The over-expression of human hydrolase hMTH1 modulates metabolism and fat composition in mice exposed to high fat diet: a MRI and MRS study

The over-expression of human hydrolase hMTH1 modulates metabolism and fatcomposition in mice exposed to high fat diet: a MRI and MRS study Synopsis Oxidative stress is implicated in cancer, neurodegeneration and aging. hMTH1 is a hydrolase able to remove oxidized precursors from nucleotide’spool, thus avoiding oxidative nucleic acids damage. Overexpression of hMTH1 in mice is protective against oxidative damage, neurodegenerationand prolongs life span. Our study showed that the overexpression of hMTH1 in mice fed with high fat diet (HFD), a dietary regimen linked toinflammation, is associated with increased brown interscapular fat (linked to protection from obesity) and with reduced perivesical fat volume(indicator of poor cardiovascular outcomes) up to four weeks. These effects seem to be reversed by prolonging HFD. Introduction The role of the oxidative stress in the pathogenesis of cancer, neurodegeneration and aging is well established. The human MutT homologue(hMTH1) is a hydrolase able to protect nucleic acids from oxidative damage, by avoiding the incorporation of oxidized precursors in both DNA andRNA. Transgenic mice, which overexpress the human MTH1 gene (hMTH1‐Tg) are protected from neurodegeneration and motor impairment andare characterized by a decreased oxidative DNA damage, a prolonged life-span and an enhanced exploratory behavior. It has been shown thathigh fat diet is one of the most frequent environmental trigger, able to cause chronic inflammation which characterizes obesity and metabolicsyndrome. Aims Aims of this study were to understand if the the oxidative DNA damage protection mediated by the over-expression of hMTH1 is able to counteractthe metabolic alterations and the inflammation induced by the exposure to a high-fat diet (HFD, 45% of fat) for 33 weeks. Methods Male C57bl6 mice 10 wk old, wild-type (wt) and hMTH1-Tg, were fed with HFD for 33 weeks (two groups of 5 mice each). Body weight, oxidative DNAdamage/repair (by Single Cell Gel Electrophoresis assay) and other clinic parameters were measured. At 4, 11 (or 16), 22 and 33 weeks of HFDfeeding and two months after the end the HFD exposure (recovery time), animals underwent to MRI and MRS. Brain metabolism, interscapularbrown fat and liver fat were assessed by MRS. The volume of perivesical fat was assessed by MRI. Experiments were performed on a VARIAN Inovasystem operating at 4.7T with a transmitter volume RF coil actively decoupled from the receiver surface coil (RAPID Biomedical, Rimpar, Germany).1H localised MR spectra were collected from the hippocampus (HIP) and prefrontal cortex (PFC) using a PRESS sequence, according to a quantitativeprotocol. A STEAM sequence was used in the interscapular brown fat and liver for water to lipid ratio determination. T1-weighted MRI wasperformed to quantify the volume of visceral fat depot (a risk factor for metabolic dysfunction) in the four different mice groups. Repeatedmeasurements ANOVA was used for statistical comparisons (significance at p<0.05). Serum Metabolomics was carried out on Bruker Avanceoperating at 9.4 T spectrometer, by using standard presaturation pulse sequence and spin echo Carr-Purcell-Meiboom-Gill 1D sequence (CPMG)according to Beckonert protocol on intact serum. Results During HFD regimen, a significant increase in the accumulation of oxidative DNA damage has been observed in wt mice (repeated measurementsANOVA: genotype effect, p=0.02), as shown in Figure 1a). Notably, hMTH1-Tg mice resulted to be protected both in basal and HFD conditions, albeita comparable weight gain has been observed (Figure 1b). Alterations in brain metabolite concentrations have been detected in both PFC and HIP atbasal level and during the HFD regimen. Quantitative results are shown in Figure 2. In the interscapular fat we observed an increase in thewater/lipid signals ratio (which corresponds to increased brown adipose tissue, BAT) in the hMTH1-tg mice group which is maintained up to 4 weeksof HFD regimen (figure 3a and b). 1H MRS of the liver revealed a reduced amount of fat for the hMTH1-tg mice after 4 weeks of HFD stimulus(p<0.05) which is no longer observed at late times, as shown in Figure 3c and d. T1-weighted MRI of the abdomen revealed a slightly reducedamount of visceral fat for the hMTH1-tg mice at basal level but an increase in the hMTH1-tg animals at late stages after the beginning of the HFDstimulus, as shown in Figure 4a and b. Analyses at the end of HFD (33 weeks) and during the recovery time are in progress. Metabolomics analyseson serum samples showed that the overexpression of hMHT1 induced a tight control of glucose and lipid homeostasis metabolism as compared towt-mice in HFD (figure 5a and b). In particular, we found significant changes in glucose, and low-density lipoprotein/very low density lipoprotein(LDL/VLDL) signals following HFD in wt mice. Despite the stimulation of a high-fat diet, MTH1 mice are not able to alter blood glucose levels, nor toincrease the levels of VLDL / LDL in circulation. Discussion and conclusions Differences have been detected between wt and hMTH1-Tg mice before the beginning of HFD, suggesting a crosstalk between genomic instabilityand metabolic dysfunction. Brain metabolism alteration highlights a direct effect on brain functionality. In spite of similar body weight increase,nuclear oxidative DNA damage is lower in hMTH1-Tg than in wt at all time points, while adipose organ extension and composition maintain thebasal differences between wt and hMTH1-Tg mice only up to 4 weeks of oxidative stimulus. The protective role of hMTH1 against oxidative damageis associated with an increase in BAT (which may provide protection from obesity ) and with a reduced abdominal perivesical fat volume (which isconsidered an independent indicator of poor cardiovascular outcomes ). The protective effect observed in the hMTH1 mice up to 4 weeks seems tobe reversed by a prolonged HFD exposition, suggesting a not-obvious link between HFD and oxidative damage modulation by hMTH1. Acknowledgements We thank the Italian National Institute of Health for financial support References 1. De Luca G et al PLoS Genet 2008;4:e1000266. 2. De Luca G et al Aging Cell 2013;12:695-705. 3. Canese R et al NMR Biomed 2012;25:632-642. 4. Beckonert O et al. Nat Protoc 2007; 2:2692–2703 5. Matsuita M et al, International Journal of Obesity 2014; 38,812–817. 6. Powell-Wiley et al, Circulation 2021;43:e984.