Modulatory Effects of Calorie Restriction on Senoinflammation Underlying Aging Process

DOI 10.17480/psk.2019.63.5.251

Review

Modulatory Effects of Calorie Restriction on Senoinflammation Underlying Aging Process

EunJin Bang*, Hee Jin Jung*, Dae Hyun Kim*, Sugyeong Ha*, Byung Pal Yu**, and Hae Young Chung*

*Department of Pharmacy, College of Pharmacy, Pusan National University

**Department of Physiology, The University of Texas Health Science Center at San Antonio (Received September 17, 2019; Revised October 7, 2019; Accepted October 8, 2019)

Abstract Age-related chronic inflammation is characterized by dysregulated inflammation that is not resolved by the multivariate low, chronic, and systemic inflammatory reactions that aggravate aging progression. A novel concept of senoinflammation proposes an age-related senescent inflammation, which is an integrated systemic view of chronic inflammation and metabolism. It discusses multiphase inflammatory networks and pro-inflammatory pathways in aging, including hyperactivation of nuclear factor (NF)-κB signaling and persistent secretion of cytokines, chemokines and induction of endoplasmic reticulum (ER) stress and lipids accumulation. Previously reporting evidence suggests that caloric restriction (CR) lowers oxidative stress and mediates anti-inflammatory action in age-related diseases, including metabolic syndrome and inflammatory diseases. This review focuses on senoinflammation and modulatory effects of CR, which is a well-known nutritional mediator of reduced energy intake. CR is known to increase maximum life expectancy and prevent age-related illnesses. Therefore, CR is well-accepted standard for aging intervention studies to discover the basic molecular mechanism of the original aging process. The current focus of CR research is on beneficial effects on decline in age- related physiological functions and preventing and delaying age-related diseases. The development of the non-human and human CR research studies provides assuring possibility for the beneficial effects of CR on healthy aging. Some major issues in understanding the anti-aging mechanism in CR studies are the importance of modulating chronic inflammation at the molecular level and consequential gene expression regulation of chromatin and histone modification. Furthermore, beneficial effects of CR mimetics (CRM) are discussed in comparison with that of CR.

Keywords aging, chronic inflammation, senoinflammation, calorie restriction (mimetics), anti-aging molecular mechanism

Introduction

Aging increases the occurrence of age-related pathological symptoms in biological aging processes characterized by time- dependence, progression, and decline in physiological function.

It is noteworthy that pro-inflammatory mediators are activated and commonly upregulated during aging progression. As is well- accepted, a chronic inflammatory response is the main cause of diverse age-related illnesses.

Recently, we proposed a novel idea of senoinflammation to suggest evidence of the molecules that connect normal non-pathological and pathological aging.

During the aging process, chronic inflammation occurs over time at low levels for the activation of leukocytes accumulated with macrophages and lymphocytes.

During the chronic inflammatory

response, both recovery and destruction are induced by redox and cell death signaling pathways to damaged cells and tissues.

Such chronic inflammatory response is closely associated with the alternating redox state that is causally related to inflammation.

The delicate interaction between inflammation and the redox environment induces aging and senescence-associated inflammatory diseases.

Chronic inflammatory conditions suppress immune function, increase the body’s self-reactivity, and are beneficial to harmful cytokines and chemokines in the early stages of life.

Furthermore, they have a detrimental effect on aging and is associated with acceleration of progression of pathological diseases.

The age-related senoinflammation concept details the systemic broad up-regulation of pro-inflammatory nuclear factor (NF)- κB transcription factor signals at the genomic level induced by oxidative stress during aging.

The gene regulation of oxidative environmental imbalance in senescent cells follows systemic cytokine and chemokine activity before altering endoplasmic reticulum (ER) stress, inflammation, lipid accumulation. Based on scientific evidence for biological analysis, senescence-associated

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Corresponding author

Hae Young Chung, Department of Pharmacy, Pusan National Univer- sity, Busan 46241, Republic of Korea

Tel: +82-51-510-2814, Fax: +82-51-518-2821

E-mail: [email protected]

secretory phenotypes (SASPs) are mainly comprised of cytokines and chemokines, which are consistently released during the aging process.

On the contrary, caloric restriction (CR) modulates inflammatory effects and suppresses cytokines and chemokines.

This result further solidifies the new concept of senoinflammation for its use in the aging process. Previous reports for a modulatory role of CR in senoinflammation and coordination generally provides an understanding of the aging mechanisms and potential therapeutic interventions and encourages healthy aging and longevity.

A new concept of the expanded view of age-related chronic inflammation and metabolic changes is called senescent- inflammation or “senoinflammation”. The NF-κB signaling pathway has been known as one of the most important core pathways responsible for inflammation.

In the previous studies, age-related inflammatory NF-κB signaling has been shown to include TNF-α/β, IL-1β, IL-6, IL-8, MCP-1 chemokines and adhesion molecules, and other major pro-inflammatory transcription factors, including p53, AP-1, STAT, and NF-κB.

Active NF- κB stimulates inflammatory-mediated production and suppresses cancer cell death.

NF-κB also interacts with signal transducers, transcription 3 activators (STAT3), and other transcription factors, such as p53.

The glycogen synthase kinase 3 (GSK3)-β and protein kinase B (PKB) involved in cancer also regulate NF-κB transcriptional activity.

The pro-inflammatory senescence- related secretomes secretion and age-related secretory phenotype SASP add significant stress at the tissue, organ, and systemic levels, as well as on the intracellular organelles affecting metabolic disorders, such as diabetes.

The vicious cycle of SASP secretion and metabolic regulation is the basis of the aging progression and age-related pathological diseases, such as arteriosclerosis, diabetes, obesity, osteoporosis, and Alzheimer’s disease.

To maintain an immune response during age-related inflammation, an important role of NF-κB is to increase secretion of various cytokines and chemokines and activation of pro-inflammatory cells. Alder et al. demonstrated that genes that are upregulated in senescence contained transcription factor motif region that highly associates with NF-κB transcription factor, indicating its close link to senescence.

Sustained hyperactivation of NF-κB was observed in tissues such as the skin, kidney, heart, brain, and muscle.

Several aging studies for NF-κB and its associated signaling pathways have presented important molecular insights into the changed cellular signaling system underlying chronic inflammation during aging.

These altered signaling molecules and pathways include the insulin and insulin-like growth factor (IGF) pathway, AMP-activated protein kinase (AMPK) and its mechanistic target

mTOR pathway, and Forkhead box O (FOXO) family, sirtuin 1 (SIRT1) and PPARs pathways.

The most prominent cellular changes in the inflammatory response in aging-associated chronic diseases are related to macrophages. Macrophages exert biological activities such as M1/

M2 polarization, phagocytic activity, toll-like receptor signaling, and wound healing.

In aged mice, a decrease in the expression of macrophage cell surface receptors, such as Major Histo- compatibility Assessment (MHC)-II protein, and reduction of antigen-presenting capability of IFN-γ was reported. Moreover, a significant discrepancy in TLR signaling, M1/M2 polarization, and NF-κB signaling was observed in senescent macrophages in comparison to young macrophages.

The increase in innate immune macrophages is accompanied by an increase in robust immune cells, such as B cells, T cells, natural killer (NK) cells, and neutrophils. In aging tissues, it was commonly observed that immune cell infiltration increases and induces chronic inflammation in the aging process. During aging, aged macrophages show M2 polarized phenotype, decreased secretion level of pro-inflammatory cytokine and, damaging phagocytosis.

Macrophages are programmed to remove aged cells that release senescence-related SA, generally referred to as SASP.

As mentioned previously, age-related systemic inflammation is different from the acute inflammation as it is characterized by a high existing level of pro-inflammatory mediators. It is well known that adipose tissue is one of the major organs that play a role in the systemic inflammatory response. In adipose tissue, elevated macrophage infiltration not only causes inflammation but also induces the production of various inflammatory cytokines that leads to the occurrence of systemic inflammation.

Macrophage infiltration into adipose tissue aggravates age-related systemic inflammation as aging is associated with an increased amount of fat.

In fact, numbers of experimental evidence suggest the continuous existing conditions of lower, chronic inflammation in the elderly population

This observation and experimental data support senoinflammation concept (Table 1).

CR not only reduces the rate of biological aging in various

organisms but also protects against age-related pathological

symptoms, for example, cardiovascular diseases, diabetes, obesity,

rheumatoid arthritis at both molecular levels.

Age-associated

hyperactivation of NF-κB, IL-1β, IL-6 TNF-α, cyclooxygenase 2

(COX-2), and inducible nitric oxide synthase (iNOS) are all

decreased by CR.

The establishment of the main metabolic

pathways modulated by CR has led to the finding of molecular

targets that can be regulated by CR mimetics.

This review

describes the general concept of CR and CR mimetics and

discusses supporting evidence for its beneficiary effects on chronic

inflammatory diseases during the aging process.

In particular, CR mimetics or longevity-promoting supplements appears to be a challenge, CR mimetics could provide a promising alternative solution that controls and prevents aging progress and age-related diseases.

Age-related Inflammation and Diseases

Chronic inflammatory conditions are often recognized during the aging process and diverse age-related pathological conditions.

The relevance of inflammatory response in different chronic diseases has been reported based on previous studies and its molecular details are briefly explained herein. Metabolic disorders, such as type 2 diabetes, obesity, insulin resistance, and fatty liver disease, are causally related to inflammation. Metabolically active tissues, such as the liver, muscle, adipose tissues, and pancreas, are prevalent sites of aging inflammation. Pro-inflammatory factors that act in an autocrine or paracrine way interfere with normal tissue functions, such as insulin resistance. Chronic inflammation of adipose tissue is well established among other metabolic tissues that it contributes to the induction of systemic inflammation, which proposes a critical link between obesity and pathophysiological symptoms.

And inflammation plays a critical

role in atherosclerosis development. Leukocyte recruitment and elevated pro-inflammatory cytokine levels are features of the early stages of atherosclerosis.

Recently, multiple evidence have suggested that chronic inflammation is the main cause for the development of neurodegenerative diseases during the aging process.

For example, some inflammatory cytokines are associated with dementia and cognitive decline. Similar to other inflammatory diseases, cognitive decline and Alzheimer’s disease are derived from complement factors, cytokines, Toll-like receptors (TLR), pattern recognition receptors, and lipid metabolites.

Lipid metabolites are signal proteins that are associated with the characteristic expression of inflammatory mediators.

Anti-aging Molecular Mechanism of CR

CR regulates aging and senoinflammation through many signal pathways (Fig. 1). CR and suppression of the insulin/IGF pathway are the most powerful molecular interventions accepted to increase life span in lower organisms. However, less is known about the molecular alteration in CR condition. As Insulin/IGF-1/FOXO signaling pathway is involved in ameliorating health, it has been reported that CR would alter this pathway in skeletal muscle, which is one of the major metabolic organs.

It has been Table 1. List of important parameters to induce senoinflammation and modulation by CR

Senoinflammation-related parameters Aging CR References

Reactive oxygen species ↑ ⎯| Lei et al., 2015

NF- κB ↑ ⎯| Chung et al., 2009

Inflammatory genes ↑ ⎯| Chung et al., 2009

SASP (growth factors, cytokines, chemokines, MMPs) ↑ ⎯| Chung et al., 2019

; Weichhart, 2018

Inflammasome ↑ ⎯| Bang et al., 2019

Oxidative stress ↑ ⎯| Chung et al., 2009

; Chung et al., 2019

Toll like receptors (TLRs) ↑ ⎯| Bang et al., 2019

HMGB1 (high-mobility group box 1) ↑ ⎯| Chung et al., 2019

RAGE (receptor for advanced glycation end product) ↑ ⎯| Chung et al., 2019

Macrophage polarization ↑ ⎯| Mahbub et al., 2011

Lipid accumulation ↑ ⎯| Kim et al., 2009

Insulin resistance ↑ ⎯| Kim et al., 2015

ER stress ↑ ⎯| Chung et al., 2019

Metainflammation ↑ ⎯| Chung et al., 2019

Adipose tissue inflammation ↑ ⎯| Sun et al., 2012

Autophagy ↑ ⎯| Marino et al., 2014

Proinflammatory miRNAs ↑ ⎯| Chung et al., 2019

↑, Increased; ⎯|, blunted

The major processes underlying senoinflammation are inflammatory and metabolic pathways. The increased oxidative and nutritional stresses lead

to upregulation of redox sensitive NF- κB-mediated immune, proinflammatory response and metabolic pathways as listed, while these are modulated

by CR. SASP, senescence-associated secretory phenotype; MMPs, matrix metalloproteinases; ER, Endoplasmic Reticulum.

shown that maintaining CR for a long-term suppresses the insulin/

IGF-1 pathway in the skeletal muscle. It was demonstrated that CR induces notable alterations in the skeletal muscle transcriptional profiles comparable to those in the young human subjects.

This prevalent signature transcriptional profile consisted of three critical pathways: insulin/IGF-1 signal, mitochondrial biogenesis, and inflammation in association with longevity. The data also identified target pathways for the therapeutic goals that prevent and delay age-related illnesses and improve human health.

Consistent with gene expression changes, the human skeletal muscle on CR regimen for a long-term showed a 35-50%

suppression in AKT phosphorylation level at both threonine and serine residues.

Most importantly, it was also observed that there was upregulation of other AKT downstream target transcription factors such as FOXO3.

Consistent with this finding, CR in human skeletal muscle upregulates FOXO and its downstream target genes; superoxide dismutase 2 (SOD2), a critical antioxidant enzyme; and DNA damage-binding protein 1 (DDB1), a major DNA repair molecule.

AMPK has appeared to be the principal nutrient sensor that can regulate systemic metabolism. AMPK is activated when the proportion of AMP-ATP increases, which mirrors the energy state

of the cell. Thus, AMPK’s capability to detect energy stress and act as a key modulator of mitochondrial biogenesis and metabolism led to the consideration that it could exert the beneficial effects of CR.

Mice with SIRT1 overexpression mimic these effects.

SIRT1 plays a key role in the capacity of resveratrol in a suitable volume to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.

In order to compare the effects of short-term CR on aging with that of long-term CR, anti-inflammatory responses of 10-day CR from the kidneys of aging mice were investigated. On day 0, CR inhibited lipid peroxide and COX-2 activity in the kidneys of aged rats. In addition, CR suppressed the signaling cascade upstream of NF-κB and AP-1, DNA binding activity, and thioredoxin/Ref-1 pathway. CR also inhibited the expression of NF-κB and AP-1-responsive genes, and COX-2, iNOS, VCAM-1, and ICAM-1.

It also has been reported that glutathione and thioredoxin systems are modulated by CR during aging. While GSH and GSH-associated enzyme activities decreased with the ad libitum (AL)-fed aged rat, CR rats continued to show resistance to a decrease in these activities. The observation showed that cellular Trx reductase (TrxR) levels are decreased in AL-fed aged rats, whereas CR prevents these decreases. Thus, an increase in nuclear Fig. 1. Anti-aging molecular mechanism of CR. The downstream signaling effects of CR on age-related metabolism and inflammatory signaling pathways are described. CR promotes anti-aging action by anti-oxidative mechanism through modulating ROS, cellular enzymes and key molecules in inflammatory process. Age-related upregulation of cellular critical components sensitive to redox status including IKK, MAPKs, NF- κB and expression of SASP, cytokines, chemokines and COX-2, iNOS are attenuated by CR. CR-mediated promotion of TRX/

GSH system, NAD+/NADH ratio, and LKB1/SIRT1/AMPK activities modulate core pro-inflammatory NF- κB activation. Furthermore, CR

modulates insulin receptor signaling and suppresses activation of Akt and mTOR, which respectively increases FOXO, a transcription factors

that supports oxidative stress resistance and autophagy for maintaining cellular homeostasis. And PPARs, a key regulator of pathophysiological

process related to energy metabolism is activated by CR. CR, calorie restriction; ROS, reactive oxygen species; TRX, thioredoxin; GSH,

glutathione; IKK, I κB kinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; SASP, senescence-associated secretory

phenotype; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; NAD, nicotinamide adenine dinucleotide; NADH, reduced

nicotinamide adenine dinucleotide; LKB1, liver kinase B1; SIRT1, sirtuin 1; AMPK, AMP-activated protein kinase; SOD, superoxide

dismutase; mTOR, mammalian target of rapamycin; FOXO, forkhead box O; PPARs, peroxisome proliferator-activated receptors.

Trx and Ref-1 activation during aging can cause an increase in redox-sensitive transcription factors, such as NF-κB or AP-1.

Redox imbalances occur during the aging process and redox changes are minimized by the antioxidant action of CR.

In our laboratory, state-of-the-art technologies such as Next Generation Sequencing (NGS), proteomics, lipidomics, metabolics, and epigenomics are used to analyze overall biology during aging at molecular level. Using the Leverage Omics big data approach to analyze and integrate processes and epigenetics, we gathered microarray data for aging and CR and reanalyzed them using systems biology using computer science algorithms. We collected and evaluated 478 aging and 586 CR-related mouse genes. For the analyzed gene, we investigated the biological pathways closely related to aging and CR using gene ontology. The analyzed results demonstrated that gene expression patterns are highly associated with immune function, lipid metabolism, and cell adhesion. In addition, according to transcription modularity analysis, during aging, there is an upregulation of cell adhesion- and immune response-related gene expression, whereas, there is a downregulation of lipid metabolism genes. Such changes were found to be reversed by CR. Using differentially expressed genes (DEGs), we executed PPI network analysis and betweenness centrality with a high degree of value among other differentially expressed genes that come from both aging and CR group and discovered a hub protein, lymphocyte-specific protein tyrosinase kinase (LCK).

Based on the study, it was demonstrated that inflammation and metabolism are closely associated with each other during aging process.

The RNA-sequencing method was used to better explain the regulatory role of inflammation during aging at a molecular level.

It was used to detect regulated genes in the kidneys of both young and aged mice. During aging, entire sequences of cytokines, chemokines, and TNFαs were significantly increased, and TLRs- AKT and IKK-NF-κB signaling pathway was hyperactivated.

However, these were reversed by CR. Similarly, based on CR and Omics big data analysis results, cytokine and chemokine pathways were significantly upregulated during aging, and CR was generally well-accepted as a positive control for anti-aging arbitration and regulated and suppressed SASP release. This reported evidence suggest that senoinflammation is a fundamental molecular mechanism of the aging process. Thus, it may be a potential target for anti-aging and anti-aging inflammation strategies.

Benefits of CR Mimetics

Increased life expectancy has increased the incidence of age- related pathologies beyond social and economic sustainability. As

a result, there is an urgent need for arbitration to reverse or at least prevent pathogenicity and age-related deterioration. Although CR prolongs the human lifespan, implementation of long-term CR is difficult. The compounds that exert the CR-mimicking effect are designated as CR mimetics (CRMs), which are also known as energy restriction mimetics. The novel idea of CRM was first introduced in a research study of 2-deoxy-d-glucose, in which the compound demonstrated the bioactivity in vivo rat model. It was demonstrated that CR compounds have physiological effects comparable to CR and exhibit exercise-like effects. In specifics, CRM showed the substantial anti-aging metabolic, hormonal and physiological effects and activate multiple signaling pathways that are normally induced by CR. CRM exerts effects on an intra- cellular signaling pathway in a similar way to CR. In certain types of CRM, it acts by directly targeting energy metabolism system and shows inhibition effects, particularly in glucose metabolism.

Metformin is one of representative CRMs for type 2 diabetes.

Metformin acts by inhibiting mitochondrial respiratory chain and promotes AMPK activation inducing catabolic reactions and generating ATP production. At the same time, metformin suppresses anabolism. For example, metformin attenuates gluconeogenesis, fatty acid synthesis and induces β-oxidation in liver. In other metabolic organs, for example, skeletal muscles and adipose tissue, metformin induced AMPK activation and led to translocation of glucose transporter type 4 to the cell membrane and promoted sugar uptake to intracellular compartment.

Rapamycin is a compound used as an inhibitor of mammalian target of rapamycin (mTOR) where it exerts significant modulatory effects on proliferation and inflammation by inhibiting interleukin-2 (IL-2) signaling and activation of T and B cells. The compound was demonstrated to inhibit immune response and has been clinically applied for preventing autoimmune diseases and transplantation rejection response.

Effective CRMs alter key metabolic pathways related to the

effects of CR, maintaining youth health and extending lifespan

without reducing food intake. CRM promotes autophagy, a cell

recycling mechanism, and a protective path for calorie restriction

through reduced protein acetylation.

It is expected for CRMs

to become a part of the pharmacological molecular tool for

prevention of aging and age-related cardiovascular, neuro-

degenerative, and malignant diseases. Upt to now, reported CRM

candidate compounds include resveratrol, metformin, PPAR

agonist, rapamycin, endocannabinoid receptor antagonist, α-lipoic

acid, glycolytic inhibitor, garlic sulfur component, β-hydroxy-

butyrate, and more.

Conclusion

The dangerous effects of senescent cells are associated with the release of pro-inflammatory mediators called SASP and metabolic changes in response to intracellular and extracellular stimuli. In the pathological and physiological aging process (Fig. 2), such chronic inflammatory condition is called senoinflammation. Based on in vitro and in vivo aging studies, cytokine and chemokine pathways and lipid accumulation pathways are significantly upregulated during the systemic aging process. As CR exerts modulatory effects and suppresses SASP secretion, it was generally accepted as a positive intervention control for anti-aging intervention method. Collectively, accumulating evidence suggests that seno- inflammation is a fundamental molecular mechanism of the aging process and CRMs can be used as an alternative potential therapeutic tool for aging and age-associated diseases.

Acknowledgment

This work was carried out with the support of “Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ006522132013)” Rural Development Administration, Republic of Korea. This research was supported by Bio & Medical Technology Development Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2015M3 A9B8029074)

Conflict of Interest

The author declares no conflict of interest.

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