Middle age as a turning point in mouse cerebral cortex energy and redox metabolism: Modulation by every-other-day fasting
Graphical abstract
Introduction
Brain aging is known to be accompanied by compromised bioenergetics that can be attributed to a decreased supply of glucose and oxygen, a decline in glycolysis (Hoyer, 1985; Goyal et al., 2017; Castellano et al., 2019), and impaired mitochondrial function with diminished ATP production (Mattson and Arumugam, 2018; Grimm and Eckert, 2017). Mitochondrial dysfunction is thought to be a main contributor to increased steady-state levels of reactive oxygen species (ROS) that are responsible for the intensification of oxidative stress during aging (Grimm and Eckert, 2017; Garaschuk et al., 2018; Simsek et al., 2019; Yanar et al., 2019). This increased intensity of oxidative stress is followed by activation of stress responses, albeit their protective capacity seems to be age-limited (Garaschuk et al., 2018). In particular, protection against oxidative damage in the brain relies largely on the use of NADPH as a cofactor for glutathione (GSH) and thioredoxin-dependent antioxidant mechanisms. Glucose-6-phosphate dehydrogenase (G6PDH), the key enzyme of the pentose phosphate pathway (PPP), together with the second enzyme of the PPP (6-phosphogluconate dehydrogenase), are known to be the primary contributors to cellular NADPH production (Bouzier-Sore and Bolaños, 2015). Therefore, glucose utilization via the PPP is important for maintaining brain antioxidant potential (Bouzier-Sore and Bolaños, 2015; Camandola and Mattson, 2017) as well as NADPH-dependent biosynthetic activities. Glycolytic rate of brain tissue is often estimated by the levels of glycolytic intermediates (Hoyer, 1985; Goyal et al., 2017; Castellano et al., 2019) and there are limited data indicating that the activities of certain glycolytic enzymes are lower in old compared with young animals (Leong et al., 1981; Hoyer, 1985; Steffen et al., 1991). Information regarding age-related changes in the metabolism of other brain fuel sources such as ketone bodies is also scarce (Ding et al., 2013). Therefore, understanding of mechanisms responsible for reorganization of energy metabolism in the brain during normal aging has both theoretical and practical importance.
Dietary restriction (DR) or limitation of food consumption is one of the strategies that may slow down age-related functional declines (Mattson et al., 2017; Lushchak and Gospodaryov, 2017; Simsek et al., 2019; Yanar et al., 2019). Two strategies are commonly used experimentally to achieve DR – caloric restriction (CR) and different types of intermittent fasting (IF) where periods of feeding and fasting are alternated. Among IF protocols, the every-other-day fasting (EODF) regimen is known to achieve life span and health span extension (Mattson et al., 2017; Xie et al., 2017). In the EODF regimen ad libitum (AL) access to food alternates with 24-h periods of food deprivation. This approach was found to extend life span without a substantial decrease in average daily food intake (Anson et al., 2003), or with slightly lower food intake (Xie et al., 2017). In the latter study EODF was initiated at two months old in mice and extended life span by about 20% (Xie et al., 2017). Although the effects of CR on many parameters of animal function have been extensively studied in recent decades, much less attention has been paid to EODF. Several studies have indicated that DR can decrease glucose metabolism in the brain and improve mitochondrial function (Boveris and Navarro, 2008), whereas others showed no effect of CR on respiratory characteristics of brain mitochondria (Chausse et al., 2015). The current data regarding DR effects on redox balance in the brain are also controversial (Gouspillou and Hepple, 2013; Yanar et al., 2019). In particular, reduced ROS levels and oxidative damage were observed in the brains of adult DR mice (Sohal et al., 1994; Rathod et al., 2011) whereas oxidative damage was reported in the brain of young rats subjected to an IF regimen (Chausse et al., 2015). Sex differences in brain aging are also known. For example, female brain seems to be more sensitive to age-related disorders (Zhao et al., 2016) and female brains show a higher prevalence of Alzheimer's disease (AD) (1.6–3:1) compared to men, whereas Parkinson's disease (PD) is more prevalent (3.5:1) in men compared to women (Villa et al., 2019). However, sex differences have not been fully explored, because males are much more frequently used as the model for aging studies (Sohal et al., 1994; Uzun et al., 2010; Walsh et al., 2014; Erdoğan et al., 2017; Xie et al., 2017).
In the present study, we focused on the mouse cerebral cortex addressing the following questions: (i) how does aging affect energy metabolism and redox homeostasis in the brain; (ii) does an EODF regimen alter the pattern of age-dependent changes and what effect does EODF have in mice of different ages; and (iii) do such changes also depend on the sex of the animals? To answer these questions, cerebral cortex samples from 6-, 12- and 18-month-old C57BL/6 J mice that were either fed ad libitum or subjected to an EODF protocol were analyzed for markers of oxidative stress, levels of intermediates of glycolysis and ketone bodies, activities of antioxidant, key glycolytic and PPP enzymes, and parameters of mitochondrial respiration.
Section snippets
Reagents, animals and feeding regimens
Reagents used in this study are listed in Suppl. File 1. Male and female C57Bl/6J mice were kindly provided by Dr. I. Shmarakov (Yuriy Fedkovych Chernivtsi National University, Chernivtsi, Ukraine) and then bred in our departmental facilities in order to obtain a sufficient number of mice per group. Mice received ad libitum (AL) standard rodent chow (#3336, Provimi Kliba AG, Kliba Nafag, Switzerland) containing 23.5% protein, 5.5% fats, 6.5% ash, 3.5% fiber, and 35% starch (full composition is
Results
Data for all parameters measured in this study were collected from both male and female groups of mice. For convenience in emphasizing the key results of the study, the figures presented below provide the data for male AL groups in all cases, for male AL + EODF in instances when these regimens showed substantial differences/patterns, and for male and female groups in cases where there were substantial differences/patterns between the two sexes. However, all data collected in this study (AL &
Discussion
There are at least two main findings in the current study: (i) many biochemical parameters of “old” cortex phenotype are established in middle age and (ii) redistribution of glucose catabolism fluxes between glycolysis and the PPP in the aged cortex may result from decreased activities of key glycolytic enzymes, namely PFK and PK, and enhanced activity of the key PPP enzyme, G6PDH. We also suggest that age-related intensification of oxidative stress after middle age can be prevented to some
Conclusions and perspectives
In this study, we showed that aging causes the following changes in the mouse cortex: (i) increased levels of oxidative stress markers and decreased antioxidant defenses; (ii) decreased activities of key glycolytic enzymes and increased activity of key PPP enzyme G6PDH in mice of both sexes, likely representing a molecular mechanism for age-dependent redirection of glucose metabolism from glycolysis to the PPP; (iii) aging-dependent reduction in the use of KB as energy substrates; (iv) slightly
Funding
This work was mainly supported by the grant from Volkswagen Foundation (VolkswagenStiftung, #90233), Germany, to VIL and OG, partially by a Ministry of Education and Science of Ukraine grant (#0118U003477) to VIL, and by a Discovery grant from the Natural Sciences and Engineering Research Council of Canada (#6793) to KBS.
Availability of data and material
The authors confirm that all data supporting the findings of this study are available in the supplementary material.
CRediT authorship contribution statement
Maria M. Bayliak: Supervising the experimental work, data curation, writing of methods' section, review and editing; Oksana M. Sorochynska: performance of experiments, formal analysis and data curation; Oksana V. Kuzniak: performance of experiments; Dmytro V. Gospodaryov: performance of experiments (mitochondrial respiration); data analysis; Oleh I. Demianchuk: performance of experiments (western-blotting); Yulia V. Vasylyk: performance of experiments; Nadia M. Mosiichuk: performance of
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgements
We thank Dr. E. Dufour from Tampere University (Tampere, Finland) and Prof. M. Y. Vyssokikh from Lomonosov Moscow State University (Moscow, Russian Federation) for the valuable advice on polarography. We thank also our students T. Pankiv, L. Sishchuk, A. Hrushchenko, A. Semchuk, T. Pryimak, A. Klonovsky, V. Balatskyj and M. Lylyk for technical assistance with biochemical measurements.
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