Aging: Oxidative Stress and Dietary Antioxidants

treatment.

Section 1

Oxidative stress and aging

Chapter 1

Oxidative stress and miR-200c

Alessandra Magentaa; Maria Cristina Floriob; Marco D’Agostinoa; Sara Silenoa    a Experimental Immunology Laboratory, Dermopatic Institute of Immaculate-IDI-IRCCS, Rome, Italy

b Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Biomedical Research Center, Baltimore, MD, United States

Abstract

Reactive oxygen species (ROS) levels are generated consequently to aerobic metabolism and play an important role as second messengers within the cell. Insufficient scavenging or ROS production exacerbation impairs many biological processes. microRNAs (miRNAs) are short noncoding RNA molecules that play key role in cellular homeostasis and in the regulation of redox balance. miR-200 family has been demonstrated to increase upon ROS, and in particular, miR-200c is the member most induced in endothelial cells (ECs). It increases also upon different ROS stimuli and in different cells such as fibroblast, muscle cells, neuronal cells, and tumor cells. miR-200c increase in ECs elicits cell apoptosis and senescence; it decreases the expression of ROS scavengers upregulating ROS and inhibiting NO production. miR-200c upregulation has been linked to different pathophysiological conditions associated with ROS increase, such as aging, ischemia, diabetes, cardiac hypertrophy, nonalcoholic steatohepatitis, Duchenne muscle dystrophy, atherosclerosis, and familial hypercholesterolemia.

Keywords

microRNAs; Oxidative stress; Aging; Apoptosis; Senescence; Endothelial dysfunction

List of abbreviations

BCNU 

1,3-bis(2 chloroethyl)-1-nitrosourea

CAT 

catalase

ECs 

endothelial cells

EMT 

epithelial-mesenchymal transition

eNOS 

endothelial nitric oxide synthase

FOXO1 

forkhead box O1

GPx 

glutathione peroxidase

HEI-OC1 

House Ear Institute-Organ of Corti 1

H2O2 hydrogen peroxide

MAPK 

mitogen-activated protein kinase

miRNA 

microRNA

MnSOD 

manganese superoxide dismutase

mRNA 

messenger RNA

NAC 

N-acetyl-l-cysteine

NOS 

nitric oxide synthase

NOX 

NADPH oxidase

p66Shc 

p66 isoform of ShcA protein

ROS 

reactive oxygen species

pRb 

retinoblastoma protein

RISC 

RNA-induced silencing complex

SIRT1 

sirtuin 1

SOD 

superoxide dismutase

TRBP 

TAR RNA-binding protein

t-BHP 

tert-butyl hydroperoxide

Xpo-5 

exportin 5

ZEB1 

zinc finger E-box-binding homeobox 1

Introduction

Physiological reactive oxygen species (ROS) levels play an important role as second messengers within the intracellular signaling.

ROS, which include superoxide anion, hydroxyl radicals, and hydrogen peroxide (H2O2), are generated as consequence of aerobic metabolism and are produced by several cellular sources. These include mitochondria, plasma membrane NADPH oxidase (NOX), and different enzymes such as several oxidases, peroxidase, cytochromes, mono- and dioxygenases, and uncoupled nitric oxide synthase (NOS). ¹ Enzymatic and nonenzymatic antioxidant defenses such as superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione finely modulate the amount of ROS within the cell. ²

Insufficient scavenging or ROS production exacerbation has been demonstrated to impair many biological processes.

There is a close link between NOS activity and ROS production, since uncoupling of NOS leads to the production of superoxide anion, rather than NO.³,⁴ The cellular transduction pathways induced by the increase in ROS are known to cause growth arrest and senescence, as well as cell death, both by apoptosis and necrosis, based on the level of oxidative stress experienced by the cell and its genotype. A mechanism of induction of apoptosis from oxidative stress involves the p53 tumor suppressor protein; p53, in turn, regulates the intracellular redox state and induces apoptosis through a mechanism involving ROS production. ⁵

A key role in ROS-induced apoptosis is also played by the p66 isoform of the ShcA protein (p66Shc), a fundamental regulator of mitochondrial ROS production from a variety of different stimuli. ⁶

Different studies underlined the role of microRNAs (miRNAs) in cellular homeostasis and in the regulation of redox balance.

microRNA

miRNAs are 21- to 23-nucleotide-long noncoding RNA molecules that modulate the stability and/or the translational efficiency of target messenger RNAs (mRNAs). miRNA genes can be expressed as polycistronic transcripts containing multiple miRNAs, as independent transcripts, or can be embedded in introns of protein coding mRNAs. Commonly miRNAs act as negative regulators of gene expression, although few opposite examples have been described. ⁷

miRNA biogenesis (Fig. 1) starts with a primary transcript transcribed by RNA polymerase II, a generally thousands of nucleotides long mRNA termed the pri-miRNA; only few miRNAs are transcribed by RNA polymerase III. The pri-miRNA is a stem-loop structure containing the active miRNA. This hairpin undergoes nuclear cleavage by the ribonuclease III Drosha, complexed to the RNA-binding protein DGCR8/Pasha, to generate a 70–100 nucleotides pre-miRNA. Notably, most intronic miRNAs can be processed from unspliced intronic regions before splicing catalysis; only a subset of intronic miRNAs, named mirtrons, enters in the miRNA-processing pathway without a Drosha-mediated cleavage. Afterward the pre-miRNA is transported to the cytoplasm by the nuclear export factor exportin 5 (Xpo-5) and then processed by the ribonuclease III Dicer, complexed to TRBP (TAR RNA-binding protein), to form the mature 22-nt miRNA: miRNA* duplex. The mature single-stranded form is incorporated into the RNA-induced silencing complex (RISC), while the complementary strand miRNA* is typically degraded.

Fig. 1 miRNA biogenesis. RNA polymerase II transcribes miRNA genes to generate the primary transcripts (pri-miRNAs). The cleavage is mediated by the Drosha-DGCR8 complex, located in the nucleus. The ~ 70-nucleotide-long product of the nuclear processing is a pre-miRNA, which shows a short stem plus ~ 2-nucleotide 3′ overhang. This structure is the signature motif, recognized by exportin 5 (Exp5), a nuclear export factor. Pre-miRNA constitutes a transport complex with Exp5 and its cofactor Ran. Ribonuclease III Dicer drives the second processing step (dicing) to produce miRNA duplexes in the cytoplasm. The duplex is then separated, and either of the strands is stably associated with RNA-induced silenced complex (RISC). The mature miRNA can inhibit the target genes by promoting translational repression and/or mRNA degradation.

Besides few exceptions, mammalian miRNAs base pair imperfectly with their mRNA targets and induce translational inhibition. Mature miRNAs loaded into the RISC mediate translational inhibition of target mRNAs through several different mechanisms. Moreover, RISC-miRNA complexes can shuttle mRNAs to specialized cytoplasmic compartments, the P-bodies, that are enriched in mRNA-catabolizing enzymes. Consequently, miRNAs also have an important effect on mRNA degradation. Since each miRNA can target multiple transcripts and individual transcripts may be subject to multiple miRNA regulation, it may prove difficult to find a biological process or function that is not, at least in part, under the influence of miRNAs.

The identification of miRNA targets is crucial to elucidate the role played by each miRNA in a biological function. The rules that guide miRNA/mRNA interactions are complex and still incompletely understood. ⁸ The current paradigm states that a complete pairing between the 3′UTR region of the mRNA target and the seed sequence of the miRNA, a region centered on nucleotides 2–7, is required for miRNA-mRNA-mediated inhibition. Thus seed pairing is a necessary requirement for most target prediction algorithms. However, recent studies have demonstrated that also noncanonical miRNA binding can confer target regulation. ⁹ Specifically, some mRNAs are targeted by miRNAs through recognition of 5′UTR or coding sequences. Moreover, seedless miRNA/mRNA interactions have been also demonstrated.¹⁰,¹¹

The dysregulation of miRNAs has been linked to different conditions, including oxidative stress increase; in most cases the miRNA transcription is deregulated. In this respect, recent investigations demonstrated that epigenetic modifications such as histone acetylation or DNA methylation can play an important role. ¹²

miR-200c and oxidative stress

In a miRNA screening of ECs exposed to H2O2, the entire miR-200 family was induced by oxidative stress. ¹³ The miR-200 family is composed of five members (miR-200c, miR-141, miR-200a, miR-200b, and miR-429); in humans, miR-200c and miR-141 are clustered on chromosome 12, whereas miR-200a, miR-200b, and miR-429 are clustered on chromosome 1. They can also be classified on the basis of the seed sequences: miR-200c, miR-200b, and miR-429 share the same seed sequence, whereas miR-200a and miR-141 show a different one. miR-200c is the most expressed family member and the most induced by oxidative stress in ECs; in fact, its expression increases nearly 30-fold upon 16 h treatment of H2O2, which is a very high upregulation for a microRNA; in fact, miRNAs are usually modulated around two- or threefolds. ¹³ miR-141 also is highly induced, similarly to the cotranscribed miR-200c, and the other members are all induced albeit to a lower level (around two- to eightfold maximum). miR-200c upregulation upon H2O2 occurs also in murine myoblast, myotubes and in human fibroblast. ¹³ Other interventions causing redox imbalance, such as the alkylating agent 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU), a glutathione reductase inhibitor that blocks the conversion of oxidized to reduced glutathione, is also able to increase miR-200c expression. Moreover, BCNU-induced miR-200c increase is inhibited by the free-radical scavenger N-acetyl-l-cysteine (NAC), confirming the oxidative stress dependence of miR-200c upregulation. ¹³

The upregulation of miR-200 family is also observed in different context and with different sources of oxidative stress summarized in Table 1; therefore it seems to be a conserved mechanism.

Table 1

miR-200c is upregulated by chronic oxidative stress-induced senescence in human fibroblasts and in human trabecular meshwork cells. ¹⁴ miR-200c and miR-141 upregulation is observed in a cell model of oxidative stress by treatment of House Ear Institute-Organ of Corti 1 (HEI-OC1) cells with different concentrations of tert-butyl hydroperoxide (t-BHP). ¹⁵ miR-200 family induction following H2O2 exposure has been confirmed also in different cell lines, that is, mouse primary hippocampal neurons, ¹⁶ human and mouse immortalized fibroblasts, colon carcinoma, mammary gland epithelial cells and human cell lines, melanoma cells, kidney cells, breast adenocarcinoma, and ovarian adenocarcinoma. ¹⁷ miR-141 and miR-200a, which display the same seed sequence, target p38α mitogen-activated protein kinase (MAPK). p38α is a signaling molecule that modulates cellular responses to stress ¹⁸ and is involved in proliferation and survival control of many cell types. ¹⁹ Indeed, p38α redox-sensing function is essential in the control of tumor development. ²⁰ Therefore enhanced expression of miR-200 family miRNAs not only mimics p38α deficiency and increases tumor growth in mouse models but also improves the response to chemotherapeutic agents.

In keeping the modulation of miR-200c and other miR-200 family expression has been exploited to sensitize different tumors to therapy.²¹–²⁴

miR-200c induces apoptosis and senescence via ZEB1 inhibition

The transcriptional factor zinc finger E-box-binding homeobox 1 (ZEB1) is a direct target of the entire miR-200 family and inhibits the transcription miR-200 family in a negative feedback loop. ²⁵ This loop has been investigated in the epithelial-mesenchymal transition (EMT) of many tumors, where miR-200 family expression levels decrease by determining the increase of their target protein ZEB1, which is an inhibitor of the adhesion molecule E-cadherin. Therefore the decrease of miR-200 determines the lack of cell adhesion causing metastasis. ²⁵

The increase of miR-200c caused by oxidative stress in ECs downregulates ZEB1 that in nontumor cells elicits apoptosis and senescence, a state of permanent cell growth arrest in response to oxidative stress, recapitulating the oxidative stress-induced phenotype. ¹³ Indeed the forced expression of ZEB1 in ECs overexpressing miR-200c partially rescued this phenotypes. ¹³

The molecular mechanisms elicited by oxidative stress in ECs are depicted in Fig. 2.

Fig. 2 miR-200c upregulation in ECs occurs via p53- and pRb-dependent mechanisms. H 2 O 2 induces a rapid dephosphorylation of pRb via a serine-threonine phosphatase (PP2A)-dependent mechanism, repressing transcription factor E2F activity. ZEB1 is under pRb/E2F control. Therefore H 2 O 2 dephosphorylating pRb causes ZEB1 mRNA and protein level downregulation. ZEB1 is a transcriptional inhibitor of miR-200c; consequently, its decrease provokes miR-200c upregulation. miR-200c transcriptional upregulation is also induced by p53, which is induced by oxidative stress. p53 in turn induces p21, which sustains pRb dephosphorylation, further decreasing ZEB1. ZEB1 downregulation induces p21 transcription, being ZEB1 a transcriptional inhibitor of p21. Finally, ZEB1 demise provokes growth arrest, senescence, and apoptosis.

miR-200c upregulation by oxidative stress in ECs occurs at the transcriptional level, since H2O2 upregulates also the pre-miR-200c and this upregulation requires the tumor suppressors protein p53 and the retinoblastoma protein (pRb). ¹³ Furthermore, ZEB1 transcription is under the control of pRb/E2F. ²⁶ In ECs, H2O2 induces a rapid pRb dephosphorylation via a serine-threonine phosphatase named PP2A²⁷,²⁸; this allows pRb binding and inhibition of the E2F transcriptional activity factor on ZEB1 gene. ²⁶ Therefore, upon oxidative stress, the upregulation of miR-200c inhibits ZEB1 protein translation and induces ZEB1 mRNA degradation; in addition, ZEB1 mRNA decreases because of a pRb/E2F-dependent mechanism. ZEB1 demise induces a further upregulation of miR-200c, since it is a transcriptional inhibitor of miR-200c, ²⁵ reinforcing the upregulation of miR-200c. ¹³ Finally, oxidative stress induces p53 activity that is known to induce the cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 (p21) transcription that reinforce pRb dephosphorylation. ²⁹ Additionally, p53 induces apoptosis and miR-200c transcription, enhancing miR-200c increase. Finally, ZEB1 is a transcriptional inhibitor of p21 ³⁰; therefore its downregulation further increases p21. This molecular circuitry induces growth arrest, apoptosis, and senescence, which are all the effects induced by oxidative stress (Fig. 2).

miR-200c oxidative stress and endothelial dysfunction

A fundamental molecular circuit for vascular homeostasis is the one existing among sirtuin 1 (SIRT1), endothelial nitric oxide synthase (eNOS), and forkhead box O1 (FOXO1) proteins. These proteins play a key role in endothelial function, since they are involved in oxidative stress resistance (Fig. 3). SIRT1 plays an important role in cell senescence, having an antiinflammatory and antioxidant role. ³¹ Indeed, it is known that ROS and aging cause a reduction in the expression of SIRT1, causing senescence and endothelial dysfunction, and the activation of SIRT1 improves the response to oxidative stress. ³² Furthermore, SIRT1 promotes mitochondrial biogenesis and NO production released by eNOS.³¹,³²

Fig. 3 miR-200c disrupts the regulatory loop existing among eNOS/SIRT1/FOXO1. miR-200c inhibits situin1 (SIRT1)/endothelial nitric oxide synthase (eNOS)/forkhead box O1 (FOXO1) regulatory loop, by targeting directly all of them. This loop controls different cell functions. Specifically, miR-200c increases ROS production by two mechanisms: (i) it decreases ROS scavengers by targeting peroxiredoxin 2 (PRDX2) and forkhead box O1 (FOXO1), a transcription factor required for catalase (CAT) and manganese superoxide dismutase (MnSOD) expression; (ii) it sustains ROS production via p66Shc phosphorylation in serine 36. miR-200c decreases NO production by targeting eNOS. miR-200c induces senescence targeting SIRT1 deacetylase that causes the following: (i) eNOS acetylation increase further reducing the bioavailability of NO that is a molecule that stabilizes SIRT1 mRNA and protein, thus provoking a further SIRT1 decrease, and (ii) acetylation of FOXO1 increase causing FOXO1 transcriptional activity inhibition. In addition, miR-200c targets FOXO1 both directly and indirectly via a p66Shc phosphorylation-dependent mechanism that inhibits FOXO1 transcriptional activity, decreasing CAT and MnSOD expression, further sustaining ROS production. In conclusion, ROS-induced miR-200c disrupts SIRT1/FOXO1/eNOS loop promoting cell senescence and activating a complex molecular pathway that sustains oxidative stress and miR-200c upregulation.

In turn, NO increases the stability of mRNA and SIRT1 protein, confirming the existence of a positive circuit between eNOS activity and SIRT1 expression levels. ³² In the molecular circuit existing between eNOS and SIRT1, the transcription of FOXO1 plays a very important role.

In fact, FOXO1 is a direct target of SIRT1 deacetylation; deacetylation of FOXO1 induces its transcriptional activity on the promoter of SIRT1 and of CAT and manganese superoxide dismutase (MnSOD) antioxidant enzymes. ³²

The correct functioning of the SIRT1/eNOS/FOXO1 molecular circuit plays a fundamental role in endothelial survival and in vasodilation (Fig. 3).

An important player in oxidative stress response and modulation is the p66 isoform of ShcA protein (p66Shc), a key regulator of mitochondrial ROS production by a variety of different stimuli. ³³ Oxidative stress induces the phosphorylation of p66Shc protein in serine 36 residue (Fig. 3), which causes an increase in ROS in three cellular districts:

-in the nucleus, where it inhibits FOXO1³⁴;

-in the mitochondria, where it binds to cytochrome c, acting as an oxidoreductase enzyme that generates ROS³⁵;

-in the plasma membrane, where it activates NADPH oxidase.³⁶

miR-200c has been shown to target directly SIRT1, eNOS, and FOXO1 ³⁷; ROS-upregulated miR-200c decreases NO and increases the acetylation of SIRT1 protein targets, such as FOXO1 and p53. p53 acetylation increases its transcriptional activity on miR-200c and also its ability to induce senescence and apoptosis. FOXO1 acetylation inhibits its transcriptional activity, decreasing the expression of antioxidant enzymes such as CAT and MnSOD, therefore increasing ROS production. Consequently, miR-200c increases ROS and induces the phosphorylation of p66Shc protein in Ser-36, which in turn inhibits the transcriptional activity of FOXO1, reinforcing this molecular circuit (Fig. 3).

Moreover, miR-200c has been demonstrated to target directly also peroxiredoxin 2, ³⁸ a selective scavenger for H2O2, ³⁹ further inducing oxidative stress.

Aging is a process of functional deterioration of an organism that can occur at different levels (cellular, tissue, and organelle), bringing life to end. One of the most relevant players in the aging process and age-related disorders is cellular senescence. In humans and in many animal models, an age-dependent increase in oxidative stress has been demonstrated, showing that increased ROS can be both a consequence and a cause of aging.

In keeping, the in vitro results obtained in ECs are also recapitulated in different in vivo oxidative stress models, namely, in human skin fibroblasts from elderly donors, femoral arteries of old mice, and in a mouse model of ischemia of the hind limbs. ³⁷ In all cases, miR-200c was higher than the control, and its targets, that is, SIRT1, eNOS, and FOXO1, were decreased. In the mouse model of hind limb ischemia, treatment with anti-miR-200c restored the expression of these proteins and limb perfusion. ³⁷

Aging increases miR-200c expression also in other tissues different from the aforementioned described and all summarized in Table 2. These include skeletal muscle from rhesus monkeys ⁴⁰ and human liver. ⁴¹

Table 2

Applications to other diseases or conditions

miR-200c is increased in different pathological conditions associated to an increase of oxidative stress (summarized in Table 3) such as acute hind limb ischemia in skeletal muscle,¹³,³⁷ ischemia and ischemia/reperfusion in the brain, ⁴² in different tissues in diabetes,⁴³–⁴⁷ high glucose-induced cardiac hypertrophy, ⁴⁸ nonalcoholic steatohepatitis, ⁴⁹ and Duchenne muscle dystrophy. ⁵⁰ Moreover, miR-200c and the entire miRNA family are deregulated in many different types of cancers, since they are deeply involved in the EMT of tumor cells. ⁵¹

Table 3

In many of the previously described disease conditions, the inhibition of miR-200c was able to recover most of thedeleterious effects caused by miR-200c increase.³⁷,⁴⁸–⁵⁰

miR-200c expression is also increased in cardiotoxicity induced by anthracyclines, such as doxorubicin (DOX) treatment in mice and in human cardiac mesenchymal progenitor cells. ⁵²

Furthermore, circulating miR-200c levels are increased in children with familial hypercholesterolemia ⁵³ and in adult patients with carotid artery plaques, ⁵⁴ both conditions associated with enhanced ROS.

Moreover, miR-200c increases also in atherosclerotic carotid plaques in human patients, and it is more enhanced in unstable than stable plaques. ⁵⁴

In conclusion, miR-200c increases in an age-dependent manner and in different pathological conditions that display an increase of ROS. miR-200c upregulation causes a decrease of antioxidants, an increase of ROS, and a decrease of NO, all features associated with aging.

Therefore the comprehension of miR-200c roles and targets may support the analysis of new therapeutic strategies to delay and treat age-related modifications and aging signs.

Summary points

•ROS play a causative pathological role in different cellular processes implicated in growth arrest, senescence, and cell death.

The cells have developed a series of enzymatic and nonenzymatic antioxidant defenses to maintain the intracellular redox state homeostasis.

miRNAs are short noncoding RNAs that modulate the stability and/or the translational efficiency of target messenger.

Each miRNA can regulate multiple mRNAs, and each mRNA can be regulated by many miRNAs.

miRNAs can act as key regulators in cell proliferation and cell death, early development, apoptosis, metabolism, and cell differentiation.

Dysregulation of miRNAs has been associated with different diseases.

•miR-200 family is induced by different sources of oxidative stress and in many different nontumor and tumor cells.

miR-200 family is composed of five members: miR-200a, miR-200b, miR-200c, miR-141, and miR-429. miR-200c is the most upregulated family member in ECs upon H2O2 treatment.

miR-200c upregulation by H2O2 in ECs is transcriptional and involves p53 and pRb.

ZEB1 is a direct target of the entire miR-200 family and inhibits the transcription of miR-200 family in a negative feedback loop.

ZEB1 plays important functions in the tumor progression process and in metastatic diffusion inhibiting E-cadherin.

ZEB1 demise induced by oxidative stress in ECs is caused by miR-200c- and pRb/E2F-dependent mechanisms.

ZEB1 downregulation in nontumor cells is responsible for cell growth arrest, apoptosis, and senescence induction.

•The loop existing among SIRT1, eNOS, and FOXO1 is a molecular regulatory circuit involved in the maintenance of vascular homeostasis.

These proteins are involved in resistance to oxidative stress and play fundamental roles in EC survival and vasodilation.

miR-200c destroys the loop existing between SIRT1, eNOS, and FOXO1 targeting all of them.

miR-200c disrupting this loop increases ROS and decreases NO, causing endothelial dysfunction.

Moreover, miR-200c enhances oxidative stress inducing p66Shc protein phosphorylation in Ser36 that sustains ROS increase.

miR-200c is upregulated in different pathophysiological conditions associated with an increase of ROS including, aging, diabetes, nonalcoholic steatohepatitis, diabetic cardiac hypertrophy, hind limb ischemia, brain ischemia reperfusion, Duchenne muscle dystrophy, familial hypercholesterolemia, and atherosclerosis.

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Chapter 2

Caloric restriction, reactive oxygen species, and longevity

Miyuki Kobara; Hiroe Toba; Tetsuo Nakata    Department of Clinical Pharmacology, Division of Pathological Science, Kyoto Pharmaceutical University, Kyoto, Japan

Abstract

The aging population in developed countries is markedly increasing, and the elongation of life and health spans is an emergent need. An inevitable process in all living organisms is aging, in which oxidative stress plays critical roles. Organ functions gradually decrease, and the prevalence of age-related diseases, including diabetes, cardiovascular diseases, cancer, and neural degenerative diseases, increases. Caloric restriction (CR) is a powerful nongenetic intervention for the aging process, which reduces susceptibility to age-related diseases and extends the life span of many species. Multiple signaling pathways contribute to the beneficial effects of CR. In nutrient restriction, growth and protein synthesis signaling pathways, such as the insulin/insulin growth factor 1 (IGF-1) and target of rapamycin (TOR) pathway, are suppressed. Furthermore, as a metabolic adaptation, AMP-dependent protein kinase (AMPK) and sirtuin signaling pathways are activated. Direct evidence for CR-induced life span elongation in humans is difficult to obtain; however, reductions in susceptibility to age-related diseases by CR have been demonstrated in clinical trials. In this chapter, we outline the beneficial effects of CR in a number of species, including humans, and describe the mechanisms responsible with a focus on oxidative stress.

Keywords

Aging; Caloric restriction; Food intake; Life span; Health span

List of abbreviations

AMPK 

adenosine 3′,5′-monophosphate-dependent protein kinase

CR 

caloric restriction

FoxO 

forkhead transcription factor O

GPx 

glutathione peroxidase

hsCRP 

high-sensitivity C-reactive protein

IGF-1 

insulin growth factor 1

IL-1β 

interleukin-1β

NFkβ 

nuclear factor kappa β

PGC1-α 

peroxisome proliferator-activated receptor-γ coactivator 1-α

ROS 

reactive oxygen species

SIRT1 

silent information regulator 1

SOD 

superoxide dismutase

TNF-α 

tumor necrosis factor α

TOR 

target of rapamycin

Introduction

Aging is inevitable and progressive phenomena in all living organisms. The accumulation of damage leads to functional decline and structural deterioration in organs in association with increases in susceptibility to diseases, ultimately resulting in death as the final stage. Age-related diseases, such as cancer, cardiovascular diseases, stroke, and neurally degenerative diseases, are now the leading causes of mortality worldwide. ¹ In antiaging research conducted to date, numerous interventions to retard the aging process have been examined. Among antiaging interventions, caloric restriction (CR), a reduction in caloric intake without malnutrition, is a powerful nongenetic intervention that prolongs maximum and mean life spans and suppresses age-related diseases.²–⁴ These favorable phenomena are conserved in many species from yeast to mammals.²–⁵ Therefore, elucidation of the mechanism responsible for the beneficial effects of CR may reveal targets for age-related disease retardation and healthy life span elongation.

Adequate CR extends healthy life span, whereas excessive CR with malnutrition is harmful. Therefore, CR to an appropriate extent and for a sufficient duration to maximize healthy life span is important. Treatment costs for age-related diseases are now markedly increasing in the aging society, and thus the cost-effective prevention of age-related diseases is desired. CR has favorable cost benefits. In this chapter, we summarize the effects of CR in a number of organisms and describe the main proposed signaling pathways for the beneficial effects of CR, with a focus on oxidative stress.

CR in animals

CR was initially reported by McCay in 1935, who showed that a reduced food intake extended the life span of rats. ⁶ Subsequent studies in this field confirmed favorable life span elongation by CR in many organisms from worms to nonhuman primates. ² CR also retarded age-related cancers, cardiovascular diseases, stroke, diabetes, dyslipidemia, and neural degenerative diseases in these short-lived organisms. ⁷ These age-related diseases are major causes of mortality, and neural disorders decrease the quality of life and shorten the health span of the elderly.

As a bridge from these short-lived animals to humans, prospective examinations on the effects of CR in rhesus monkeys were initiated by two independent associations in the United States from the late 1980s: the Wisconsin National Primate Research Center based at the University of Wisconsin-Madison (WNPRC) study and the National Institute on Aging (NIA) study. In the WNPRC study, 30% CR in adult rhesus monkeys significantly delayed age-associated pathological conditions and improved all-cause mortality. ⁸ On the other hand, in the NIA study, all-cause mortality did not improve in CR monkeys even though similar CR was conducted; however, measurements on the metabolic health index and overall function showed that old-onset CR exerted beneficial effects. ⁹ Although the impact of CR on mortality differs, comparisons of important evidence from these long-term studies demonstrated that CR resulted in a favorable lipid profile and reduced susceptibility to cancer, type 2 diabetes, and cardiovascular diseases. ¹⁰

Mechanisms responsible for beneficial effects of CR in animals

Oxidative stress and inflammation

In the aging process, tissue and cellular damage induced by oxidative stress and inflammation accumulate. ¹¹ Oxidative stress also enhances the progression of age-related diseases, including cancer, neurodegeneration, cardiovascular diseases, and diabetes. ¹² CR studies using animal models and humans demonstrated that CR reduced the production of ROS, resulting in decreases in lipid peroxidation, protein carbonylation, and DNA/RNA oxidation, and also increased the antioxidant capacity, including glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase.⁷,¹²–¹⁶ CR was shown to suppress the age-related and oxidative stress-related diseases of cancer, neurodegeneration, and cardiovascular diseases. ¹² It also inhibited inflammation and reduced the levels of inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β). ² Among several sources of ROS production in the aging process, damaged mitochondria are important. ¹⁷ Mitochondrial ROS were shown to induce mitochondrial DNA mutations, leading to an impaired mitochondrial respiratory chain, while a deficiency in mtDNA polymerase promoted premature aging in mammals. ¹⁸ Previous studies found that CR attenuated mitochondrial ROS production from the respiratory chain and preserved its function.⁷,¹⁹,²⁰ Therefore, life span elongation by CR may be due to, at least in part, antioxidant activities and the preservation of mitochondrial function. ¹⁹ However, the importance of antioxidants for CR-induced life span elongation remains controversial. One of the reasons for this is the absence of an effect of the deletion of NF-E2-related factor 2, a well-known transcription factor of antioxidant enzymes, on CR-induced life span elongation. ²¹

Moreover, Wang et al. reported that CR-induced mild oxidative stress by mitochondria plays positive and integrative roles as adaptive phenomena, which are beneficial rather than detrimental in the aging process. ¹⁷ Previous studies reported that CR-induced ROS also slowed aging and prevented the progression of cancer. ²² Therefore, the roles of ROS in the effects of CR may be beneficial and detrimental depending on their source and volumes.

Insulin/IGF-1 pathway

During nutrient restriction, growth signaling by the insulin/IGF-1 pathway is suppressed as an adaptation, ² and animal models of the genetic suppression of the insulin/IGF-1 pathway showed an extended life span.²,²³ The CR-induced suppression of the insulin/IGF-1 signaling activates the forkhead family of transcription factors (FoxO) through phosphatidylinositol-3-kinase (PI3K) and Akt inhibition.²⁴,²⁵ Since FoxO3 is a transcription factor of several antioxidant genes, ²⁶ this signaling pathway contributes to the CR-induced inhibition of oxidative stress. However, the contribution of this signaling pathway to CR-induced life span elongation has not been fully clarified, because CR could elongate life span in genetic knockout animals of IGF-1 signaling. ² Therefore, the insulin/IGF-1 signaling pathway is enhanced during CR and contributes to its beneficial effects; however, the dependency of CR-induced life span elongation on this signaling pathway is limited.

The TOR pathway and autophagy

The TOR signaling pathway is another nutrient sensor that is suppressed by CR. ²⁷ As another candidate pathway for CR-induced longevity, the suppression of TOR signaling has been confirmed, and its deletion has been shown to extend the life span of many organisms.²,²⁷ TOR suppression under nutrient starvation conditions enhanced autophagy, which removes damaged mitochondria, a major source of ROS production, and preserves their function.²⁸,²⁹

The AMPK pathway

AMPK is an energy sensor of nutritional starvation. CR activates AMPK in association with declines in the AMP/ATP ratio in many organisms, and the deletion of AMPK blunts CR-induced life span elongation. ³⁰ AMPK regulates many intracellular signaling pathways, including the activation of FoxO, ³⁰ SIRT1, peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α), and the suppression of TOR. ⁴ These factors collaborate with the suppression of oxidative stress. Another pathway for AMPK-mediated ROS reductions is the preservation of NADPH levels through the suppression of acetyl CoA carboxylases. ³¹

Sirtuins

Sirtuins are a family of NAD+-dependent protein deacetylase that regulate the activities of many enzymes by deacetylation. Under nutrient restriction states, sirtuins are activated as nutrient sensors in association with increments in the NAD+/NADH ratio and mediate CR-induced life and health span elongation.³²,³³ In a mouse model the attenuation of aging-related hearing loss by CR was blunted in SIRT3 deletion mice. ³⁴ In mammals, CR has also been shown to activate sirtuins, particularly SIRT1, in many organs, leading to an enhanced antioxidant capacity by FoxO3.²⁶,³³ In addition, CR activated mitochondrial SIRT3, which preserved the activity of Mn-SOD ³⁵ and enhanced the mitochondrial glutathione antioxidant defense system, leading to mitochondrial protection. ³⁶

Mitochondrial maintenance

In the aging process the abundance of mitochondrial DNA and its functions were found to be impaired in human skeletal muscle. ³⁷ Mitochondria are central to energy metabolism in cells and nutrient restriction induces metabolic reprogramming in mitochondrial energy metabolism. The AMPK/PGC1-α pathway and mitochondrial sirtuin SIRT 3 play critical roles in maintaining mitochondrial function, and both signaling pathways were shown to be activated by CR.³⁰,³³ The genetic deletion of SIRT3 abolished protection of mitochondria by CR. ⁴ Changes in membrane lipid compositions and peroxidation, which are involved in the aging process, ³⁸ were recently shown to regulate mitochondrial function and affect CR-induced longevity. ³⁹ Collectively, these finding show that mitochondrial maintenance and biogenesis play important roles in the beneficial effects of CR.

In summary, multiple signal transduction pathways are activated in nutritional restriction, and cross talk among each signaling pathway influences the beneficial effects of CR. Since the majority of these signaling pathways reduce oxidative stress, the suppression of ROS production and protection of mitochondria by CR have critical roles in life span elongation and the retardation of age-related diseases. On the other hand a small amount of ROS functions as a trigger for mitochondrial maintenance and has beneficial roles (Fig. 1).

Fig. 1 Relationships between signal transduction in CR and oxidative stress. A number of nutrient sensors are modified in CR and contribute to CR-induced life span elongation and the retardation of age-related disorders in organisms.

CR in humans

The most important question in this research field is whether the beneficial effects of CR, such as life and health span elongation, are conserved and if CR is feasible and safe in humans. However, the human life span is long, and the strict restriction of nutrient intake for a long period is very difficult. Therefore direct evidence for the beneficial effects of CR on life span in humans is difficult to obtain. Therefore the majority of studies being conducted on humans in the CR research field involve the effects of short-term CR on candidates, which are assumed to achieve life span elongation in animal models.

The first human study on CR was performed by Strom et al. in 1951. ⁴⁰ In this study, Scandinavians were placed on a semistarvation diet in the 1940s as a consequence of World War II, and this was associated with a reduction in the prevalence of heart diseases. ⁴⁰ Several clinical trials were subsequently performed. The main randomized clinical trials on human CR were conducted in the United States in the early 21st century. Three independent pilot studies (comprehensive assessment of the long-term effects of reducing intake of energy [CALERIE]-1) were performed at Tufts University, Washington University, and the Pennington Biomedical Research Center, using different protocols. To confirm the long-term feasibility and effectiveness of CR, a multicenter single protocol study was also conducted (CALERIE-2) (Table 1).

Table 1

Data from Das SK, Balasubramanian P, Weerasekara YK. Nutrition modulation of human aging: the calorie restriction paradigm. Mol Cell Endocrinol 2017;455:148–57. Il'yasova D, Fontana L, Bhapkar M, Pieper CF, Spasojevic I, Redman LM, Das SK, Huffman KM, Kraus WE, CALERIE Study Investigators. Effects of 2 years of caloric restriction on oxidative status assessed by urinary F2-isoprostanes: the CALERIE 2 randomized clinical trial. Aging Cell 2018;17:e12719, with permission from Publishers.

CALERIE-1 study

In the CALERIE-1 study conducted at Tufts University, 30% CR in obese individuals for 1 year significantly reduced body weight, improved glucose-insulin dynamics, increased the plasma GPx capacity, an antioxidant, and decreased protein carbonyl levels, an indicator of oxidative stress. ⁴¹ On the other hand, SOD and catalase levels were not affected by this CR. ⁴¹ However, 30% CR was not feasible for more than 1 year.

In the CALERIE-1 study conducted at Washington State University, nonobese individuals were included and separated into two groups: 20% dietary CR (CR group) or 20% increased energy expenditure by exercise (EX group) for 1 year. Energy reductions were similar between the CR and EX groups. Insulin sensitivity, blood pressure, and lipid profiles showed similar improvements in the EX and CR groups,⁴²,⁴³ and DNA and RNA oxidation in white blood cells was reduced. ⁴⁴

In the CALERIE-1 study conducted at the Pennington Biomedical Research Center (PBRC), overweight individuals were enrolled. Participants in the CR alone group (25% reduction in nutritional caloric intake alone) and CR + EX group (mild nutritional caloric reduction [12.5%] with 12.5% increase in energy expenditure by exercise) showed a lower core body temperature, a previously reported index of longevity, reduced body weight, and a favorable lipid profile and insulin sensitivity. ⁵ Furthermore, the expression of mitochondrial function-preserving genes, such as PGC1-α, TFAM, and SIRT1, and the mitochondrial DNA content in skeletal muscle were preserved in both CR groups.