Adult-onset, short-term dietary restriction reduces cell senescence in mice

Abstract

Dietary restriction (DR) extends the lifespan of a wide variety of species and reduces the incidence of major age-related diseases. Cell senescence has been proposed as one causal mechanism for tissue and organism ageing. We show for the first time that adult-onset, short-term DR reduced frequencies of senescent cells in the small intestinal epithelium and liver of mice, which are tissues known to accumulate increased numbers of senescent cells with advancing age. This reduction was associated with improved telomere maintenance without increased telomerase activity. We also found a decrease in cumulative oxidative stress markers in the same compartments despite absence of significant changes in steady-state oxidative stress markers at the whole tissue level. The data suggest the possibility that reduction of cell senescence may be a primary consequence of DR which in turn may explain known effects of DR such as improved mitochondrial function and reduced production of reactive oxygen species.

Figure 1.. DR reduced frequencies of senescent hepatocytes and intestinal crypt enterocytes.

(A) Frequencies of γ-H2A.X positive enterocytes per crypt, immunohistochemistry on paraffin sections. ** p<0.005. (B) Correlation between sen-β-Gal and γ-H2A.X positive enterocytes (p=0.002). Data points are means per animal (DR: pink; Al: blue). Linear regression (solid line) and 95% confidence intervals (dashed lines) are given. (C) Representative images (left) and quantitative evaluation (right) of PCNA and γ-H2A.X double immunofluorescence of intestinal crypts from AL and DR mice. Blue: DAPI; red: γ-H2A.X; green: PCNA. (D) Representative images of γ-H2A.X immunohistochemistry in livers from AL (left) and DR (right) mice. Examples of centrilobular (top) and periportal (bottom) areas are shown. CV: central vein; PV: portal vein. Boxed areas are shown at higher magnification. Arrows indicate nuclei containing γ-H2A.X foci (red). (E) Quantification of γ-H2A.X positive hepatocytes. * p<0.05. (F) Representative images for sen-β-Gal activity. Pink: nuclei; blue: cytoplasmic sen-?-Gal staining. All data are from 5 animals/group, mean±S.E.M.

Figure 2.. DR improves telomere maintenance.

(A) Representative Q-FISH images (left panels, red: telomeres, blue: nuclei) and distribution of enterocyte telomere fluorescence intensity per nucleus (right panels, n≥2230 nuclei, 5 animals) in intestinal crypts. Mean nuclear telomere fluorescence intensity is indicated by blue vertical lines. p<0.001, Mann-Whitney rank sum test. (B) Representative Q-FISH images (left panels, red: telomeres, blue: nuclei) and distribution of hepatocyte telomere fluorescence intensity in centrilobular (CV, top, n≥560 nuclei) and periportal (PV, bottom, n≥650 nuclei) in liver areas. Mean fluorescence intensities are indicated for AL (blue) and DR (pink). P-values for AL vs DR were calculated by Mann-Whitney rank sum test. (C) Telomerase catalytic activity (% of TRAP activity in 3T3 cells) in whole liver (left, n=4) and intestinal mucosa (right, n=5) homogenates. Data are mean±S.E.M. n.s.: not significant (T-test).

Figure 3.. DR decreased lipid peroxidation in liver.

(A) Representative 4-HNE immunohistochemistry in small intestine from AL (left) and DR(right) mice. Brown: 4-HNE staining; blue: nuclei. (B) Representative 4-HNE images from centrilobular areas in liver. Brown: 4-HNE, Blue: nuclei. Arrows indicate examples of positive cells. (C) Frequencies of 4-HNE-positive hepatocytes in periportal and centrilobular areas of liver. Data are mean±S.E.M. * p<0.05, n=5 animals/group. (D) Co-localisation of γ-H2A.X (green) and 4-HNE (red) in AL liver. Representative image, double immunofluorescence, cryosection. Cells with nuclei (DAPI, blue) positive for γ-H2A.X are marked by arrows. Cells were scored as either single positive (H2AX+ HNE - or H2A.X- HNE +), double positive (H2A.X+ HNE+) or double negative (H2A.X- HNE -). Data are from four animals from the AL group.

Figure 4.. DR decreased the intensity of broad-band autofluorescence.

(A) Representative autofluorescence images (left) and quantitative data (right) in small intestinal crypts under AL (left) and DR (right). (B) Representative autofluorescence images from centrilobular areas in liver (AL left, DR right) and quantitative data in periportal and centrilobular areas. All data are mean±S.E.M from 5 animals/group. *p<0.05; n.s. not significant (T-test).

Figure 5.. DR does not change oxidative damage markers measured in whole liver homogenates.

(A) 8-oxodG levels in liver homogenates from AL and DR mice measured by HPLC with electrochemical detection. n=9 animals/group. (B) Nitrotyrosine levels in liver homogenates from AL and DR mice measured by ELISA; n=6 animals/ group. (C) Steady state hydrogen peroxide release from liver homogenates from AL and DR mice measured by Amplex Red fluorimetry; n=12 animals/ group. All data are mean ±S.E.M.; n.s.: not significant (T-test).