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IntroductionReducing sugars, such as glucose in basic solutions,

and lipids by β-oxidation or peroxidation generate formyl (aldehyde) and ketone groups. Aldehydes and ketones have a highly polarized carbonyl (C=O) group, the oxygen atom of which is electronegative and may react with nucleophiles in proteins. As a result, aldehydes and ketone groups may react non-enzymatically with cell proteins by glycation, and degrade protein function. Although similar reactions have been observed in vivo, we have little information on the relationship between these reactions and age-related changes in skin.

Many age-related regressive changes are actually due to protein degradation, such as posttranslational modification (proteomic denaturation), accumulation of degenerated wastes, deterioration of functional proteins, functional disorder of the tricarboxylic acid (TCA) cycle, or activation of inflammatory pathways by intracellular signals 1). All of these changes are symptomatic of “glycation stress”.

Review Article

Glycation Stress and Photo-Aging in Skin

Masamitsu Ichihashi, Masayuki Yagi, Keitaro Nomoto, Yoshikazu Yonei

Anti-Aging Medical Research Center and Glycation Stress Research Center, Graduate School of Life and Medical Sciences, Doshisha University

AbstractThis article reviews glycation stress as a factor linked to age-related diseases and functional and structural alterations of the skin.

Photo-aging and glycation stress are major causes of skin deterioration. Glycation occurs when a reducing sugar, such as glucose or fructose, combines with a protein in a non-enzymatic reaction forming a glycated protein. Further reactions form advanced glycation end products (AGEs), which may accumulate in tissue. AGEs may also bind to a receptor for AGEs (RAGE), thus inducing inflammatory changes in skin and other tissues. The concept of glycation stress includes cellular and tissue responses to reducing sugars and aldehydes, not just to the production of AGEs.

Glycation stress may be induced and affected by many factors, including exposure to ultraviolet light, which drastically intensifies AGE accumulation. AGEs modify skin collagen by reducing skin elasticity, and one result is wrinkle formation. AGEs accumulated in skin may be detected using auto-fluorescence (AF); our data from 136 healthy Japanese females shows the AF level is correlated with age.

Glycation stress, and subsequently skin aging, may be reduced by managing levels of blood glucose, low-density lipoprotein cholesterol, and triglyceride through an appropriate diet, lifestyle, and intake of anti-glycation materials such as AGE generation inhibitors, AGE decomposers, and AGE receptor antagonists. Study of glycation stress may help identify new anti-aging treatments.

KEY WORDS: AGEs (advanced glycation end products), elasticity, ultra violet, pentosidine, Nε- (carboxyl)methyllysine (CML)

Received: May. 17, 2011 Accepted: Jun. 2, 2011Published online: Jun. 13, 2011

Anti-Aging Medicine 8 (3) : 23-29, 2011(c) Japanese Society of Anti-Aging Medicine

Prof. Yoshikazu Yonei, M.D., Ph.D.Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences Doshisha University

1-3, Tatara Miyakodani, Kyotanabe city, Kyoto Prefecture, 610-0321 JAPANTel: +81-774-65-6382 / Fax: +81-774-65-6394 / E-mail: yyonei@mail.doshisha.ac.jp

Glycation stressGlycation stress includes a number of different chemical

reactions. For example, a Maillard reaction is defined as a non-enzymatic/irreversible reaction of reducing sugar and protein. This reaction may generate advanced glycation end products (AGEs) by a chain of various intermediate reactions that produce Schiff bases and Amadori products. A Schiff base (general structure R1R2C=N-R3, where R3 = alkyl or aryl or similar group, but not H) is a reversible compound generated from the interaction of a reducing sugar, such as glucose, and lysine or arginine amino acids or the N-terminal amino group in a protein 1). A Schiff base may be further modified by Amadori rearrangement. For example, hemoglobin A1c is an irreversible ketoamine formed by glycation of hemoglobin. The overall reaction is divided into three parts, an initial reaction, intermediate product generation, and an advanced reaction (Fig. 1).

Inter mediate products include 3-deoxyglucosone (3DG) 2), glyoxsal 3), methylglyoxal (MG) 4), glycolaldehyde (GA) 5), glyceraldehyde, are also carbonyl compounds that may generate AGEs. For example, 3DG is an α-dicarbonyl compound 10,000 times as reactive as glucose generated

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Glycation Stress and Photo-Aging in Skin

from an Amadori product. When plasma 3DG concentration rises above 100 nmol/l, the risk of diabetic retinopathy and nephropathy doubles 6). Several other AGEs have also been implicated in the pathology of age-related diseases.

In other reactions, excess levels of reducing sugars, such as intracellular glucose, may affect mitochondria and disturb the TCA cycle by inducing production of excess fumaric acid. In turn, fumaric acid reacts rapidly with the amino acid cystein in proteins, finally forming S-(2-succinyl)cysteine (2SC) 1). The resulting protein modifications provoke functional disorders in vivo. Proteins that are susceptible to this reaction include cytoskeleton protein, heat shock protein 7), and adiponectin 8).

Once AGEs are generated, they are metabolized by protease and oxidized protein degradation enzymes, such as oxidized protein hydrolase (OPH), in the proteasome and then excreted. OPH is a serine protease widely distributed in vivo that preferentially degrades oxidized or glycated protein by proteolysis 9). OPH is known to be an acylamino-acid-releasing enzyme (AARE) which separates acylated amino acids from protein at the N-terminal 10). Although the precise functions and activation of AAREs are unclear, OPH may modify protein carbonylation. OPH serum levels are higher in diabetic rats than in non-diabetic rats, and the amount of protein modified by carbonylation is lower in serum with elevated OPH activity than in relatively low-activity samples 11).

Other enzymes are also known to modify AGEs and intermediate compounds. For example, glyoxalase 1 (GLO1) metabolizes methylglyoxal (MG), an intermediate product of glycation stress. In experimental animals, renal damage induced by ischemia and reperfusion was inhibited by gene over-expression of 1 (GLO1) 12).

Although OPH and GLO1 are integral components of the excretion pathway for AGEs and intermediate compounds, the activity of proteolytic enzymes decreases with age 13). For this reason, therapies that reduce AGE accumulation by activating the proteasome, OPH, or GLO1 may prove effective in treating age-related disease or other diseases linked to glycation, such as diabetes mellitus.

Glycation stress also includes secondary or derivative reactions (Fig. 1), and these reactions can be implicated in three major diabetic complications: neuropathy, nephropathy, and retinopathy. These diseases are characterized by a marked accumulation of AGEs in tissue. Glycation stress is extremely

high in hyperglycemia patients, where plasma glucose concentration exceeds 160 mg/dl, and may also be elevated in response to aldehydes generated by excessive alcohol consumption, hyper-triglyceridemia, or hyper-low-density lipoprotein (LDL)-cholesterolemia.

Glycation stress markersGlycation generates various substances that can be used

to identify glycation stress. For example, glycated hemoglobin (HbA1c) and glycoalbumin are Amadori products generated via Schiff base formation and are used as markers for diabetic evaluation. Other AGEs that may be used as glycation stress markers, including Nε-(carboxymethyl) lysine (CML) 14), pentosidine 15), pyrraline 16), clossline 17), GA-pyridine 18), Nε-(carboxyethyl)lysine (CEL) 19), Nω-(carboxymethyl) arginine (CMA) 20), and 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole 21).

CML is a non-f luorescent and non-cross-linked AGE generated from intermediate glyoxsal and can be observed in patients with diabetes or high oxidative stress. CML may react with collagen, and CML-ized collagen added to a culture of human dermal fibroblast induces apoptosis 22). CML is present in the skin, including the epidermal layer where metabolic turnover is higher than in deeper layers 23). CML attaches to f luorescent anti-CML polyclonal rabbit antibody and can be observed on fluorescent microscopy (Fig. 2).

Pentosidine is another common AGE, f luorescent and cross-linked and generated from ribose, arginine, and lysine. This compound is recognized as an early clinical marker for nephropathy. Recently, pentosidine has been used as a marker for detecting osteoporosis (bone aging) 24). Pentosidine is also found in dermal collagen, and concentrations increase with age. After adjusting for age, the accumulated concentration of pentosidine is much higher in diabetic patients than in healthy persons 25).

Fig. 1. Concept of glycation stress. AGEs: advanced glycation end products, RAGE: receptor for AGEs, TCA: tri-carboxylic acid.

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Glycation Stress and Photo-Aging in Skin

25

Receptors for AGEIn addit ion to disrupt ing cell funct ion by protein

modification, AGEs may also combine with a specific receptor for AGEs (RAGE). RAGE may play a pathogenetic role as AGE receptors by modifying intracellular signals and responses, such as activating cell signals that generate inf lammatory cytokines 1). Many other cell surface receptors are reported to recognize AGEs as a ligand besides RAGE. One such intracellular signaling pathway amplifies oxidation stress and activates the transcription factor NF-κB, which is mediated by the ras/MAP kinase pathway. In vascular endothelial cells, AGEs stimulate a RAGE-induced signal that executes gene expression of vascular endothelial growth factor (VEGF). VEGF augments vascular permeability and angiogenesis and generates vascular cell adhesion molecule-1 (VCAM-1), which enhances local inflammation 26).

Although RAGE is usually bound to cell membranes, some may be soluble outside cells (soluble RAGE) 1). Soluble RAGE may bind with AGEs, acting as a decoy receptor and thereby inhibiting activation of RAGE on cell membranes 27). Presence of soluble RAGE may therefore indicate resistance to glycation stress.

Glycation stress in skinAccumulation of AGEs such as CML in dermal tissue

greatly influences glycation stress in the skin. Recent reports have shown that carbonylated protein deposited in the outer layer of epidermis changes the optical characteristics of dermal cells by reducing skin transparency 28). AGEs formed in the corium cause skin yellowing 29). In the corneum, where cell turnover is rapid, K10 protein generated by differentiation of keratinocytes can be susceptible to AGE formation 30).

AGEs accumulate in skin partially by binding with collagen protein. A collagen fiber forms a triple-helical structure in vivo and maintains skin elasticity in combination with elastin fiber. Lysine and arginine amino acids in collagen may be glycated to AGEs and form cross-links between fibers, thus reducing elasticity 31) (Fig. 3). Glycation of collagen may also cause loss of skin elasticity and wrinkle formation, although this has yet to be confirmed.

A second example of AGE-induced glycation stress arises from pentosidine. This compound is a strong activator of NF-κB, and induces cytokines generation and causes inflammatory skin changes.

Photo-aging and AGEsSymptoms of photo-aging, such as spots (lentigo seniles),

wrinkles, or tumors, frequently develop in skin repeatedly exposed over many years to ultraviolet (UV)-A, UV-B, or infrared light from the sun. After age 40, small spots may develop on the back of the hands, and the number of spots may increase with age 32). These spots also develop in patients with xeroderma pigmentosum, a rare hereditary skin disorder caused by a defect in the enzymes that repair DNA damaged by ultraviolet light, a few months after birth. These previous findings suggest that this spot formation may result from mutation of a gene involved in melanin formation in epidermal keratinocytes and melanocytes 33). UV-A exposure-induced mutation of a gene controlling a transcription factor, such as stem cell factor (SCF), might alter gene expression in keratinocytes. One possible SCF receptor found in melanocytes, c-kit, is implicated in spot development, but the precise mechanism is unknown.

Fig. 2. Distribution of CML in human skin. Fluorescent-immunohistochemistry using anti-CML polyclonal rabbit antibody 23). Green fluorescence indicates presence of CML in the epidermal layer. CML:Nε-(carboxyl)methyllysine. Cross-section of human skin. Bar indicates 20 µm.

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Glycation Stress and Photo-Aging in Skin

A further cause of changes in skin color and transparency may be delayed turnover in the epidermis. UV-A light may delay epidermal turnover by inactivating enzymes that promote keratinocyte detachment and peeling of the horny layer, although this mechanism is also unclear. All of these alterations are induced by photo-aging and intensified by AGEs accumulation 22,23).

Reducation of skin elasticityThe reduction in skin elasticity in humans with age

(Fig. 4) 34) is assumed to be caused by a reduction in elastic fiber production, i.e. collagen, elastin, and extracellular matrix ingredients such as fibronectin, possibly due to some disorder in fibroblast function. However, collagen protein also deteriorates due to oxidation or glycation. The skin elasticity curve is

shifted downwards in patients with type 2 diabetes compared with that of healthy subjects, implying that glycation stress is a major factor in the reduction of skin elasticity.

Wrinkles appear on the face after age 30 as proteins, elastic fibers and collagen generated by fibroblasts in the dermis change, mainly due to solar irradiation 35-37). Low-dose solar irradiation elevates metalloproteinase concentration, by activating AP-1 and NF-κB which degrade collagen and elastin 35), and reduces the production of procollagen type I 36) .Shallow winkles may be caused by structural changes, such as skin dryness; deeper wrinkles form on the face and neck of persons chronically exposed to sunlight, such as agricultural workers. In some of these workers, deep, triangle-shaped wrinkles called “diamond-shaped skin” appear on the neck by age 50, and the skin may appear yellowish and feel like hard cloth. In diamond-shaped skin, anti-CML antibody-positive substances are deposited in clumps in the middle and upper layers of the dermis, a condition known as solar elastosis. The clumps are detectable by staining elastin via van Gieson’s method 23). These

Fig. 3. Glycation of collagen and AGEs accumulation in the skin. Fluorescent AGEs in the skin are detected by AGE Reader. AGEs: advanced glycation end products.

Fig. 4. Changes in skin elasticity with age in diabetic patients and a control group. Data shows elasticity index R2 and R7 in healthy Japanese measured at left inner upper arms measured with a cutometer (Courage & Khazaka) 34). R2: biological elasticity (Ua1/Uf1), R7: elastic recovery (Ur1/Uf1), DM: diabetes mellitus. Data are expressed as mean ± standard error mean (SEM).

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Glycation Stress and Photo-Aging in Skin

27

characteristic wrinkles are not seen in facial skin before the age of twenty or in older persons not exposed to UV light.

UV-B light also stimulates production and release of inflammatory cytokines, such as IL-1α, IL-6, and TNFα from keratinocytes in the epidermis. In turn, the cytokines stimulate f ibroblasts in the dermis and keratinocytes through the autocrine and paracrine systems and elevate mRNA formation and synthesis of matrix metalloproteinase (MMP)-1, MMP-3, or MMP-9, enzymes which degrade collagen and elastic fibers 38) .MMP-1 is known to cut fibrous protein and may be responsible for wrinkle formation. Elastase is another enzyme which breaks down elastin, and inhibition of elastase has been found to reduce UV exposure-induced skin wrinkle formation in experimental animals. Taken together, these previous findings indicate that wrinkle formation is due to quantitative and qualitative changes in elastic fibers 37), which may be stimulated by UV light.

UV-A may also act directly on fibroblasts in the corium, enhancing mRNA formation and protein enzyme synthesis 39). Reactive oxygen species (ROS) induced by UV-A, UV-B, and infra-red light may also elevate levels of MMP-1 mRNA 40) ,in turn leading to further collagen degradation. Given that mitogen-activated protein kinases (MAPK) have been implicated in this intracellular signal pathway 41), anti-oxidants may effectively prevent wrinkle formation.

Non-invasive skin AGEs examination (AGE Reader)

The AGE reader™ (DiagnOptics, Groningen, Netherlands) is designed to non-invasively evaluate the level of AGEs accumulated in sk in 42- 44), ut i l izing the pr inciple that accumulated AGEs emit characteristic autof luorescence (AF) when excited by UV light (Fig. 3). The AF of immuno-histochemical skin biopsies from patients with diabetes mellitus and nephropathy receiving dialysis is correlated with the level of accumulated AGEs, such as pentosidine or CML, although

CML is a non-fluorescent AGE 45).Measurements of skin AF in healthy Japanese females

show that AF intensity increases with age (Fig. 5). In Europe and the USA, it is common practice to measure the skin AF for Caucasian patients from the upper forearm. However, results from the present authors’ previous research showed that precise measurement of AF in suntanned Asian subjects was difficult. Clinical practice in Japan is to have the subject rest his or her cheek on his or her hands and measure the AF from a 10-cm length of skin, on the back of the arm above the elbow 44). Further data will be required to validate this method of measuring skin AF for Asian patients.

Precisely which part of the skin fluoresces in response to the AGE reader or the types of AGE detected and their locations within the dermis remain unclear at present using this method. We assume that the AGEs measured by the AGE Reader are pentosidine, crossline, and pyrropiridine.

Prevention and treatment of glycation stressTo prevent skin aging, potential treatments must be

effective against photo-aging, oxidative stress, and glycation st ress. Primary prevention is achieved by encouraging maintenance of reasonable skeletal muscle mass, moderate exercise, and proper eating habits. These recommendations will help ensure even concentrations of blood sugar and reduce insulin resistance.

Muscle load training is particularly important. More than 70% of blood glucose is consumed in skeletal muscle, and reduction of muscle mass increases insulin resistance. Eating habits that reduce glycation stress are to eat slowly, chew food well, choose foods that do not raise blood sugar rapidly (i.e. foods with a low glycemic index), and avoid high sugar content foods, such as juice, carbonated drinks, and sweets. We also recommend moderate or minimal alcohol intake, as alcohol metabolites enhance glycation stress in vivo.

Persons with excessive weight or waist size should

Fig. 5. Changes in skin AF derived from AGEs with age. Data shows AF values measured using an AGE Reader in healthy Japanese females (n = 136, mean age: 49.4 ± 20.6 years). Measurements were taken from a 10-cm length of skin on the back of the arm above the elbow. Regression line; Y = 0.017X + 1.0503, r = 0.603, p < 0.001. AF: auto fluorescence.

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Glycation Stress and Photo-Aging in Skin

receive medical checkups and manage their blood sugar, LDL-cholesterol, and triglyceride levels. Numerical targets for each measure should follow the guidelines published by the Japan Diabetes Society or Japan Society of Ningen Dock.

Further treatment with anti-glycation materials may be provided if necessary after lifestyle factors have been corrected. In the future, we expect that the following therapies may be provided to treat AGE-related disorders: AGE generation inhibitors, AGE breaker that enhance AGE metabolism 46,47), and AGE receptor antagonists 48). Aminoguanidine is one such AGE generation inhibitor 49-51) but is associated with a high frequency of side effects and is not used clinically in Japan. A mixture of herbal extracts from chamomile (Anthemis nobilis), hawthorn berry (Crataegus oxyacantha), doku-dami (Houttuynia cordata), and grape leaf (Vitis vinifera) has been found effective in inhibiting AGE formation in vitro 56), in experimental animals 56), and in clinical trials 49,57). Astragaloside, which is der ived f rom Astragalus radix, is reported to significantly inhibit generation of CML

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