Young transgenic hMTH1 mice are protected against dietary fat-induced metabolic stress-implications for enhanced longevity

Abstract

hMTH1 protects against mutation during oxidative stress. It degrades 8-oxodGTP to exclude potentially mutagenic oxidized guanine from DNA. hMTH1 expression is linked to ageing. Its downregulation in cultured cells accelerates RAS-induced senescence, and its overexpression in hMTH1-Tg mice extends lifespan. In this study, we analysed the effects of a brief (5 weeks) high-fat diet challenge (HFD) in young (2 months old) and adult (7 months old) wild-type (WT) and hMTH1-Tg mice. We report that at 2 months, hMTH1 overexpression ameliorated HFD-induced weight gain, changes in liver metabolism related to mitochondrial dysfunction and oxidative stress. It prevented DNA damage as quantified by a comet assay. At 7 months old, these HFD-induced effects were less severe and hMTH1-Tg and WT mice responded similarly. hMTH1 overexpression conferred lifelong protection against micronucleus induction, however. Since the canonical activity of hMTH1 is mutation prevention, we conclude that hMTH1 protects young mice against HFD by reducing genome instability during the early period of rapid growth and maximal gene expression. hMTH1 protection is redundant in the largely non-growing, differentiated tissues of adult mice. In hMTH1-Tg mice, expression of a less heavily mutated genome throughout life provides a plausible explanation for their extended longevity.

Keywords: Comet assay; DNA damage; ageing; life span; metabolic rate; micronucleus; mitochondria; mouse models; oxidative stress.

FIGURE 1

Growth curves and markers of oxidative stress in wild‐type (WT) and hMTH1‐Tg mice. (a) Mean weights of WT (n = 5) and hMTH1‐Tg (n = 5) animals maintained on a standard diet (SD) were recorded weekly from birth up to 42 weeks. (b) Mean weights of 2‐month‐old animals (n = 6 for each experimental condition) during 5 weeks on a SD or high‐fat diet (HFD). Body weights were recorded weekly. (c) As in panel B but showing body weights of 7‐month‐old animals. (d) Serum MDA levels at the end of a 5‐week period on SD or HFD in 2‐ and 7‐month‐old WT and hMTH1‐Tg mice (n = 4 per group). (e) As in panel D but showing data for serum AOPP levels. All values are mean ± SE. Asterisk indicates a significant difference (p < 0.05, unpaired t test)

FIGURE 2

PCA results (score plots). (a) PC1 scores for 2‐month‐old WT and hMTH1‐Tg mice on the standard diet (SD) or a high‐fat diet (HFD). Average values for each genotype are indicated by square symbols. (b) PC2 scores for SD and HFD in 2‐month‐old WT and hMTH1‐Tg mice. Average values for each genotype are indicated by square symbols. (c) PC1 values as in panel A for 7‐month‐old animals. (d) PC2 values as in panel B for 7‐month‐old animals

FIGURE 3

Liver metabolic profiles in WT and hMTH1‐Tg mice as a function of diet and age. (a) The mean values of single metabolites measured in 2‐month‐old animals maintained on the standard diet (SD) or high‐fat diet (HFD) are represented as hMTH1‐Tg:WT ratios. Metabolites belonging to common metabolic pathways are grouped in boxes. (b) As in panel A for 7‐month‐old animals. The absolute quantification of m‐Ins (at 4.01 ppm in proton NMR spectra) was not reported due to the presence of other signals overlapping in the same spectral interval. The indicated metabolites were analysed in the liver of 3–7 animals per group. Abbreviations grouped for metabolic pathway: (a) glucose metabolism: D‐glucose (Glc) and lactic acid (Lac); (b) nucleotide metabolism: adenosine monophosphate (AMP) + adenosine diphosphate (ADP) + adenosine triphosphate (ATP), AXP = (AMP + ADP + ATP), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), uridine monophosphate (UMP) + uridine diphosphate (ADP) + uridine triphosphate (UTP), UXP = (UMP + UDP + UTP); (c) one carbon metabolism: formic acid (Form); (d) amino acid metabolism: L‐phenylalanine (Phe), L‐tyrosine (Tyr), L‐histidine (His), L‐glycine (Gly), L‐glutamic acid (Glu), L‐valine (Val), L‐glutamine (Gln), L‐isoleucine (Ile), L‐Aspartic acid (Asp) and L‐alanine (Ala); (e) redox balance metabolism: glutathione (Gs) and taurine (Tau); (f) lipid metabolism: glycerophosphocholine (GPC), phosphocholine (PCho), free choline (Cho) and ethanolamine (Etn); (g) lipid and amino acid metabolism: acetic acid (Acet); (h) tricarboxylic acid (TCA) cycle: fumaric acid (Fum) and succinic acid (Succ); (i) other pathways: total creatine (creatine + phosphocreatine; Creat)

FIGURE 4

Levels of the most relevant metabolites in WT and hMTH1‐Tg mice as a function of diet and age. (a) Succinate/fumarate ratios in 2‐month‐old animals maintained on the standard diet (SD) or high‐fat diet (HFD). (b) Levels of aromatic amino acids in 2‐month‐old animals maintained on the SD or HFD. (c) As in panel A for 7‐month‐old animals. (d) As in panel B for 7‐month‐old animals. The indicated metabolites were analysed in the liver of 3–7 animals per group. L‐Phe: L‐phenylalanine; L‐Tyr: L‐tyrosine; and L‐His, L‐histidine. All values are mean ± SE

FIGURE 5

Effects of a brief fasting period on body weight, metabolic profiles and markers of oxidative stress in 2‐month‐old animals. Scheme of the fasting experiment is shown on the top of the figure. After 35 days in high‐fat diet (HFD), mice were fasted for further 20 h. Animals were sacrificed on Day 36. (a) Mean body weights were recorded at days 35 (end of HFD) and 36 (end of fasting). (b) Values of PC2 scores for HFD and fasting in WT mice and hMTH1‐Tg mice. Average values for each genotype are indicated by square symbols. (c) Discrimination of WT and hMTH1‐Tg mice by weight loss and PC2 scores. (d‐e‐f‐g‐h) Comparison of metabolite levels in HFD and fasted WT and hMTH1‐Tg mice. (d) Succinate/fumarate ratios. (e) L‐Phe, L‐Tyr and L‐His levels. (f) Comparison of GPC/PCho ratios. (g) Comparison of AOPP levels. (h) Comparison of MDA levels. Mean values of 4 animals/group are reported ± SE. Asterisks indicate significant changes (**p < 0.01, unpaired t test)

FIGURE 6

Analysis of DNA damage by alkaline comet assays on peripheral blood cells of WT and hMTH1‐Tg mice as a function of age and diet. (a) Percentage of tail DNA in blood cells from 2‐month‐, 7‐month‐ and 18‐month‐old WT and hMTH1‐Tg mice maintained on the standard diet (SD). Cells from WT mice (n = 3) exposed to 2 Gy of 137Cs γ radiation were used as a positive control. (b) Calibration curve relating the percentage of tail DNA to single‐strand breaks (SSBs) per genome. A dose–response curve was obtained by treatment of blood cells with ionizing radiation. The background levels of the percentage of tail DNA revealed by the comet assay (red line) correspond to the damage induced by 1.1 Gy gamma‐rays or around 1300 DNA SSBs per genome (c) Percentage of tail DNA in blood cells from 2‐month‐old WT and hMTH1‐Tg mice maintained on the SD, high‐fat diet (HFD) and 20‐h fasting following a 5‐week HFD. (d) Percentage of tail DNA in blood cells from 7‐month‐old WT and hMTH1‐Tg mice on the SD or HFD (e) DNA 8‐oxodG measured by the comet assay in the presence of the Fpg DNA glycosylase/AP endonuclease. DNA 8‐oxodG levels are calculated as the increase in the percentage of tail DNA induced by Fpg over background level in 2‐month‐ and 7‐month‐old WT and hMTH1‐Tg mice. All values are mean ± SE. The comet assay parameters in different experimental groups were compared by two‐tailed Student's t test. Asterisks indicate significant differences (**p < 0.01)

FIGURE 7

Analysis of micronucleus frequency in peripheral blood reticulocytes of WT and hMTH1‐Tg mice as a function of age and diet. (a) Peripheral blood cells were stained with acridine orange allowing the simultaneous detection of RNA (stained red) and DNA (stained yellowish green). In the picture, two immature reticulocytes can be identified by the red‐fluorescing reticulum structure (red cells); one reticulocyte contains a yellowish green‐fluorescing micronucleus (arrow). Erythrocytes are seen in the background as green‐bordered unstained black cells. A green‐fluorescing micronucleus can also be observed in one erythrocyte. (b) Frequency of micronucleated reticulocytes in 2‐month‐, 7‐month‐ and 18‐month‐old mice maintained on the standard diet (SD). (c) Frequency of micronucleated reticulocytes in WT and hMTH1‐Tg mice maintained on the SD and high‐fat diet (HFD). (d) Effects of fasting on the frequency of micronucleated reticulocytes. Micronuclei were analysed in 2‐month‐old WT and hMTH1‐Tg mice before and after an overnight fast (20 h) following a 5‐week HFD. The experimental design is represented in Figure 5. All values are mean ± SE. Different experimental groups were compared by two‐tailed Student's t test

FIGURE 8

Graphical scheme. Long‐lived transgenic hMTH1 mice are protected against dietary fat‐induced metabolic stress and DNA damage only when they are young