Silent information regulator, Sirtuin 1, and age-related diseases


  • Authors' contribution: L. Z. and R. C. contributed equally to this study. L. Z. and R. C. were responsible for study concept and design, interpretation of data, and preparation of the manuscript. F. L., H. T., H. M., T. N., K. I. and T. K. were involved in data collection and entry. S. M. and T. T. were responsible for study concept and design, analysis of data, and preparation of the manuscript.

Professor Shigeto Morimoto MD PhD, Department of Geriatric Medicine, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku-gun, Ishikawa 920-0293, Japan. Email:


Sirtuin 1 (SIRT1), a member of the silent information regulator 2 in mammals, has recently been found to be involved in age-related diseases, such as cancer, metabolic diseases, cardiovascular disease, neurodegenerative diseases, osteoporosis and chronic obstructive pulmonary disease (COPD), mainly through deacetylation of substrates such as p53, forkhead box class O, peroxisome proliferator activated receptor γ co-activator 1α, and nuclear factor-κB. It is widely reported that SIRT1 can promote not only carcinogenesis but also metastasis and insulin resistance, andhave beneficial effects in metabolic diseases, mediate high-density lipoprotein synthesis and regulate endothelial nitric oxide to protect against cardiovascular disease, have a cardioprotective role in heart failure, protect against neurodegenerative pathological changes, promote osteoblast differentiation, and also play a pivotal role as an anti-inflammatory mediator in COPD. However, there are controversial results suggesting that SIRT1 has an effect in protecting against DNA damage and accumulation of mutations, and preventing tumorigenesis. In addition, a high level of SIRT1 can induce cardiomyopathy and even heart failure. This article reviews recent developments relating to these issues.


Sirtuin 1 (SIRT1), the family member with the greatest homology to the silent information regulator 2 (Sir2), has recently been pinpointed as one good candidate to regulate the process of caloric restriction, a beneficial regimen for aged people to increase the resistance to oxidative and stress, inhibit fat storage, ameliorate neurodegeneration and so on, thereby mitigating disease processes in many tissues and extend lifespan.1 Its gene encodes a nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase (HDAC).2 Several lines of evidence suggest that the SIRT1 protein plays a role in regulating different cellular processes through deacetylation of important substrates such as p53, forkhead box class O (FOXO) transcription factors, peroxisome proliferator activated receptor (PPAR)γ co-activator 1α (PGC-1α), nuclear factor (NF)-κB and others, which are closely linked to some age-related diseases (Table 1). This paper reviews the published work dealing with issues surrounding these effects and the mechanisms of actions of SIRT1 in age-related diseases such as cancer, metabolic diseases, cardiovascular disease, neurodegenerative diseases, osteoporosis and chronic obstructive pulmonary disease (COPD).

Table 1.  Substrates of Sirtuin 1 and their main effects on age-related diseases
SubstrateRegulation typeMain effectsReference
  1. +, Positive regulation; −, negative regulation. hTERT, human telomerase reverse transcriptase; FOXO, forkhead box class O transcription factor; NF-κB, nuclear factor-κB; PGC-1α, peroxisome proliferator activated receptor γ co-activator 1α; IRS-2, insulin receptor substrate 2; UCP-2, uncoupling protein-2; LXR, liver X receptors; HDL, high-density lipoprotein; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; PARP, poly(ADP-ribose) polymerase.

Ku70+DNA repair activity/suppression of apoptosis3,4
hTERTSuppression of cell growth5
p53Inhibition of p53-dependent apoptosis6,7
FOXO-3+/−Stress resistance/reduction of apoptosis8
NF-κBPromotion of carcinogenesis etc.9,10
PGC-1α+Promotion of gluconeogenesis13
IRS-2+Regulation of insulin signaling pathway14
UCP-2Enhanced insulin secretion by pancreas15
LXR+Promotion of HDL synthesis16
eNOS+Regulation of NO and vascular tone17
PARPProtection of cardiac myocytes in heart failure18,19


Cancer is an age-related disease caused mainly by age-related accumulation of gene mutations due to errors during DNA replication. In addition, tumor growth mainly depends on loss of control of differentiation and inhibition of apoptosis. Normal expression of SIRT1 has a protective effect against DNA damage, enhancing DNA repair capacity and guarding against accumulation of mutations, to prevent tumorigenesis.20 However, overexpression of SIRT1, such as in human prostate cancer cells21 and colon carcinoma,22 which allows rapid proliferation and loss of checkpoints, promotes continued propagation of the progress of cancer. In order to clarify the effect of SIRT1 in cancer cells in detail, several aspects should be considered carefully.

Beneficial effects in carcinogenesis

Sirtuin 1 has a protective effect on the limited replicative lifespan, protecting against DNA damage, enhancing DNA repair capacity and guarding against accumulation of mutations and against genomic instability through its normal expression. Loss of SIRT1 expression, activity or regulation can bypass replicative senescence, allow cell division to proceed without the proper repair of DNA, and promote accumulation of mutations and genomic instability, leading to tumor development.20

Its protective effect against DNA damage is associated with Ku70, a heterodimer of a polypeptide of approximately 70 kDa that binds strongly to DNA double-strand breaks. Its homolog, yKu70, promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.23 Once DNA is damaged, SIRT1 physically complexes with Ku70, leading to subsequent deacetylation and DNA repair activity to prevent the formation of tumor cell.3

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of the mammalian telomerase. It is the first tumor antigen identified to have a global expression in more than 85% of human cancer cells and its continuing expression is necessary to the oncogenic process.24 Narala et al.5 found that there was a small increase in hTERT mRNA level and a significant increase in levels of hTERT protein in some cell lineages when SIRT1 was inhibited. Therefore, SIRT1 might act as a growth suppressor cooperating with hTERT. Nevertheless, it is still uncertain whether SIRT1 cooperates with hTERT to suppress the cell growth in tumor cells.

Hence, resveratrol, a plant polyphenol that stimulates SIRT1 activity, was purified and shown to have cancer chemopreventive activity in assays representing three major stages of carcinogenesis. It was found to act as an antioxidant, antimutagen and anti-initiation.25 This was supported by the study of Bickenbach et al.26 who found that resveratrol can activate the radio-and chemo-inducible cancer gene therapy vector Ad.Egr.TNF, a replication-deficient adenovirus that expresses human tumor necrosis factor-α (TNF-α) under control of the Egr-1 promoter, which suggested its anti-neoplasia activity.

Adverse effects on tumor growth

In several human cancers, overexpression of SIRT1 has been found to stimulate rapid proliferation and promote continued propagation.21,22 This adverse effect is mainly associated with the negative regulation of apoptosis-dependent factors and the promotion of neovascularization in tumors.

The most widely known substrate of SIRT1 is p53, a tumor suppressor with a critical role in cancer cell-cycle regulation and apoptosis that responds to various biological signals. The key role of p53 in regulating cell proliferation and stress response is highlighted by inactivation or loss-of-function of p53 in over 50% of human tumors.27 It regulates expression of genes, including p21, p53-upregulated modulator of apoptosis and Bax, to initiate cell-cycle arrest, senescence or apoptosis. SIRT1 binds to and deacetylates p53 on lysine 382 (K379 in mouse p53), thereby negatively regulating p53-mediated transcriptional activation, repressing p53-dependent apoptosis and promoting tumor growth.6,7

Forkhead box class O transcription factors, including FOXO-1, FOXO-3a, FOXO-4 and FOXO-6, respond to DNA damage and oxidative stress and regulate expression of cell-cycle, DNA repair and apoptosis genes.28 FOXO proteins can regulate cell fate by modulating the expression of genes involved in apoptosis, cell-cycle transitions, DNA repair, and oxidative stress, as well as cell differentiation in cancer, after regulation by phosphorylation and acetylation.29 During the process of tumor growth, SIRT1 appears to shift FOXO-induced responses away from apoptosis towards cell survival by deacetylation.8

Nuclear factor-κB is a dimeric transcription factor that regulates the expression of numerous genes controlling immune and inflammatory responses, cell proliferation, differentiation and apoptosis. Recent in vivo data have indicated that inhibition of NF-κB in hepatocytes may actually promote hepatocarcinogenesis.30,31 In addition, SIRT1 can inhibit NF-κB-mediated transcription through interacting with transducin-like enhancer of split-1.9 Moreover, gankyrin is an oncoprotein commonly overexpressed in some human carcinomas. It directly binds to RelA (p65), one of the NF-κB subunits. In human uterine cancer HeLa and embryonic kidney 293 cells, overexpression of gankyrin suppresses the basal as well as TNF-α-induced transcriptional activity of NF-κB, whereas downregulation of gankyrin can increase it. Importantly, the inhibitory effect of gankyrin is abrogated by nicotinamide as well as downregulation of SIRT1.10

E2F1 also has important roles in regulating cell proliferation and apoptosis in neoplasia, mainly by stimulating the transcription of several genes in the apoptotic pathway.32 Similarly to p53, E2F1 is stabilized and activated by DNA damage.33 Once DNA is damaged, E2F1 is overexpressed to induce premature S-phase entry and often results in apoptosis.34 SIRT1 is a direct transcriptional target of E2F1 and its expression protects cells from death by regulating E2F1.35

Though Ku 70 can promote genomic stability and prevent the change from normal cells to cancer cells, Ku70 can also suppress Bax-mediated apoptosis. The acetylation level of Ku70 is regulated by the result of a dynamic equilibrium between the activity of acetyltransferases and the opposing deacetylases. According to the report of Cohen et al.,4 treatments by increasing Ku70 acetylation, either by treating cells with SIRT1 inhibitor nicotinamide or by overexpressing acetyltransferases CBP or PCAF, are capable of abrogating the ability of endogenous Ku70 to suppress Bax-mediated apoptosis. Hence, SIRT1 might increase the anti-apoptotic ability of endogenous Ku70 and play as harmful role in the treatment of tumor.

Neovascularization is one of the important characteristics of neoplasia. SIRT1 is highly expressed in the vasculature during blood vessel growth in tumors, where it promotes the angiogenic activity of endothelial cells. Loss of SIRT1 function blocks sprouting angiogenesis and branching morphogenesis of endothelial cells, with consequent downregulation of genes involved in blood vessel development and vascular remodeling. Disruption of SIRT1 gene expression in zebrafish and mice results in defective blood vessel formation and blunts ischemia-induced neovascularization.36

Harmful effects in metastasis

Sirtuin 1 plays a very important role in metastasis, mainly by activating FOXO-1 to induce transcription of vascular endothelial growth factor-C (VEGF-C). VEGF-C has been identified as being involved in lymph node metastasis of several cancers including colorectal cancer, human pancreatic endocrine tumors, esophageal carcinoma, head and neck squamous cell carcinoma, uterine cervical cancer, primary non-small-cell lung cancer, gastric carcinoma, and laryngeal squamous carcinoma. FOXO-1 is a potential transcription factor for VEGF-C and is activated by SIRT1.11

Now that SIRT1 has been shown to have such adverse effects on tumor growth and metastasis, SIRT1-specific inhibitors may be useful chemotherapeutic agents for some SIRT1-dependent tumors. Cambinol, a SIRT1 inhibitor, inactivates the critical oncogene B-cell lymphoma 6 protein (BCL6) in Burkitt's lymphoma cells by promoting its acetylation, and leads to induction of apoptosis. In mouse xenograft models, cambinol alone was effective specifically against tumors expressing BCL6. Interestingly, inhibition of SIRT1 by cambinol sensitizes cells to DNA-damage-induced apoptosis independently of p53.37 BML-210, another specific SIRT1 inhibitor, can abrogate FOXO-1-dependent VEGF-C transcription, which is beneficial for inhibiting metastasis.11 Nevertheless, SIRT1-specific inhibitors have not yet been used in the clinical treatment of carcinoma, perhaps mainly because of SIRT1's protective effect against DNA damage and gene mutation.

Metabolic diseases

In low and middle income countries, the majority of people with diabetes are in the age range of 45–64 years, according to World Health Organization reports. Other metabolic diseases are mostly associated with age-related changes of body function as well, for instance, the imbalance of food intake and energy expenditure, which results in abdominal fat accumulation, causing insulin resistance. It is known that the SIRT1 protein level is increased after fasting and returns to nearly the control level upon re-feeding.13 Resveratrol can protect mice against diet-induced obesity.38 All these findings indicate that SIRT1 may be a regulator of energy and metabolic homeostasis, and may even regulate some key points in age-related metabolic diseases, such as insulin resistance.

To define the role of SIRT1, Milne et al.39 identified and characterized novel small molecule activators of SIRT1 both in vitro and in vivo. These SIRT1 activators ablated insulin resistance and diabetes in diet-induced obese mice fed a high-fat diet and in diabetic Lepob/ob mice. In addition, these new SIRT1 activators ameliorated the metabolic disturbances in Zucker fa/fa rats. Moreover, SIRT1 activators improved glucose homeostasis and insulin sensitivity in key metabolic tissues including liver, muscle and fat.

The mechanisms of SIRT1 in ameliorating insulin resistance and improving glucose and lipid homeostasis are mainly associated with PGC-1α, PPARγ and FOXO-1. PGC-1α is a key regulator of glucose production in the liver through activation of the entire gluconeogenic pathway.40–42 SIRT1 induces gluconeogenic genes and hepatic glucose output through PGC-1α. In addition, SIRT1 modulates the effects of PGC-1α repression of glycolytic genes in response to fasting. Thus, SIRT1 acts as a modulator of PGC-1α in regulating glucose homeostasis.13 Moreover, SIRT1 and PPARγ bind to the same DNA sequences and SIRT1 is a co-repressor of PPARγ. Adipogenesis in a cell model, 3T3-L1, with lipid accumulation is promoted by the nuclear receptor PPARγ. SIRT1 acts a negative modulator of adipogenesis in this cell model by docking with the PPARγ cofactor, nuclear receptor co-repressor (NcoR).43 FOXO-1 is not only involved in insulin's inhibition of hepatic glucose production and stimulation of β-cell proliferation in insulin-resistant mice,44 but also plays an important role in coupling insulin signaling to adipocyte differentiation.45 The regulation of adipocyte differentiation by FOXO-1 can potentially affect insulin sensitivity by regulating adipocyte size.45 Furthermore, FOXO-1 and PGC-1α interact in insulin-regulated gluconeogenesis.41 SIRT1 binds FOXO-1, decreases its acetylation and inhibits its transcriptional activity,12 which might be another pathway for intervention in metabolic diseases.

As a critical component of overall energy homeostasis, the insulin signaling pathway has been well studied and the key steps have been characterized.46 The insulin signaling pathway is initiated by auto-tyrosine phosphorylation of the insulin receptor upon insulin binding, and subsequently tyrosine phosphorylation of several key adaptor proteins including insulin receptor substrate 1 (IRS-1) and IRS-2. The phosphorylated IRS proteins further transmit insulin signaling to downstream events, mainly through two kinase cascades, the mitogen-activated protein kinase cascade and the phosphatidylinositol 3-kinase-Akt cascade. SIRT1 protein may, through regulation of the acetylation level of IRS-2 protein, directly regulate insulin-induced IRS-2 tyrosine phosphorylation and its downstream Akt activation.14

Insulin secretion by pancreatic β cell plays a very important role in the pathophysiology of type 2 diabetes. An age-associated impairment of β-cell function has also been demonstrated in rodents.47 Increasing SIRT1 dosage or activity in pancreatic β cells can provide life-long beneficial effects of enhanced β-cell function on glucose homeostasis in the process of aging. One of the mechanisms is that SIRT1 mediates the repression of uncoupling protein (UCP)2 expression and thereby increases adenosine triphosphate (ATP) content, resulting in the enhancement of glucose-stimulated insulin secretion.15

Additionally, adiponectin is secreted by adipose tissue in response to metabolic effectors in order to sensitize the liver and muscle to insulin. Reduced circulating levels of adiponectin that usually accompany obesity contribute to the associated insulin resistance.48 Adiponectin secretion is regulated by SIRT1. A lower level of SIRT1 increases adiponectin transcription by activating FOXO-1 and enhancing FOXO-1 and CCAAT/enhancer binding protein (C/EBP)α interaction in adipocytes in patients with obesity or type 2 diabetes.49 However, in the study of Qiang et al., the secretion of high molecular weight adiponectin was decreased by the treatment of SIRT1 activator resveratrol, whereas it was enhanced by SIRT1 inhibitor nicotinamide.50

Heart disease

Cardiovascular disease

Sirtuin 1 has protective effects against cardiovascular disease. Genetic analysis of SIRT1 haplotypes revealed a tendency for decreased cardiovascular mortality in haplotype 2 carriers.51 This observation is in accordance with other genetic and clinical studies. It is well known that plasma high-density lipoprotein (HDL) level is inversely associated with risk of cardiovascular events, as HDL-mediated reverse cholesterol transport can protect against atherosclerosis by clearing excess cholesterol from arterial cells.52,53 SIRT1 activates transcription of the liver X receptors (LXR) target gene encoding the ATP-binding cassette transporter A1, which mediates HDL synthesis, reverses cholesterol transport and decreases the risk of atherosclerosis and cardiovascular events.16

Moreover, SIRT1 plays a fundamental role in regulating endothelial nitric oxide (NO) and endothelium-dependent vascular tone by deacetylating endothelial nitric oxide synthase (eNOS), which is closely associated with blood pressure. SIRT1 and eNOS co-localize and co-precipitate in endothelial cells, and SIRT1 deacetylates eNOS, stimulating eNOS activity and increasing endothelial NO. Inhibition of SIRT1 in the endothelium of arteries inhibits endothelium-dependent vasodilation and decreases bioavailable NO.54 On the contrary, resveratrol, a SIRT1 activator, can activate eNOS,17 improve endothelial function, prevent elevation of blood pressure and restore vascular eNOS activity in animal models of endothelial dysfunction.55

Heart failure

Increasing lines of evidence suggest that the balance between growth and death of cardiac myocytes plays an important role in determining long-term cardiac function in heart failure patients.56 SIRT1 plays a very important role in cardiac development and the growth of myocardial cells to maintain cardiac function. In SIRT1-null embryos, developmental defects in the heart have been observed in a previous study,57 while in wild-type embryos, the expression pattern of SIRT1 protein and mRNA is high during cardiogenesis in embryogenesis.58 Moreover, in isolated neonatal cardiomyocytes, inhibitors of SIRT1 activity cause a moderate increase in basal cell death and upregulation of the expression of the hypertrophy-associated gene, atrial natriuretic factor, even though cell size actually decreases. Likewise, an increased SIRT1 level protects myocytes from serum starvation-associated cell death, while also increasing overall cell size.59

In addition, overexpression of SIRT1 in the heart of dogs with heart failure protected cardiac myocytes from apoptosis in response to serum starvation and significantly increased the size of cardiac myocytes, which suggest that endogenous SIRT1 plays an essential role in mediating cell survival and maintaining modest hypertrophy.60 Hence, an increase in SIRT1 expression may have a cardioprotective role in pathological hearts.

Pillai et al.18 reported more evidence for the protective effect of SIRT1 against heart failure. Poly(ADP-ribose) polymerase-1 (PARP) is a multifunctional DNA-bound enzyme located in the nuclei of various cells, including cardiac myocytes. Robust activation of PARP by oxidative stress and other factors has been demonstrated to be a major cause of myocyte cell death contributing to heart failure. In both cultured cardiac myocytes and failing hearts, increased activity of PARP was associated with reduced SIRT1 deacetylase activity, and myocyte cell death induced by PARP activation was prevented only when SIRT1 was intact, which indicates that SIRT1 is a beneficial factor leading to cardiac myocyte protection in heart failure.19

Nevertheless, the opposite result was reported, that a high level of SIRT1 can induce cardiomyopathy and even heart failure, possibly through induction of mitochondrial dysfunction in the heart in vivo.61 The mechanism may be linked to NAD+ consumption. A high level of SIRT1 causes depletion of NAD+ which is required for mitochondrial respiration. Depletion of NAD+ could lead to deficiency of ATP and, consequently, myocardial cellular dysfunction and eventual cardiac cell death. Hence, although stimulation of SIRT1 may be considered as anti-aging therapy for the heart, careful evaluation regarding the dosage seems essential to best use the therapeutic potential of SIRT1.

Neurodegenerative diseases

Neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis are increasingly prevalent in aging societies because of a progressive loss of neurons with age. Recent studies demonstrated that activation of SIRT1 could attenuate neuronal degeneration and death in animal models of neurodegenerative disease and exert a neuroprotective effect.

According to the study of Kim et al.,62 SIRT1 is not only enriched in the nucleus but also localized in the cytoplasm in AD patients and mouse models, acting as a protective response to neurodegenerative conditions. Its activating molecule, resveratrol, can slow in vitro neuron death as well as in vivo neurodegeneration. In a mouse model of Parkinson's disease, SIRT1 also protects against neurodegenerative pathological changes.63 The mechanisms of the neuroprotection by SIRT1 may be associated with its downregulation of the pro-apoptotic factors, p53,7,64 FOXO8,65 and NF-κB,66 and the deacetylation and activation of PGC-1α.62 Furthermore, SIRT1 may be involved in the axonal protection observed in the wallerian strain of mice, which have a translocation that increases the level of the NAD biosynthetic enzyme nicotinamide mononucleotide adenylyl-transferase 1 and renders peripheral axons more stable after a neuronal insult. One study showed that the effects of NAD and the wallerian strain are dependent on SIRT1, leading to the conclusion that SIRT1 is a neuroprotective factor.67

There have been many studies focusing on mitochondrial dysfunction in the etiology and pathogenesis of neurodegenerative diseases.68–70 SIRT1 acts as a functional regulator of PGC-1α that induces a metabolic gene transcription program of mitochondrial fatty acid oxidation and promotes mitochondrial function in skeletal muscle.71 Its activator, resveratrol, can also improve mitochondrial function through the SIRT1/PGC-1α pathway in muscle.38 However, the modulation of the SIRT1/PGC-1 pathway in the central nervous system is not well documented, and requires further study.


Osteoporosis is widespread in elderly people, and age-related deficiency of osteoblast differentiation is one well-known pathogenetic mechanism. Inhibiting adipocyte formation and promoting osteoblast differentiation to enhance bone formation is a promising therapy for osteoporosis.72 PPARγ is an important regulator of adipocyte differentiation. p53 is one of the key factors in osteoblast differentiation. Both of their activities are regulated by SIRT1. Activation of SIRT1 in mesenchymal stem cells can decrease adipocyte and increase osteoblast differentiation.73


Chronic obstructive pulmonary disease is also a major cause of disability, morbidity and mortality in elderly patients. It is characterized by progressive and largely irreversible airflow limitation, which is associated with an abnormal inflammatory response in the lung.74 Increased NF-κB activation and acetylation of histone proteins have been identified as important inducers of local secretion of pro-inflammatory cytokines. SIRT1 is an important protein involved in deacetylation of histone proteins and negatively regulates NF-κB activation to decrease pro-inflammatory cytokine release.75


The core role of SIRT1 in age-related diseases is associated with different substrates in different organs (Fig. 1), which regulate carcinogenesis, metastasis, metabolic homeostasis, anti-inflammatory effects, vascular tone, cardiac function, neurodegeneration and others. Sometimes, SIRT1 has dual effects on its substrate, such as FOXO-38 and FOXO-1,11,12 and induces conflicting promotion. Additional insights into the biological actions of SIRT1 are required to identify the precise roles of different members of its substrate family in different age-related diseases, especially in vivo. Then, its inhibitors or activators can be safely used for the treatment of age-related diseases.

Figure 1.

Different substrates of Sirtuin 1 (SIRT1) in different target organs. SIRT1 plays an important role in age-related diseases, such as cancer, metabolic diseases, cardiovascular disease and neurodegenerative diseases, mainly through deacetylation of substrates such as p53, FOXO, PGC-1α and NF-κB. AMPK, adenosine monophosphate-activated protein kinase; AR, androgen receptor; eNOS, endothelial nitric oxide synthase; FOXO, forkhead box class O transcription factor; LXR, liver X receptors; NF-κB, nuclear factor κB; PARP, poly (ADP-ribose) polymerase-1; PPARγ, peroxisome proliferator activated receptor γ; PGC-1α, PPARγ co-activator 1α; UCP-2, uncoupling protein-2; VEGF-C, vascular endothelial growth factor-C. +, Positively regulated by SIRT1; −, negatively regulated by SIRT1; ?, uncertain regulation type by SIRT1.


We express our gratitude to the reviewers and editorial staff of GGI for their insightful discussion and checking of this paper. This study was funded in part by grants from the Ministry of Health, Labor and Welfare of Japan (to S. M.).