Abstract

Introduction
Gradual decline in hormones due to aging is a critical factor that indicates aging process. Gradual decreases due to aging are recognized in growth hormone/insulin-like growth factor-1 system (GH/IGF-1), dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), and testosterone, which are designated as somatopause, adrenopause, and andropause, respectively 19 , although they are not as drastic as estrogen at menopause. These alternations in hormones are related to aging phenomena such as a subjective loss of sense of well-being and cognitive impairments. Furthermore, the decrease of hormones is considerably related to diverse symptoms that often occur in the middle and old aged, such as obesity, the onset and progression of diabetes and the decline in bone mineral density. From the above perspective, replacement therapies of these hormones are expected to be effective for anti-aging medical treatments. Therefore, studies on hormone replacements have been developed in recent years. The present study, focusing on Corresponding to: Toshihiko Yanase MD, PhD Seiwakai Muta Hospital 3-9-1 Hoshikuma, Sawara-ku, Fuokuoka 814-0163, Japan TEL: +81-92-865-2211  FAX: +81-92-865-5556  Email: t-yanase@seiwakai-hp.jp Co-authors; Kazuo M, info@sciwakai-muta-hp.or.jp ; Hajime N, nawata-h@mx22.tiki.ne.jp DHEA, describes the anti-oxidative and anti-glycative actions, which are considered to be mechanisms for anti- aging. DHEA has properties for anti-obesity, anti-diabetes, and anti-arteriosclerosis 1-4 as has been suggested mainly in research on the cellular level and in animal experiments 1-4). The current states of these studies are introduced, placing anti-diabetic effects as the central focus. Furthermore, we introduce a target gene that we identified in DHEA mechanism. At the end of this report, we introduce the meaning of DHEA in sarcopenia from the viewpoint of cortisol/DHEA-S ratio.
What are DHEA and DHEA-S ?
DHEA is an adrenal-derived androgen, and also exists as a sulfate conjugate, DHEA-S in the body. The body can mutually transform DHEA and DHEA-S. They are produced mainly in the adrenal gland and gonad, and some are produced in peripheral tissues. DHEA-S is the steroid that exists at the highest concentration in the body. The half- life of DHEA is short at one to three hours and its levels are high in the morning and low in the evening, showing diurnal variations as cortisol levels in the blood do. Contrarily, the half-life of DHEA-S is long at 10 to 20 hours, and does not exhibit as clear diurnal variations as DHEA. However, the alternation of serum DHEA levels in the daytime usually synchronizes to serum DHEA-S levels. Measurement of DHEA-S is common in daily medical practice, as it is easier to measure. While serum cortisol level, which is also an adrenal steroid, does not alter throughout the lifetime, serum DHEA-S levels start to increase at six to seven years of age, reach its peak around 12 to 13 years old, and remain at the highest level until 13 to 25 years old. After that, its levels gradually decrease in a linear fashion along with aging 19 . In this sense, DHEA-S can be considered a biomarker for aging. Its decline, functionally, is suggested to have a relationship with geriatric syndrome with a functional decline in mental and physical functions due to aging ¹-³) . Deposition of lipofuscin, a lipid peroxide, in adrenal reticularis is observed along with aging. There are diverse theories regarding mechanisms of the decline in DHEA and DHEA-S along with aging. It has been reported that the lipid peroxidation of adrenal microsomes impairs P450c17 activity, which is responsible for DHEA synthesis, and an antioxidant, vitamin E, protects against this impairment 5). There is a possibility that the acceleration of adrenal oxidative stress with aging is one of the mechanisms. In fact, recent studies on humans have clarified that the genetic mutations of nicotinamide nucleotide transhydrogenase (NNT) and thioredoxin reductase 2 (TXNRD2), which are anti-oxidative enzymes of mitochondria, induce the onset of adrenal insufficiency 6,7) . We have observed that ascorbic acid (vitamin C), an anti-oxidative substance, specifically accumulates in adrenal gland 89 . Also, this suggests the importance of the defense system preventing the adrenal gland from oxidative stress. Serum DHEA-S concentration level decreases with aging in rhesus monkeys as well as humans. It was reported in an experiment of rhesus monkeys that calorie restriction promoted the extension of lifespan and the group which showed extended longevity had comparatively higher DHEA-S values 91 . However, afterward, a study reported that calorie restriction did not necessarily change the DHEA-S level in the blood of rhesus monkeys 101 . Furthermore, it was reported that administration of DHEA did not restore the daily activities of aged rhesus monkeys 11 ). There was a report that six-month calorie restriction on humans did not affect concentration levels of serum DHEA-S 12) . Further investigations are required to verify the meaning of DHEA-S as a biomarker for longevity assessment under calorie restrictions. Interestingly, there have been studies regarding longevity, including a research report on inhabitants in Baltimore, U.S.A. Inverse correlations have been identified in male subjects between serum DHEA-S values, and the mortality and the onset of cardiovascular diseases in the follow-up study conducted in Baltimore 91 and most (not to say all) studies which were conducted on regional inhabitants for long-term periods 13-16 . In Japan, a retrospective cohort study, which examined inhabitants in Tanushimaru-cho, Fukuoka Prefecture, Japan for 27 years, clarified that survival ratios of males were high in groups with high serum DHEA-S (200 µg/dL or higher) and were the lowest in the group with low serum DHEA-S (lower than 200 μg/dL). No such relation was observed in female subjects ¹⁷ ). DHEA is not only a biomarker for aging in both men and women but also a potential biomarker for longevity at least in men.
Mechanisms of DHEA Action
DHEA is a precursor of sex steroids such as estrogens and testosterone. One of the potential mechanisms is the transformation of DHEA into active sex steroids. There is a hypothesis that DHEA exerts its action through a specific mechanism. However, including the existence of its receptor molecules, mechanisms of DHEA action are largely unknown. As another mechanism, DHEA has inhibitory effects against excessive cortisol, which induces neurotoxicity, muscular atrophy, and bone loss. It is advocated that the local glucocorticoid is converted into an inactive form, which is promoted by DHEA via the decrease in 11β-HSD-1 activities and the acceleration of 11β-HSD-2 activities 18,19) . It is reported that 7α-hydroxy- DHEA, a metabolite of DHEA, is involved in the decrease in 11β-HSD-1 activities due to DHEA 201. In sex steroid hormone-sensitive tissues such as bones, prostate and mammary glands, DHEA, which is a steroid precursor for testosterone and estrogen, plays a significant role. DHEA, which is formed in the adrenal gland and the peripheral tissues, is converted to sex steroids in the local tissues of bones, prostate and mammary glands by the so- called intracrinology system. We confirmed that aromatase activity in the osteoblastic cells transformed DHEA into sex steroids and clarified that in fact, the bone mineral density of postmenopausal women had a higher association with serum DHEA-S concentration than with estradiol ²¹ ) . In recent years, CYP17A inhibitor, abiraterone, is an innovative drug for prostatic carcinoma, blocking the provision of androgen from the human adrenal gland and gonad to prostatic carcinoma. The importance of intracrinology has been proven in humans (22) It remains unclear regarding the existence of receptors of DHEA(-S) and the mechanism of signal transduction. However, from the viewpoint of outcomes from genome projects, it is considered to be a negative at present that DHEA receptors would exist as nuclear receptors which play a genomic role via genetic transcription. Contrarily, DHEA receptors are firmly suggested to play a non-genomic role as a membrane bound receptor. DHEA(-S) is synthesized as a neurosteroid in the brain. It has been reported mainly in animal experiments that DHEA(-S) enhances memory retention and works as an anti-depressant through neuroprotective and neurotrophic actions. The effects of neurosteroids including DHEA are relatively rapid and receptors in the brain such as NMDA (N-methyl-d-aspartate) receptor, GABA ( γ -aminobutyric acid A) receptor and sigma receptors ²³) are reported as molecular targets of DHEA. Although a high degree of affinity of DHEA binding activity has been reported on peripheral blood mononuclear cells ² 4 ) and vascular smooth muscle cells ² ⁵ ) , no conclusive evidence of the existence of specific receptor molecules has been shown. It is recognized that DHEA has vasodilator actions via nitric oxide (NO) 26 . It was proven that high affinity DHEA receptors, which are a membrane binding type and G protein coupled type, existed (Kd 49pM) and linked with endothelial nitric oxide synthase (eNOS) activation ² ⁷ ) . T lymphocyte cell line, Peer cells, shows high DHEA binding activity under the condition of T cell receptor activation by antigen ² ³ . We have identified DHEA-induced dual specificity protein phosphatase (DDSP) from the Peer cells 291 . DDSP was induced by DHEA as a target gene of DHEA. DDSP was expressed extensively throughout tissues in the whole body, and phosphatase activities were observed for phosphotyrosine and phosphoserine/threonine, and specific binding to p38 was observed. The MAP kinase system is deeply involved in cell proliferation, canceration, apoptosis, and immune response. There is a possibility that DHEA, through DDSP, exerts diverse physiological activities via inhibitory controls on p38 MAP kinase system ( Fig. 1 ). Interestingly, we indicated that in mice with excessive DDSP expression, the weight increase of a male mouse under high- fat diet was significantly inhibited compared with wild type mice 301 . DDSP could explain a part of the anti-obesity mechanism of DHEA. It is assumed that DHEA exerts its physiological activities via multiple mechanisms, such as the action system with membrane receptors of DHEA and its metabolite, the transformation from DHEA to sex steroids, and the counteraction to cortisol.
Anti-atherosclerotic, anti-obesity and anti-
diabetic effects of DHEA
(1) Anti-atherosclerotic effects
Studies on anti-atherosclerotic effects of DHEA in humans, both in cross-sectional and longitudinal studies, have suggested that the onset of coronary artery disease is more frequent with lower serum concentrations of DHEA and DHEA-S 14,16,31). At present, there are no studies to verify the effects of DHEA administration on human arteriosclerosis. However, studies with male rabbits indicated that DHEA showed effects on HCD (high cholesterol diet)-induced arteriosclerosis model, where such anti-atherosclerotic DHEA effects were confirmed with repeatability and reproducibility ³²·³⁴ . Furthermore, the anti-arteriosclerosis DHEA effects were also observed in research with HCD- induced arteriosclerosis models of Apo E knockout mouse 35 ). We identified and reported an anti-atherogenic mechanism in actions of DHEA, clarifying that DHEA markedly inhibited macrophage foam cell formation (cholesterol ester accumulation) induced by modified LDL in cell levels ³ ⁶ ). Interestingly, previous studies suggested that DHEA regulated proinflammatory cytokines and inflammatory signal transductions in immune cells, indicating possibilities of anti-inflammatory actions of DHEA 37) , which seems to partly contribute to the anti-atherosclerotic effects. Oxidative stress is accelerated in type 2 diabetes mellitus (T2DM). The increase of reactive oxygen species
Fig. 1. Schematic representation of the action of DDSP.
DDSP is a serine/threonine phosphatase which binds p38 and suppresses its activity. DHEA, dehydroepiandrosterone; DDSP, DHEA-induced dual specificity protein phosphatase. The figure is based on the results of reference 29). (ROS) in the blood and the decrease of glutathione (GSH) and vitamin E, which are a defense system against oxidative stress, are observed in T2DM patients. An administration of 50 mg DHEA decreased the levels of serum ROS and a biomarker of advanced glycation end products (AGEs), pentosidine, and increased the levels of GSH and vitamin E. That is, DHEA mitigated oxidative stress in T2DM, enhanced the defense system against oxidative stress, and simultaneously, inhibited the accumulation of AGEs ( Fig. 2 ) ³⁸ ). These findings also suggest another mechanism for anti-atherosclerotic effects of DHEA.
(2) Anti-obesity effects
We reported that DHEA had remarkable anti- obesity effects on Zucker fatty rats, which are rat models with genetic obesity 391 . HCD-induced obesity has been also reported to be improved by DHEA 40 . As possible mechanisms, DHEA is known to increase the level of anti- obesity hormones like estrogen and IGF-1, and increase the fatty acid β-oxidation via activation effects of peroxisome proliferator-activated receptor α (PPARα) in the muscles and the liver 41) . Furthermore, there is a hypothesis that accelerated expression of 11β-HSD-1 in adipose tissues would increase the cortisol production in local fat, and then, induce metabolic syndromes. It was reported that DHEA reduced the cortisol levels in tissues via the modification of adipose cells, 11 β - HSD activities 18-20 ) A randomized, controlled, double-blind clinical trial in 2004 with a target of 56 subjects at 65-78 years of age (28 men and 28 women) reported the outcome that a significant decrease of visceral and subcutaneous fat and the improvement of insulin resistance were observed in both men and women in groups with DHEA replacement 421 . However, most studies on DHEA administration to humans, excluding this trial, suggested that DHEA effects were neutral in terms of body composition including body weight. For example, in two trial results of influences on body composition including fat, there were no significant differences between groups with DHEA administration and groups without administration of DHEA; one clinical trial which was reported in 2005, providing  DHEA  administration (50 mg  per  day)  to  140 healthy aged subjects for one year 43) , and another in 2006 that administered 50 mg per day to 87 healthy aged female subjects for two years 44 ).
(3) Effects on skeletal muscles
Research of rat models has suggested that DHEA has the effect of an increase in muscle mass via protein anabolism. That is, DHEA at physiological concentration inhibits the expressions of MuRF-1 mRNA, which is the proteolysis system of rat myoblasts, increases the expressions of myosin heavy chain of muscle contractile proteins, and maintains the muscle mass 459 . In rat strains, exercise and DHEA administration promote de novo steroidogenic enzymes in muscles and increases the muscle mass with the increase of testosterone, dihydrotestosterone, and estradiol 46) . There is a positive correlation in humans between the serum DHEA-S concentration and the muscle strength and mass 47) . DHEA increase decrease Fig.2. Schematic representation of the action of DHEA on oxidative stress, antioxidant system and AGEs accumulation in T2DM. diabetes mellitus. The figure is based on the results of reference 38). administration to patients with muscular dystrophy has exhibited an increase in muscle strength and has improved daily activities 48) . For the elderly, exercise and DHEA administration increased muscle strength and mass 49 , and the muscle strength of the lower limbs 50 . On the other hand, glucocorticoid including cortisol, shows protein catabolism in most tissues which contain muscles. Glucocorticoid activates ubiquitin-proteasom system in muscles, inducing the degradation of skeletal muscle 51,52) We performed a logistic regression analysis on 108 patients with T2DM (age: 65 years old or older, mean age: 76.2 years old) with an objective variable as sarcopenia diagnosed by the Asian version of the diagnostic criteria for sarcopenia. We clarified that the independent risk factor for sarcopenia was an elevation of the serum cortisol/DHEA-S ratio (high value of serum cortisol and low value of serum DHEA-S), in other words, the serum cortisol/DHEA-S ratio ≥ 0.2. The high- level cortisol induces the catabolism of skeletal muscles, while the low-level DHEA reduces the anabolism of skeletal muscle, as is shown in Fig. 3 53).
(4) Anti-diabetic effects
Most cross-sectional studies targeting relationships between serum DHEA concentration and diabetes have suggested that the control of diabetes is unfavorable when DHEA levels are lower. However, there are few results of longitudinal studies which suggest significant relations between the onsets of diabetes and serum levels of DHEA or DHEA-S 54-57) . Only one trial, a Rotterdam Study with 5,189 male and female subjects, reported that the incident rate of T2DM was higher when the serum concentration levels of DHEA were lower but there was no association with serum DHEA-S concentration 58) . Results of studies concerning the effect of DHEA administration on insulin resistance in humans were suggested to be controversial; improvement 42, 59) no alternation 44, 60) and deterioration 61). In particular, a trial regarding DHEA replacement therapy for a relatively long term of two years with a target of 112 elderly subjects, reported that DHEA replacement did not show clear effects in terms of insulin secretion, insulin resistance, and postprandial blood glucose level 609 . A small- scale investigation in Japan reported that oral administration of DHEA to humans (25 mg per day) improved functions of human vascular endothelial cells and insulin resistance ² ⁶ ). On the other hand, a variety of studies regarding improvement effects of DHEA on insulin resistance and glucose tolerance have been reported, using various types of impaired glucose tolerance models, such as cultured cell models and rodent models. These studies have reported on beneficial glucose tolerance effects with reproducibility 62-67) since Coleman et al . reported that DHEA had remarkable effects on db/db mice 629 . Its mechanisms are the inhibition of enzymes responsible for gluconeogenesis like glucose 6-phosphatase (G6Pase) and the enhancing effects of insulin signaling via the activation of the PI3K-AKT system. These studies reported that in cell levels, DHEA had an enhancing effects on glucose uptake via protein kinase C (PKC) in adipose cells, and an enhancing effects on the glucose transportation via glucose transporter 1 and 4 (GLUT1 and 4). a) C
Fig.3. Relation between (a) cortisol, (b) DHEA-S,
(c) Cortisol/DHEA-S ratio and severity of
sarcopenia.
The graphs are plotted as mean ± SD. p values were determined by the Jonckheere–Terpstra test for increased or decreased tendency of continuous variables. *p < 0.05, **p < 0.01, ***p < 0.001 vs non-sarcopenia (non sarco) as determined by a multiple comparison method (Fisher least significant difference test) after ANOVA. Pre sarco, presarcopenia; Sarco, sarcopenia; Severe sarco, severe sarcopenia; DHEA-S, dehydroepiandrosterone-sulfate; SD, standard deviation; ANOVA, one wat analysis of variance. The figure is based on the results of reference 53).
Conclusion
Clinical trials on DHEA replacement remain small in number. Clear-cut clinical effects are difficult to observe as long as an administration period of one or two years is concerned. However, few negative aspects such as adverse side effects have been recognized, which is an advantage of DHEA. Further evaluations are required from a long-term perspective. Reductions of serum DHEA-S level are observed in patients with diabetes. However, such reductions can be recovered by the improvement of diabetes control. Thus, voluntary efforts or some medical intervention to improve metabolic and diabetic abnormalities will help persons to raise serum DHEA-S levels and to speed down the slope of DHEA-S decline with aging, leading in the direction of anti-aging.
Reference
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Conflict of interest
Conflict of interest The author claims no conflict of interest in this study.
Acknowledgments
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