Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : June 24, 2016
Accepted : December 1, 2016
Published online : December 31, 2016

Original article

Melanin synthesis induction by advanced glycation end-products (AGEs) without
α-melanocyte stimulating hormone (α-MSH ) or UV exposure
Yumi Abe 1, 2), Wakako Takabe 1), Masayuki Yagi 1), Akemi Uwaya 2), Fumiyuki Isami 2),
Shoichi Yamagishi 3), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center, DoshisAha University, Kyotanabe, Kyoto, Japan.
2) Quality Assurance & Research, Morinda Worldwide, Inc., Tokyo, Japan.
3) Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications Kurume University School of Medicine,
Kurume, Fukuoka, Japan

Abstract

Ultraviolet (UV) radiation causes irregular skin pigmentation known as freckles. The mechanism behind UV-induced
melanin production has been studied extensively, with the resulting knowledge being used to develop commercial skincare
products. Some freckles, such as senile lentigo (age spots), may appear independently of UV exposure. The role of aging in
the development of these freckles is not well-studied. We hypothesized that advanced glycation end-products (AGEs) induce
age spots and, therefore, examined their effects on melanogenesis in B16 murine melanoma cells (B16F10). Significant
melanin production occurs in melanoma cells, after incubation with AGE-collagen (collagen-glucose or collagen-fructose).
We further investigated the active compounds in AGE-collagen and identified methylglyoxal (MGO) as having the strongest
activity. But melanin production by collagen-fructose is less active than collagen-glucose, even though it contains more
MGO. This discrepancy suggests that AGEs-induced melanogenesis is caused by multiple factors. This is the first report ofmelanin synthesis being stimulated by AGEs without exposure of UV orα-melanocyte stimulating hormone (α-MSH). As
AGEs-induced melanogenesis can result in brown spots or freckles on the skin (AGE pigment freckle), managing AGEs may
be as important as UV care for the maintenance of healthy, bright skin.

KEY WORDS: Advanced glycation end-products (AGEs), AGE pigment freckle, collagen, glucose, fructose,
B16 melanoma cell.

Introduction

Aging deteriorates cellular function and structure andfrequently leads to organ dysfunction. Morphological changes
in skin are obvious. As such, skin aging receives a lot of
medical and commercial attention. With increasing age,dermal and epidermal layers become thin, less resilient, andprone to damage. While such changes seem inevitable, theskin is often more affected by environmental factors than
genetics. Among these factors, ultraviolet (UV) irradiationis the most deleterious and is responsible for photoaging 1, 2).
Photoaging is defined as a “characteristic morphologicalchange such as wrinkles and freckles induced by chronic
UV exposure” 2). However, senile lentigo may also develop
independently of UV exposure. The cause is still unknown,
but it is likely due to age-related decline in, and/or abnormal

cellular function.
Glycation stress has been reported as a skin aging factor 3).
It results from the accumulation of advanced glycation endproducts (AGEs) that are formed by non-enzymatic reactions
between reducing sugars and proteins. The accumulation of
AGEs in tissues induces a number of structural and functionalchanges, including skin aging. Glycated collagens lose theirresilience, leading to wrinkles and inflexible skin 4). Ogura
et al. reported that lipid peroxidation-induced protein
carbonylation was responsible for dull and yellowish skintones 5). However, it is not known if AGEs or glycation stresscontributes to freckling. Therefore, we investigated therole of AGE accumulation on melanogensis in B16 murine
melanoma cells.

Corresponding author: Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1- 3 Tatara Miyakodani, Kyotanabe City, Kyoto 610-0394, JAPANTEL: +81-774-65-6382 FAX: +81-774-65-6394 E-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Abe Y, Yumi_Abe@jp.morinda.com ; Takabe W, wtakabe@mail.doshisha.ac.jp ;Yagi M, myagi@mail.doshisha.ac.jp ; Uwaya A, Akemi_Uwaya@jp.morinda.com ;Yamagishi S, shoichi@med.kurume-u.ac.jp ; Isami F, Fumiyuki_Isami@jp.morinda.com.

_ 22 9 _Glycative Stress Research 2016; 3 (4): 229- 235(c) Society for Glycative Stress ResearchMaterials and methods
Reagents

Synthetic melanin, [ Nle 4, D-Phe 7 ]-α-melanocyte
stimulating hormone trifluoroacetate salt (α-MSH), 3,
4-Dihydroxy- L -phenylalanine ( L -DOPA) and methylglyoxal(MGO) were purchased from Sigma-Aldrich Japan (Tokyo,
Japan). Glyoxal (GO) was purchased from Wako Pure
Chemical Industries, Ltd. (Osaka, Japan). 3 - Deoxyglucose(3DG) was purchased from Dojindo Laboratories(Kumamoto, Japan). N ε-(carboxymethyl) lysine (CML), N ε(carboxyethyl) lysine (CEL) and pentosidine were purchasedfrom Kurabo Industries Ltd. (Osaka, Japan). Collagen type I(bovine skin, pepsin-solubilized) was purchased from Nippi,
Incorporated (Tokyo, Japan). Other reagents were purchasedfrom Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Dulbecco’s modified Eagle’s medium (DMEM) was
purchased from Sigma-Aldrich (Tokyo, Japan).

Preparation of AGE-modified collagen

AGE-modified collagen (AGE-collagen) was preparedaccording to the method of Hori et al. 6), with minor
modifications. Briefly, the reaction solution contained 250
μL of 200 mM phosphate-buffer (PB; pH 7.4), 200 μL of
2.0 M sugar (glucose; Glu or fructose; Fru) solution, 200 μL
of 3.0 mg/mL collagen and 350 μL water. Control solutions
were prepared without collagen and/or without sugar. Thenthe reactions were incubated at 60 ℃ for 0, 1, 10, 24, 72
and 168 h. When conducting a test, reaction samples werediluted to 20 times in DMEM.

Fluorescence AGEs measurement

The fluorescence intensity of AGEs was measured witha microplate reader (Thermo scientific Varioskan™ FlashMultimode Reader, Thermo Fisher Scientific K.K., Kanagawa,Japan) at an excitation of 370 nm and emission at 440 nm.
Each fluorescence value was calculated as a ratio between
the fluorescence intensity of the sample and 5 μg/mL quinine
sulfate in 0.1 N sulfuric acid, multiplied by 1,000.

Quantification of 3-deoxyglucose (3DG),
methylglyoxal (MGO) and glyoxal (GO)3DG, MGO and GO were measured by HPLC according
to Hori et al. 6), with slight modification. Briefly, 200 μL ofreaction solution was combined with 130 μL water and 170
μL 6% perchloric acid, stirred, then centrifuged at 13,800 ×g (12,000 rpm) for 10 min. Next, 400 μL of the supernatant
was combined with 350 μL saturated sodium bicarbonate
solution and 50 μL 2, 3-diaminonaphthalen (DAN) labeling
reagent (1mg/mL), stirred, then incubated for 24 h. Afterwards,
chromatographic separation involved a Unison UK-Phenyl,
75 × 3 mm I.D., column (Imtakt, Kyoto, Japan). The mobile
phase was 50 mM phosphoric acid: acetonitrile (89:11)
and was eluted at 1.0 mL/min. Detection was at excitation
wavelength 271 nm and detection wavelength 503 nm.
Concentrations of 3DG, MGO and GO in samples were
determined with standard curves, based on peak areas frompreviously analyses of chemical reference standards.

Cell culture

B16 murine melanoma cells (B16F10) were purchasedfrom Riken BioResource Center (Ibaragi, Japan) in February
2015. The B16 cells were cultured in DMEM supplemented
with 10 % (v/v) fetal bovine serum (FBS, Nichirei
Biosciences Inc., Tokyo, Japan) and 1% Pen Strep (a mixtureof 10,000 U/mL penicillin and 10,000 μg/mL streptomycin,
Life Technologies Japan Ltd., Tokyo, Japan) at 37℃ in ahumidified, CO2-controlled (5%) incubator.

Melanin content assay

Melanin synthesis in B16 cells was evaluated according
to methods described by Masuda et al. 7) and Lee et al. 8), withslight modification. Briefly, 900 μL cell culture in phenol red
free DMEM were seeded on 24 well-plates (2 × 10 4 cells/well), then treated with 100 μL sample 24 h after seeding.
The samples evaluated were AGE-collagen and its
chemical components, including MGO, GO, 3DG, CML, CEL
and pentosidine. α-MSH was used as the positive control.
α-MSH was dissolved in aqueous acetic solution (5%, v/v),
then diluted with DMEM to 0.1 μM.
After treatment for 72 h, the cell cultures were centrifuged.
The supernatants were collected and transferred to a 96 wellplate for measurement of secreted melanin. Cell pellets were
washed with phosphate-buffered saline (PBS, pH 7.4) andlysed with 1N NaOH for 1 h at 60 ℃. After centrifugation,
the supernatants of lysed cell pellets were transferred to a
96 well-plate for measurement of intracellular melanin.
Absorbance was measured at 405 nm with using a microplate
reader (Thermo scientific Varioskan™ Flash Multimode
Reader, Thermo Fisher Scientific K.K., Kanagawa, Japan),and melanin concentration was determined using a standard
curve prepared with synthetic melanin.

Tyrosinase activity assay

Intracellular tyrosinase activity in B16 cells was evaluatedaccording to the slightly modified methods of Masuda et al. 7)
and Li et al. 9). Cells were seeded and α-MSH used as apositive control, as described above. After treatment for 72h, the cells were washed with ice-cold phosphate-bufferedsaline (PBS, pH 6.8) and then lysed with 0.1% triton in PBS(pH 6.8). The lysates were centrifuged at 10,000 rpm and
4 ℃ for 10 min to obtain a supernatant that contained
tyrosinase. Protein concentration of the supernatant wasquantified by DC protein assay (Bio-Rad Laboratories, Tokyo,
Japan) and adjusted with lysis buffer. The reaction mixture,containing the supernatant (tyrosinase) and 0.1% L-DOPA
solution in PBS, was incubated at 37 ℃ for 20 min. Afterincubation, dopachrome formation was assayed by measuring
absorbance at 475 nm with a microplate reader. Tyrosinase
activity in treated cells was calculated as a percentage withrespect to activity in pretreated cells.

Cell viability assay

Cell viability was determined by the Cell Counting Kit- 8(Dojindo Laboratories, Kumamoto, Japan), which measuresdehydrogenase activity using 2-(2-methoxy-4-nitrophenyl)3-(4-nitrophenyl)-5-( 2, 4-disulfpphenyl)-2H-tetrazolium(WST-8) as a substrate. Test samples were dissolved DMEM
to an appropriate concentration. In the control group, DMEM
solution was used instead of the sample solution. Briefly, 2.8
× 10 3 cells in the DMEM (90 μL) were placed on 96 -wellplate. After 24 h of incubation at 37 ˚C, 5 % CO 2, AGEs or
α-MSH in DMEM (10 μL) were added. After incubation_ 23 0 _Glycative Stress Researchfor 72 h more, DMEM was replaced with a WST solution(WST: culture medium = 1:10, 100 μL). After incubation for
1 h, the resulting formazan was determined by measuring
absorbance at 450 nm with a microplate reader. Cell viability
was expressed as a percentage of the control group value.

Statistical analysis

The mean ± standard deviation (SD) was calculated forthe data obtained from experiments. Intergroup differenceswere evaluated by one-way analysis of variance (ANOVA)
followed by Dunnett’s multiple comparison test using Statcel
Ver. 3 (OMS Publishing Inc., Saitama, Japan, 2011).

Results

Effect of AGE-collagen (collagen-glucose or- fructose)
on melanogenesis in B16 murine melanoma cells

We investigated the conditions of AGE-induced
melanogenesis in B16 murine melanoma cells. Both collagenGlu and collagen-Fru induced melanin formation after 72 h
incubation without affecting cell proliferation ( Fig. 1 ). The
photoemission from fluorescent AGEs may confound melanin
absorption. However, the absorbance of AGE-collagen after
72 h treatment at 37 ˚C was less than 0.02 at 405 nm (data notshown). Therefore, AGEs did not influence the quantificationof melanin. Compared to collagen-Fru, collagen-Glu promoted

cell proliferation(% of control )Stimulant time Sugar

140 120 100 80 60 40 20

Melanin contents (% of control )0Cont
Collagen
(- )

0h

50

100

150

200

250

Secreted (Supernatant)Glu

Intracellular (Pellet)

Fru
−
0h

Glu
Fru
−

1h

Glu
Fru
−

10 h

＊＊

Glu
＊

Fru
Collagen
( +)

−
24 h

Glu

＊＊

＊＊
＊＊

Fru
−
72h

Glu

＊＊

＊＊
＊＊

Fru
−
168 h

Glu
Fru

α-MSH

−

＊＊

＊＊

＊＊

＊＊
＊＊

−

Fig. 1. Effect of AGE-collagen on melanin synthesis in B16 cells＊＊

Melanin content is provided as the mean ± SD of 3 experiments. Significantly different from the control group: *; p<0.05, **; p<0.01. Cell
proliferation values are mean ± SD of 3 experiments. AGE, advanced glycation end-product; Cont, control; Glu, glucose; Fru, fructose;
α-MSH, α-melanocyte stimulating hormone; SD, standard deviation.

_ 231 _

melanin formation in a shorter amount of time. CollagenGlu, incubated for over 24 hours, was able to stimulate melanin
production as much as α-MSH. When incubated for over 7days, collagen-Fru also stimulated melanocytes as much as
α-MSH did.

Effect of AGE-collagen (collagen-glucose or- fructose)
on tyrosinase activity of B16 murine melanoma cells

Since AGE-collagen stimulated melanin production, we
measured tyrosinase activity in B16 murine melanoma cells.
While α-MSH significantly enhanced tyrosinase activity,

AGE-collagen only had a minor effect ( Table 1 ).

Effect of AGE-collagen (collagen-glucose or- fructose)
on melanogenesis of B16 murine melanoma cells

We further investigated the active principle responsible
for AGE- collagen-induced melanogenesis. Melanogenesis
capacity was evaluated for three AGEs (CML, CEL, andpentosidine) and three AGE intermediates (3DG, GO, MGO).
CML, CEL, and pentosidine had no activity, while 3DG and
GO displayed weak activities. However, MGO exhibitedstrong melanogenic activity ( Fig. 2 ).

Table 1. Effect of AGE-collagen on tyrosinase activity in B16 cells

Stimulant
Glu or Fru

Tyrosinase activity (%)(n = 3)Cell proliferation (%)(n = 3)0

100.0 ± 29.4

100.0 ± 2.8

0121.2 ± 7.2

106.8 ± 14.6

0120.7 ± 6.2

105.7 ± 8.0

0101.1 ± 0.7

110.2 ± 5.3

0116.4 ± 12.9

107.7 ± 11.8

0117.1 ± 14.9

106.3 ± 6.0

1108.7 ± 0.7

102.3 ± 3.5

1126.7 ± 4.2

105.7 ± 3.8

1118.0 ± 5.6

105.7 ± 5.9

10109.1 ± 1.6

110.1 ± 7.8

10126.7 ± 20.9

109.6 ± 7.2

10128.9 ± 14.1

109.5 ± 7.2

24114.4 ± 4.8

106.0 ± 3.4

24131.4 ± 9.8

107.4 ± 7.8

24130.8 ± 14.0

105.7 ± 9.4

72117.5 ± 4.2

105.9 ± 1.2

72131.7 ± 8.5

105.4 ± 2.7

72122.3 ± 13.7

107.4 ± 6.2

168113.2 ± 1.0

110.3 ± 7.7

168122.6 ± 13.4

109.6 ± 8.1

168120.3 ± 5.2

110.6 ± 8.7−

220.7 ± 12.7**

110.0 ± 0.6

Time (h)Incubation without collagen
Control
Glu
Fru
Incubation with collagen
−
Glu
Fru
−
Glu
Fru
−
Glu
Fru
−
Glu
Fru
−
Glu
Fru
−
Glu
Fru
α- MSH−

Significantly different from the control group, **; p＜0.01. Results are expressed as mean ± standard deviation. AGEs, advanced
glycation endo-products; Glu, glucose; Fru, fructose; α-MSH, α-melanocyte stimulating hormone.

_ 232 _Glycative Stress Researchcell proliferation(% of control )Sample(μg/ml)140 120 100 80 60 40 20

Melanin contents (% of control )
0

50

100

150

200

250

Cont
＊

0.1
MGO

＊＊

0.5

＊＊

＊＊

2.5

＊＊

0.1
GO

0.5
＊＊

2.5
0.02
3DG

0.1
＊＊

0.5

＊＊

0.1
CML

0.5
2.5
0.1

CEL

0.5
2.5
0.02

Pentosidine

α-MSH

0.1

Secreted (Supernatant)

0.5

Intracellular (Pellet)＊＊

0.1 μM＊＊

Fig. 2. Effect of AGEs (CML, CEL and pentosidine) and reaction intermediate (MGO, GO and 3DG) on melanin synthesisin B16 cells
Melanin content is provided as the mean ± SD of 3 experiments. Significantly different from the control group: *; p<0.05, **; p<0.01. Eachvalue in cell proliferation represents the mean ± SD of 3 experiments. AGEs, advanced glycation end-products; CML, N ε- (carboxymethyl)
lysine; CEL, N ε- (carboxyethyl)lysine; MGO, methylglyoxal; GO, glyoxal; 3DG, 3-deoxyglucosone; Cont, control; α-MSH, α-melanocyte
stimulating hormone; SD, standard deviation.

Measurement of fluorescent AGEs, 3DG, MGO and
GO in the AGE-modified collagenWe then quantified the formation of fluorescent AGEs
and AGE intermediates in AGE-collagen. Crossline,
pyrrolopyridine, and pentosidine are examples of fluorescent
AGEs. AGE intermediates and fluorescent AGEs occurred
less and were more slowly formed in collagen-Glu than incollagen-Fru ( Fig. 3 ).

Discussion

Melanogenesis causes irregular pigmentation, such as skinblotches and freckles. Stressors, such as UV light andinflammation, stimulate the production of α-MSH, endothelin-1(ET-1), stem cell factor (SCF), and secretion of other cytokines
in epidermal keratinocytes. As melanocytes react with these
activating factors, cellular signals initiate melanin production 10).
UV induced mutation of these genes in keratinocytes may_ 233 _

A

B
1.4
1.2

Collagen-Glu

1.0

Collagen-Fru

0.8
0.6
0.4

0

1.0

Collagen-Fru

0.8
0.6
0.4

0
0

1

10

(day)

24

72

168

(hour)

1

3

7

(day)

C

0

1

10

24

72

168

1

3

7

24

72

168

1

3

7

D
100

Collagen

800

Collagen-Glu

700

Fluorescence intensity120

3DG (µg/ml)

Collagen-Glu

0.2

0.2

(hour)

Collagen

1.2

GO (µg/ml)

MGO (µg/ml)

1.4

Collagen

Collagen-Fru

80
60
40
20
0
(hour)(day)

Collagen
Collagen-Glu

600

Collagen-Fru

500
400
300
200
100
0

0

1

10

24

72

168

(hour)

1

3

7

(day)

0

1

10

Fig. 3. MGO, GO, 3DG and fluorescent AGEs in AGE-modified collagen

A: MGO, B: GO, C: 3DG, D: fluorescent AGEs. Each value represents the mean ± SD of 3 experiments. MGO, methylglyoxal;
GO, glyoxal; 3DG, 3-deoxyglucosone; AGEs, advanced glycation end-products; Glu, glucose; Fru, fructose; SD, standard
deviation.

cause freckles on the skin. Tyrosinase generates dopaquinone
by oxidizing tyrosine. Dopaquinone then undergoes nonenzymatic reactions which lead to precursors of melaninformation. Tyrosinase inhibition has been a main target ofthe cosmetic industry for the prevention of dark spots or
freckles 11). Many types of skincare products have beendeveloped through the in vitro evaluation of ingredients forinhibitory activity against tyrosinase, melanin production,and α-MSH stimulated gene expression.
In our study, we discovered that AGE-collagen is capableof stimulating melanogensis in B16 murine melanoma cells,with equal or greater activity than α-MSH. This suggeststhat AGE accumulation may stimulate melanin synthesis andpossibly contribute to freckle formation on the skin. However,
AGE accumulation only slightly increased tyrosinase activity,

an effect which was not statistically significant.
Since over expression of the receptor for advanced
glycation end products (RAGE) is reported in various cancer
cells 12), B16 murine melanoma cells (B16F10) may also overexpress RAGE. AGEs/RAGE interaction reportedly elevates
ROS levels through RAGE-NADPH oxidase activation 13, 14).
UV-induced free radical/reactive oxygen species (ROS)
production is strongly correlated with melanin content theskin 15), ROS, such as secreted hydrogen peroxide, cause AGE
formation 1), including CML 16). As such, it is possible thatmelanin production was enhanced by ROS that were generated
by RAGE-AGEs interaction. But further study is necessary
to confirm this hypothesis.
Wondrak et al. reported that UV exposure generates
hydrogen peroxide and hampers cellular proliferation of_ 23 4 _Glycative Stress Researchkeratinocytes and fibroblasts. A combination of UV exposureand AGE accumulation synergistically accelerates hydrogen
peroxide generation and inhibits cellular growth 1). The
accumulation of AGEs may not be the sole cause of skindamage. But it may be a synergistic enhancer. Epidermal
keratinocytes are differentiated regularly and are replacedby new cells. This epidermal turnover causes the excretionof melanin from the epidermis. But AGEs increase integrin
expression which slows turnover 17). AGEs deteriorate healthyskin via three potential mechanisms: (1) AGE accumulation
leads to the production of stress stimulants, (2) AGEs directly
promote melanin production, and (3) AGEs disrupt cellularepidermal turnover and retard melanin excretion. Amongthese mechanisms, the third seems most likely as a cause ofdull skin and partly a cause of freckles, due to AGEs causing
irregular retention of pigment in skin cells.
While investigating potential active compounds in AGEcollagen, we found that CML, CEL, and pentosidine had
no effect on melanin production. On the other hand, AGE
intermediates (MGO, GO, and 3DG) enhanced melanin
production. This indicates that these AGE intermediates arelikely melanogenic stimulants. However, these intermediates

are also present in collagen-Fru, which had very weak activity.
Additionally, the intermediates were less active than whole
AGE-collagen itself. This seems to suggest that glycation
associated melanin production is influenced by multiple
factors to be clarified in future.

Conclusion

This study revealed that AGE-collagen enhances melanin
production. While MGO was the most active of the principle
components, the various compounds in AGE-collagen work
synergistically together. Further research is necessary tobetter understand the mechanistic details of, and the active
compounds responsible for, AGE-induced melanogenesis.

Conflict of interest

The authors have no conflict of interest in this study.

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