overlap in a random distribution. “P”, p-value of the observed overlap vs. the expected one (Pearsons's chi-squared test). Additional details are in the Materials and Methods section. B. Detailed analysis of the overlap between genes from the Eps8KO list (column “Gene”; in red, upregulated genes, in green, downregulated genes) with CR lists from other studies (CR-I, CR-II and CR-III as in A). Only concordant overlaps are shown and are indicated by a red box if the gene was upregulated, or by a green box if the gene was downregulated. Genes not present in a specific array experiment are indicated as NP C. Kaplan-Meyer survival curves for Eps8KO mice and WT littermates. D. ROS production as measured by DCFDA fluorescence in primary embryo fibroblasts (PEF) from Eps8KO and p66KO mice and WT littermates. p66KO fibroblasts were used as positive controls. E. Insulin signaling. Left, PEF from Eps8KO or WT mice were treated with Insulin 10 µg/ml for the indicated times, followed by immunoblot (IB) as shown. Right, 3T3-L1 adipocytes were subjected to Eps8 silencing (eps8 siRNA) or to mock-silencing (ctrl siRNA) followed by Insulin treatment and IB as in left.

Figure 6. Eps8 is required for correct microvillar morphogenesis in the mouse.

A. IB analysis of Eps8 in the intestine. Duod., duodenum, Jejen., jejunum, Ile. B. Small intestines (jejunum) from WT or KO mice were stained with hematoxylin-eosin (H&E, top) to assess general morphology, or Alcian blue (bottom) to visualize goblet cells. Bar, 100 µm. C. Morphometric analysis of Alcian blue positive cells (top), villus height (middle) and crypt depth (bottom) in the indicated intestinal segments. D. Confocal images of Caco-2 cells, transfected with Eps8 (EGFP-Eps8, right) or control EGFP (EGFP, left). Cells were allowed to differentiate for 10 days on collagen-coated coverslips, and then stained for F-actin using rhodaminated phalloidin. The merged images (merge. Bottom panels) show the colocalization (yellow) of EGFP-Eps8 or EGFP (green) with F-actin (red). Bar, 10 µm. E. Transmission electron microscope analysis of the intestinal brush border demonstrates shortened and irregular shaped microvilli (dashed red brackets) in the duodenal tracts of KO compared to WT mice. Bar, 0.5 µm. F. Morphometric analysis of duodenal microvilli length.