Middle age as a turning point in mouse cerebral cortex energy and redox metabolism: Modulation by every-other-day fasting

https://doi.org/10.1016/j.exger.2020.111182Get rights and content

Highlights

  • Aging intensifies oxidative stress and weakens antioxidant defense in the mouse cerebral cortex.

  • Aging decreases activities of phosphofructokinase and pyruvate kinase and increases activity of glucose-6-phosphate dehydrogenase.

  • The “old” phenotype as determined with the parameters studied is mainly established at middle age.

  • Every-other-day fasting slightly slows down age-related changes.

  • Brain mitochondrial respiration is slightly affected by either aging or every-other-day fasting.

Abstract

Normal brain aging is accompanied by intensification of free radical processes and compromised bioenergetics. Caloric restriction is expected to counteract these changes but the underlying protective mechanisms remain poorly understood. The present work aimed to investigate the intensity of oxidative stress and energy metabolism in the cerebral cortex comparing mice of different ages as well as comparing mice given one of two regimens of food availability: ad libitum versus every-other-day fasting (EODF). Levels of oxidative stress markers, ketone bodies, glycolytic intermediates, mitochondrial respiration, and activities of antioxidant and glycolytic enzymes were assessed in cortex from 6-, 12- and 18-month old C57BL/6J mice. The greatest increase in oxidative stress markers and the sharpest decline in key glycolytic enzyme activities was observed in mice upon the transition from young (6 months) to middle (12 months) age, with smaller changes occurring upon transition to old-age (18 months). Brain mitochondrial respiration showed no significant changes with age. A decrease in the activities of key glycolytic enzymes was accompanied by an increase in the activity of glucose-6-phosphate dehydrogenase suggesting that during normal brain aging glucose metabolism is altered to lower glycolytic activity and increase dependence on the pentose-phosphate pathway. Interestingly, levels of ketone bodies and antioxidant capacity showed a greater decrease in the brain cortex of females as compared with males. The EODF regimen further suppressed glycolytic enzyme activities in the cortex of old mice, and partially enhanced oxygen consumption and respiratory control in the cortex of middle aged and old males. Thus, in the mammalian cortex the major aging-induced metabolic changes are already seen in middle age and are slightly alleviated by an intermittent fasting mode of feeding.

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.

References (76)

  • L.K. Gilmer et al.

    Age-related changes in mitochondrial respiration and oxidative damage in the cerebral cortex of the Fischer 344 rat

    Mech. Ageing Dev.

    (2010)
  • L.A. Gómez et al.

    Age-related decline in mitochondrial bioenergetics: does supercomplex destabilization determine lower oxidative capacity and higher superoxide production?

    Semin. Cell Dev. Biol.

    (2012)
  • G. Gouspillou et al.

    Facts and controversies in our understanding of how caloric restriction impacts the mitochondrion

    Exp. Gerontol.

    (2013)
  • E.H. Heiss et al.

    Glucose availability is a decisive factor for Nrf2-mediated gene expression

    Redox Biol.

    (2013)
  • S. Hoyer

    The effect of age on glucose and energy metabolism in brain cortex of rats

    Arch. Gerontol. Geriatr.

    (1985)
  • A.-G. Lenz et al.

    Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride

    Anal. Biochem.

    (1989)
  • V.I. Lushchak

    Free radicals, reactive oxygen species, oxidative stress and its classification

    Chem. Biol. Interact.

    (2014)
  • V. Lushchak et al.

    Possible role of superoxide dismutases in the yeast Saccharomyces cerevisiae under respiratory conditions

    Arch. Biochem. Biophys.

    (2005)
  • O.V. Lushchak et al.

    The effect of potassium dichromate on free radical processes in goldfish: possible protective role of glutathione

    Aquat. Toxicol.

    (2008)
  • M.P. Mattson et al.

    Hallmarks of brain aging: adaptive and pathological modification by metabolic states

    Cell Metab.

    (2018)
  • M.P. Mattson et al.

    Impact of intermittent fasting on health and disease processes

    Ageing Res. Rev.

    (2017)
  • A.E. McDonald et al.

    Alternative NAD(P)H dehydrogenase and alternative oxidase: proposed physiological roles in animals

    Mitochondrion

    (2019)
  • C.E. Robinson et al.

    Determination of protein carbonyl groups by immunoblotting

    Anal. Biochem.

    (1999)
  • H.M. Semchyshyn et al.

    Fructose compared with glucose is more a potent glycoxidation agent in vitro, but not under carbohydrate-induced stress in vivo: potential role of antioxidant and antiglycation enzymes

    Carbohydr. Res.

    (2014)
  • R.S. Sohal et al.

    Oxidative damage, mitochondrial oxidant generation and antioxidant defenses during aging and in response to food restriction in the mouse

    Mech. Ageing Dev.

    (1994)
  • V. Steffen et al.

    Age-dependent changes in the activity and isoenzymatic pattern of the phosphofructokinase in different areas of the central nervous systems

    Neurosci. Lett.

    (1991)
  • H. Uzun et al.

    The chance of gender dependency of oxidation of brain proteins in aged rats

    Arch. Gerontol. Geriatr.

    (2010)
  • A. Villa et al.

    Sexual differentiation of microglia

    Front. Neuroendocrinol.

    (2019)
  • M.E. Walsh et al.

    The effects of dietary restriction on oxidative stress in rodents

    Free Radic. Biol. Med.

    (2014)
  • L. Zhao et al.

    Sex differences in metabolic aging of the brain: insights into female susceptibility to Alzheimer’s disease

    Neurobiol. Aging

    (2016)
  • R.M. Anson et al.

    Intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and neuronal resistance to injury from calorie intake

    Proc. Natl. Acad. Sci. U. S. A.

    (2003)
  • Y. Benjamini et al.

    Controlling the false discovery rate: a practical and powerful approach to multiple testing

    J. R. Stat. Soc. Ser. B Methodol.

    (1995)
  • H.U. Bergmeyer et al.

    Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides

    Biochem. J.

    (1967)
  • A.K. Bouzier-Sore et al.

    Uncertainties in pentose-phosphate pathway flux assessment underestimate its contribution to neuronal glucose consumption: relevance for neurodegeneration and aging

    Front. Aging Neurosci.

    (2015)
  • A. Boveris et al.

    Brain mitochondrial dysfunction in aging

    IUBMB Life

    (2008)
  • M.M. Bradford

    A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal. Biochem.

    (1976)
  • D.A. Butterfield et al.

    Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease

    Nat. Rev. Neurosci.

    (2019)
  • V. Calabrese et al.

    In vivo induction of heat shock proteins in the substantia nigra following L-DOPA administration is associated with increased activity of mitochondrial complex I and nitrosative stress in rats: regulation by glutathione redox state

    J. Neurochem.

    (2007)
  • Cited by (21)

    View all citing articles on Scopus
    View full text